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Quantification of the Extent of Diagenesis in Biogenic Apatite of Cenozoic Shark Centra

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Title:
Quantification of the Extent of Diagenesis in Biogenic Apatite of Cenozoic Shark Centra
Copyright Date:
2008

Subjects

Subjects / Keywords:
Apatites ( jstor )
Bones ( jstor )
Carbonates ( jstor )
Diagenetic processes ( jstor )
Fossils ( jstor )
Oxygen ( jstor )
Rare earth elements ( jstor )
Sea water ( jstor )
Sharks ( jstor )
Signals ( jstor )

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University of Florida
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University of Florida
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4/17/2006

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QUANTIFICATION OF THE EXTENT OF DIAGENESIS IN BIOGENIC APATITE
OF CENOZOIC SHARK CENTRA
















By

JOANN LABS HOCHSTEIN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Joann Labs Hochstein
































This dissertation is dedicated to my parents, Robert and Denise, and my Husband, Jason
for all their love and support.















ACKNOWLEDGMENTS

Firstly, I would like to thank my advisor, Bruce MacFadden, for his insights, expert

guidance, support, patience, and continual enthusiasm for this project. I also wish to

thank my committee members, Ellen Martin, Phil Neuhoff, Neil Opdyke, and John

Krigbaum, for their patience and support throughout this project. George Kamenov,

Jason Curtis, and Penny Higgins gave invaluable insight during sample preparation and

analysis. I am grateful for Clifford Jeremiah for providing the inspiration of this project

with the donation of Otodus obliquus specimens analyzed in this study. I would also like

to thank Michael Gottfried, Gordon Hubbell, Dirk Nolf, O. Sakamoto, and Sabine

Wintner for allowing me to borrow and sample specimens.

I would like to thank all my friends, especially Helen Evans and Steve Volpe, for

their continual support and comic relief. The staff of the Geological Sciences

Department, Ron Ozbun, Jody Gordon, and Mary Ploch, has been very helpful during my

study at the University of Florida. I would like to thank my parents and sister for their

constant support and patience. Finally, I would like to thank my husband, Jason, for the

encouragement to accomplish my dreams and his unconditional love.

This research was supported by Geological Society of America Grant number

2009018 and National Science Foundation grant EAR 0418042.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .............. ..... ..................................... .......... .. .. ............. .. vii

LIST O F FIG U R E S ......................................................... ......... .. ............. viii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 IN T R O D U C T IO N ............................................................................. .............. ...

2 DIAGENSIS AND VARIATION OF THE OXYGEN ISOTOPIC SIGNATURES
IN VERTEBRAL CENTRA FROM Otodus obliquus............... ............... 3

Intro du action ..................................................................................................... .... .. 3
M materials and M methods ................................................................. ....................... 8
G ross X -ray A nalyses ................... .......... .... .......... .. ...... ........... ... ...... 9
Fourier Transform Infrared Spectroscopy Preparation and Analysis..................9
Stable Isotope A nalyses................................................ ............ ............... 10
R results and D discussion ................................... .. ...... ... ........ .... ............ 12
Fossil and Recent Shark Centra Mineralization and Diagenesis......................12
Physical Increments and Variation.................. ...... ...............15
Stable Isotope (6180) Signal Archived in Eocene Otodus obliquus centra........17
C o n c lu sio n s..................................................... ................ 2 1

3 QUANTIFICATION OF DIAGENESIS IN CENOZOIC SHARKS:
ELEMENTAL AND MINERALOGICAL CHANGES ....................... .................23

Introdu action ................. .............. ................... ............. ................. 2 3
B one Chem istry and D iagenesis...................................... ........................ 24
R are Earth Elem ents ........................................................................... 26
M materials and M ethods ......................................................................... ............... 30
Fourier Transform Infrared Spectroscopy (FT-IR) ..........................................31
Elem ental A analysis (ICPM S) ........................................ ......................... 33
R results ...........................................................................................33
M ineralogical Changes ......................................................... ............... 33
E lem ental C concentration ........................................................... ....................34









D iscu ssion ........................................................................................ .. 4 1
M ineralogical Characterization of Centra ....................................................41
Implications for Diagenetic and Biological Signal Reconstruction ....................42
C o n clu sio n s..................................................... ................ 4 6

4 OXYGEN ISOTOPIC AND RARE EARTH ELEMENTAL ANALYSIS OF
MODERN LAMNID SHARKs: DETERMINATION OF LIFE HISTORY? ...........48

Intro du action ............. ......... .. .. ......... .. .. ............................................. 4 8
R are Earth Elem ents ........................... ........... ...................... ...............50
B background ............. .. .. .... .. ..........................................................................51
Great White (Carcharodon carcharias) ..................... ..................... 51
Longfin Mako (Isuruspaucus) ............................ ........................52
Shortfin M ako (Isurus oxyrinchus) ........................................ ............... 53
M materials and M methods ....................................................................... ..................54
X-radiograph Analyses ............... ........................... ............... 54
Oxygen Isotopic Preparation and Analyses......... ............................. 55
Bomb Carbon Dating Preparation and Analysis ...........................................55
Inductively Coupled Plasma Mass Spectroscopy (ICPMS) .............................57
Results and D discussion .................................... ..... .. ...... .............. 58
O ntogenic A ge D eterm nations ........................................ ....................... 58
R are Earth Elem ents .......................................................................... 61
C o n c lu sio n s..................................................... ................ 6 3

5 SUMMARY AND CONCLUSIONS......................................................................68

L IST O F R EFE R E N C E S ............................................................................. ............. 72

B IO G R A PH IC A L SK E TCH ..................................................................... ..................82















LIST OF TABLES


Table page

2-1. Comparison of crystallinity index (CI) and carbonate content (C/P) from FT-IR
spectrum for samples treated with acetic acid and samples that were not treated
with acetic acid and from Eocene and modern samples ............................... 13

2-2. Stable isotopic data for three vertebral centra of Otodus obliquus, UF 162732,
from the Early Eocene of Morocco.................................................20

3-1. Some possible substitutions in the apatite crystal structure. .....................................25

3-2. Lamnid shark specimens used in this study ...... ......... ....................................... 31

3-3. Elemental and mineralogical data of nine shark vertebral centra. Elemental
concentration are in ppm .. ............................. .... .......................................36

4-1. Lamnid specimens used in this study. ............................................ ............... 53

4-2. Ontogenic age estimates based growth ring counts (GR), oxygen isotopic (6180)
cyclicity, and bom b carbon (A13C)................................................ ............... 59

4-3. Elemental data (in ppm), and oxygen isotopic and bomb carbon dating ages ..........64
















LIST OF FIGURES


Figure page

2-1. Modern white shark, Carcharodon carcharias (UF211352, left), and Eocene
Otodus obliquus (UF162732, right) showing well defined growth increments.........5

2-2. Infrared spectrum of O. obliquus (UF162732A, sample j102-53) between 500 and
7 0 0 c m 1 .......................................................... ................ .. 7

2-3. Fourier transform infrared spectra of (a) Otodus obliquus centrum (UF162732C,
sample j102-58) (scale on y-axis 0 to 1.4) and (b) Isurus paucus centrum
(UF211353, sample j102-319) (scale on y-axis 0 to 1.2) ........................................ 14

2-4. Graph representing decrease in the amount of carbonate with an increase in
crystallinity ............................................................................................ 15

2-5. Contact prints of the three Otodus obliquus (UF162732) x-rays. The "." symbols
indicate dark growth bands on centra............................................ ...............16

2-6. Centrum of Otodus obliquus, UF 162732A, from the early Eocene of Morocco
showing exact sampling locations (grooves, top) and plot of variation in 6180c
(b o tto m )............................................................................................... 19

3-1. Nine vertebral centra used in this study........................................................ ........ 32

3-2. FT-IR spectra of all nine shark vertebral centra illustrating the differences from
modern (C. carcharias) to fossil biogenic apatites. ............................................35

3-3. FT-IR spectra from 400 to 850 cm-1, illustrating the y4 PO43- band differences
between modern (solid line) to fossil (dashed line) shark centra...........................35

3-4. Carbonate content (C/P) vs. crystallinity index (CI) of the nine shark vertebral
central. ................................................................................36

3-5. Isocon plots (Grant, 1982) depicting variations in fossil elemental concentrations
and ratios due to diagenesis........................................................... ............... 38

3-6. PAAS normalized REE of the nine vertebral centra divided into four diagenetic
groups ................ ......... ............................. ........................... 39









3-7. Compilation of observed (La/Yb)N vs. (La/Sm)N in biogenic apatites of various
ag es an d ty p es................................................... ................ 4 0

4-1. Scanned contact print ofBTO433 centra ....................................... ............... 49

4-2. BTO433 bomb carbon data plotted vs. two reference curves.................................57

4-3. Oxygen isotopic data (VPDB) for the shark centra analyzed...............................61

4-4. Post Archean Australian Shale normalized rare earth element plots for the eight
sharks analyzed. .................................................... ................. 65

4-5. Depth estimates for the eight lamnid sharks. Arrow indicates direction of
increasing w after depth............ .. .................. ........ ... ... .. ........ .... 66















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

QUANTIFICATION OF THE EXTENT OF DIAGENESIS IN BIOGENIC APATITE
OF CENOZOIC SHARK CENTRA

By

Joann Labs Hochstein
December 2005

Chair: Bruce J. MacFadden
Major Department: Geological Sciences

Diagenesis of bone in the fossil record is pervasive; however, the extent of this

process varies with depositional environment. Diagenesis is any chemical or physical

change that occurs below 200C. This study quantifies the extent of diagenesis in shark

vertebral centra through analysis of a suite of physical and chemical properties including

crystallinity index, carbonate content, isotopes, and major, minor, and trace elemental

concentrations. The sharks used in this study (Family Lamnidae) range in geographic

location and geological age from the Cretaceous to Recent. Although shark skeletons are

initially cartilaginous, the cartilage of the vertebral centra is replaced with carbonate

hydroxyapatite during growth of the individual. Understanding chemical changes to

biogenic apatite informs of the extent diagenesis has altered the biological signal

preserved in vertebrate bones.

Modern lamnid vertebral centra establish a modem analog for comparison to fossil

lamnid sharks. Rare earth element (REE) compositions, A14C, and 6180, give indications









of timing of when these sharks migrate, changes in their eating habits, changes in water

depth, and determination of ontogenic age and growth rates.

Fossil shark centra used in this study have undergone diagenesis; therefore how

have the processes of diagenesis affected the original signal recorded in these centra?

Shale-normalized REE patterns indicate that diagenesis has erased the original signal;

however because diagenesis occurred at or near the seawater/sediment interface, a

seawater REE signal may still be preserved in lamnid shark centra (related to the time of

deposition and location). Therefore, with caution, geochemical data from biogenic

apatite of fossil marine vertebrates, such as lamnid sharks, may be used to understand

paleoceanography and paleoenvironment. Also, the centra from Otodus obliquus

demonstrate that the biological oxygen isotopic signal is not completely erased by

diagenesis. Therefore, biological signals and diagenetic signals of fossil lamnid sharks

can be utilized to understand paleobiology, paleoclimatology, and paleoceanography.














CHAPTER 1
INTRODUCTION

Diagenesis is a fundamental and pervasive aspect of the fossil record. Diagenesis

is defined here as all physical and chemical changes that occur to vertebrate fossils

(bones and teeth) after deposition and below 200C. For vertebrate bones, rapid physical

and chemical changes are known to occur within relatively short geological timescales.

For example, Trueman (2004) demonstrated that after death and decay of a modem

animal, their bones will uptake rare earth elements (REE) within 25 years. The changes

that occur in bones during diagenesis can affect crystallinity, organic content,

mineralogy, density, major, minor, and trace element concentrations, and stable isotopic

compositions. Any scientist interested in interpreting the fossil record must understand

how diagenesis has affected the evidence. Despite the importance of understanding how

diagenesis affects biogenic apatite, this process is poorly understood and frequently

considered to be an impediment to progress. In many cases the effects of diagenesis are

explained as being of minor importance, or insignificant, to the interpretation of

geochemical data archived in the fossil record.

In this context, the fundamental questions that I seek to answer in this research

project include:

* How does diagenesis affect the geochemistry of fossil bone?

* How can these changes be quantified?

* Can life histories of sharks (e.g., age determination and ecology) be determined by
the chemical signal preserved in modem and fossil shark vertebral centra?









Although initially cartilaginous, sharks deposit carbonate hydroxyapatite in their

vertebral centra throughout their life, and therefore their centra are prone to fossilization

(except for the ubiquitous teeth, the rest of the skeleton remains cartilaginous and usually

does not fossilize). Lamnid shark centra were chosen to understand the affects of

diagenesis on fossil bone for the following reasons:

1. Vertebral centra in sharks are incrementally calcified during an individual's
lifetime. Calcified bands deposited in concentric rings are either, dark and compact
representing periods of slow growth whereas light, less dense bands correspond to
periods of faster growth. According to Ridewood (1921) and Moss (1977), there is
no resorption or redeposition of bone during life as is the case in other vertebrates,
e.g., the limb bones of reptiles (e.g., Francillon-Vieillot et al. 1990).

2. Lamnid sharks (Family Lamnidae, sensu Gottfried et al., 1996) are widely
distributed in space, time, and different marine sedimentary (phosphate, carbonate,
and plastic) environments throughout the Cenozoic.

3. Lamnid sharks are large, including the largest shark ever to have lived, the Mio-
Pliocene Carcharodon megalodon (Gottfried et al. 1996). Consequently, their
vertebral centra are physically large and therefore can be more easily sampled for
higher resolution.

4. Modem lamnids such as, Carcharodon carcharias (the Great White Shark) and
Isurus makoss), are available for analysis of an unaltered end-member.

This dissertation will discuss the use of oxygen isotopic data as a tool for

incremental growth studies in fossil sharks, and the chemical and mineralogical alteration

of lamnid shark centra caused by diagenesis. Also, discussed is the use of growth ring

counts, bomb carbon, and oxygen isotopic data in modern analogs (i.e., modem great

white and mako centra) as ontogenic age determination tools.














CHAPTER 2
DIAGENSIS AND VARIATION OF THE OXYGEN ISOTOPIC SIGNATURES IN
VERTEBRAL CENTRA FROM Otodus obliquus

Introduction

Sharks have been an abundant part of the marine fossil record since they first

appeared in the Devonian. Sharks are usually represented in the fossil record by their

durable teeth; most of the remainder of the skeletal tissue is composed of hyaline

cartilage, and thus not prone to fossilization. However, vertebral centra, and in rare

instances the jaws, both undergo secondary calcification during ontogeny resulting in

more durable skeletal elements composed of carbonate hydroxyapatite that are frequently

preserved in the fossil record (Ridewood, 1921; Goodrich, 1930; Applegate, 1967; Moss,

1977; Compango, 1999). Shark centra grow incrementally, laying down a dark band that

represents time of slow growth and a light band that represents times of faster growth.

Most shark species deposit a set of bands (one dark, which represents winter and one

light, which represents summer) annually (called annuli), but this may vary depending on

the species, physical environment (including temperature and water depth), food

availability, and stress (Branstetter et al., 1987). Clearly, the periodicity of the growth

bands cannot be established in modem or fossil sharks just by counting the growth rings.

Therefore, the challenge for paleobiological interpretation is how to interpret the

periodicity of the growth bands. Stable isotopic analysis provides the potential to

independently determine whether growth band increments represent annual growth, and

therefore the ontogenetic age of an individual. In most modern fish 6180 in body fluids is









close to that of ambient water, and since P043- and CO32- are cogenetic oxygen-bearing

phases in isotopic equilibrium with the same oxygen reservoir at the same temperature, a

linear correlation should exist between the oxygen isotopic composition of the phosphate

and carbonate, 6180p and 6180, respectively (lacumin et al., 1996). The results below

show that isotopic 6180, are archived in mineralized bone of fossil shark centra, even

when it is diagenetically altered. These data may not represent the actual amplitude of

temperature change, but demonstrate seasonal cycles that can be used to corroborate age

determinations based on counting physical growth bands. The preservation of

incremental growth layers in fossil vertebrate skeletal tissues provides the opportunity to

assess growth rates of individual species and the evolution of developmental strategies in

ancestral and descendant species. In addition to the physical archives of bone growth

preserved in the fossil record, recent studies have also applied stable isotope analyses to

understand periodic growth and related parameters of diet and seasonality preserved in

fossil bone and teeth (Longinelli and Nuti, 1973; Kolodny et al. 1983; Cerling and Sharp

1996; Bocherens et al., 1996; MacFadden et al., 1999; and Vennemann et al., 2001).

Results from associated vertebral centra of the lamnid shark Otodus obliquus

from the early Eocene (Ypresian) of Morocco (Fig. 2-1) are presented in this chapter.

This is a cosmopolitan species and is well represented in the highly fossiliferous

phosphate mines of the Oued Zem, central Morocco (Arambourg, 1952). The current

study was undertaken to determine:

If 50-million-year-old shark centra preserve an archive of incremental growth and
isotopic data that can be interpreted in a meaningful ontogenetic and phylogenetic
context.









The extent to which secondary calcification during ontogeny and/or diagenesis
after death obscures or removes the biologically significant incremental growth
and stable isotopic variation preserved in the centra.

If there is intravertebral variation in the physical incremental growth preserved in
the centra.


















0 21n
O 5cm




Figure 2-1. Modern white shark, Carcharodon carcharias (UF211352, left), and Eocene
Otodus obliquus (UF162732, right) showing well defined growth increments.

The Eocene shark Otodus obliquus was chosen for this study for several reasons.

This species is represented by excellent specimens of intact, fossilized portions of the

vertebral column in association with teeth. Otodus obliquus is conservatively classified

within the Lamnidea, the family that includes the modern mako (Isurus oxyrinchus),

white (Carcharodon carcharias), and extinct shark species, including the Carcharodon

megalodon (Gottfried and Fordyce, 2002). Therefore, this study is a necessary

foundation for further studies of the evolution of development and body size in extinct

lamnid sharks.









Isotopic studies of bone are rare due to the potential for diagenetic alteration as a

result of large surface area and small crystal size. During fossilization, replacement and

recrystallization occurs within the crystal lattice, which changes the original composition

of carbonate hydroxyapatite to carbonate fluorapatite (sometimes referred to as

"francolite") and eventually to fluorapatite. Transformation of carbonate hydroxyapatite

to fluorapatite occurs with the loss of CO2 and OH- and addition of F-, which causes an

increase in crystallinity (Barrick, 1998; Wang and Cerling, 1994; Shemesh et al., 1983).

Fourier transform infrared (FT-IR) spectroscopy has been effectively used to evaluate

mineral characteristics of fossils. Infrared spectroscopy measures the absorption of

infrared radiation by the sample at the vibrational frequencies of its component molecular

bonds, allowing characterization of its structural sites. In addition, the magnitude of IR

absorption is proportional to the concentration of a molecular species in the sample

(Sibilia et al., 1988). The crystalline structure of bone can be determined by calculating

the crystallinity index (CI) from the extent of phosphate peak splitting at 565-605 cm-1 in

an FT-IR spectrum (Figure 2-2). The 605 cm1 peak intensity increases with respect to

the 565 cm- peak intensity with an increasing degree of fluorination. Ultimately, the CI

is influenced by the size distribution of crystallites and the degree of the substitutional

order-disorder within the crystal lattice (Shemesh, 1990). Apatites with larger, more

ordered crystals show greater separation of these peaks and a higher CI, while in poorly

crystallized apatites the peaks are closer together and therefore have a lower CI (Weiner

and Bar-Yosef, 1990; Wright and Schwarcz, 1996).









In the apatite lattice carbonate can substitute in two sites, OH-1 and P04-2

(Shemesh, 1990; Lee-Thorp and van der Merwe, 1991; Rink and Schwarcz, 1995),

indicated by the superscripts A and B in the formula:

Ca5 [(P04)3-B(CO3)B] [(OH)-A(C03)A]

which results in two sets of absorption bands in an FT-IR spectrum, corresponding to

A(1545-1450-890 cm-1) and B (1465-1412-873 cm-1). Carbonate content can be

estimated from the ratio of the absorbance of the CO3 and P04 peaks (C/P) in the FT-IR

spectrum (Shemesh, 1990; and Wright and Schwarcz, 1996). The amount of carbonate

present in apatite affects the CI, due to type B carbonate substitution for P04, which

produces smaller crystals with greater strain; therefore, highly carbonated apatites show

little peak splitting and have lower crystal indices. Fourier transform infrared spectrum

will indicate the presence of non-apatite mineral structures, such as fluorine. Francolite

(carbonate fluorapatite) has a characteristic peak at 1096 cm-1, therefore the presence of

fluorine can be determined (Wright and Schwarcz, 1996).








0.0-
to











700 650 600 550 500
Wavenumber (cm-1)

Figure 2-2. Infrared spectrum of 0. obliquus (UF162732A, sample j102-53) between 500
and 700 cm- 1. The crystallinity index, (CI) is calculated by (A + B)/C, where
A, B, and C represent the peak height from the baseline.









It has been found that recrystallization during diagenesis does not necessarily affect

the isotopic composition (Barrick, 1998). When bone recrystallizes in a closed system,

there is no alteration of the isotopic value (Stuart-Williams et al. 1996). Studies have

shown (see Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996) that when

a sample is treated with buffered 1M acetic acid any apatite that has been diagenetically

enriched in CO32- and secondary carbonate minerals can be removed (Lee-Thorp and van

der Merwe, 1991; Wright and Schwarcz, 1996). It is known that when CO3-2 substitution

increases apatite solubility, rendering it more susceptible to diagenesis (Krueger, 1991;

Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996). As a consequence,

6180, values may not be a reliable paleothermometer in fossil bone. However, fossils that

have been affected by diagenesis the oxygen isotope composition may have some

biological information preserved, as will be discussed below.

Materials and Methods

Three associated fossil shark precaudal centra were analyzed from a single

individual catalogued in the Vertebrate Paleontology Collection, Florida Museum of

Natural History (FLMNH), University of Florida (UF) 162732. These centra are

identified as Otodus obliquus based on association with a diagnostic dentition. This

specimen was collected from the Early Eocene (Ypresian) unit within the phosphate

mines at Oued Zem, central Morocco. For comparison with the fossil shark, four modern

shark centra representing Carcharodon carcharias (great white, UF Environmental

Archaeology specimen 31648 and UF Vertebrate Paleontology specimen 211351), Isurus

oxyrinchus (shortfin mako, UF Environmental Archaeology specimen 47943), and Isurus

paucus (longfin mako, UF Vertebrate Paleontology specimen 211353) were analyzed.









Gross X-ray Analyses

Traditional x-radiographic ("x-rays") photography can reveal physical differences

in bone density, such as those representing incremental growth bands. X-rays of the

centra were taken at the C.A. Pound Human Identification Laboratory at UF. The x-rays

are set at 78 kV for 2 minutes. The x-rays are used to make contact prints, which are a

reversed pattern of the x-ray (i.e. dark lines on the x-ray are the light lines on the contact

print). The dark and light alternating growth rings are easily seen on the contact prints

and are marked to indicate the location of the samples used for oxygen isotope analysis.

These contact prints were scanned digitally and modified for presentation using Adobe

PhotoshopTM

Fourier Transform Infrared Spectroscopy Preparation and Analysis

For the intended study presented here, Fourier transform infrared spectroscopy has

its advantages over x-ray diffraction (XRD), including: (1) only a small amount of

sample is required (<1 mg); (2) preparation is easier and produces more accurate results;

and (3) carbonate content can be assessed from FT-IR.

Four -2 mg microsamples were drilled with a low speed Foredom drill from each

of the three centra. For the FT-IR analyses, two samples came from the center, and two

from the edge of each centrum. One-half of the samples were treated with the same

procedures as those analyzed for oxygen isotope composition (Table 2-1) to compare

with the results from samples not treated with acetic acid and hydrogen peroxide. The

other half of the samples were left untreated to serve as a control. The samples were

weighed out to 0.8 mg, and combined with 150 mg of spectral grade KBr, and ground

together in a ball mill. The KBr dye was put under vacuum for 5 minutes and

compressed (under vacuum) at 20,000 psi for another 8 minutes. The vacuum was









removed and the KBr dye remained under 20,000 psi for another 2 minutes, which

generated a 13 mm pellet. Infrared spectra were obtained between 4000 and 400 cm-1 on

a FT-IR Nicolet 20 SXB Bench in the Major Analytical Instrument Center in the UF

Material Science and Engineering Department. Interferences from KBr were cancelled

by subtracting a standard KBr spectra from the sample spectra. The crystallinity index,

(CI) measures the degree of P043- band splitting and is defined by:

CI= (A605 + A565)/(A595)

where Ax is the absorbance at wave number x (Shemesh, 1990), assuming a straight

baseline between 700 and 500 cm1 (Fig. 2-2). An estimate of the carbonate content is

given by the absorption ratio of the height of the carbonate peak at 1428 cm-1 to the

height of the phosphate peak at 1042 cm- of the FT-IR spectrum (Featherstone et al.,

1984; Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996; Stuart-Williams

et al., 1996), that is:

C/P=A1428/A1042.

Stable Isotope Analyses

For each of the three centra, from 24 to 28 microsamples of ~5 mg each were

drilled with a low speed Foredom drill across the growth axis starting from the center and

ending at the external margin. As far as practicable, the goal was to sample each annulus

twice, i.e., once in the dark portion and another in the light portion of the mineralized

bone. Sample powders were treated with standard isotope preparation techniques (e.g.,

MacFadden et al., 1999) used to analyze teeth. This included first washing with H202

overnight to remove organic contaminants and then with weak (0.1 N) acetic acid

overnight to remove mineral (principally CaCO3) contaminants, and then dried using

methanol. About 2 mg of each treated sample powder were then measured into









individual vials and placed in the automated Multiprep device for introduction into the

VG Prism mass spectrometer in the Stable Isotope Laboratory in the UF Department of

Geological Sciences. The sample runs were calibrated to internal laboratory and NBS 19

standards. The carbon and oxygen isotopic results are reported in the standard "6"

convention: 6-value = [(Rsample/Rstandard)-l] x 1,000 (parts per mil, %o), where R = 13C/12C

or 180/160, and standard is VPDB (Vienna Pee Dee Belemnite).

After the isotopic data were run and plotted against distance from origin of the

centra, they were the entered into a time series analysis program, AnalyseriesT version

1.2. This is necessary because each of the three centra are of slightly different sizes and

the growth lines do not match exactly, i.e., as measured by the distance from center.

Analyseries is traditionally used in paleoclimatological interpretation to construct age-

depth relations for sedimentary records. In Analyseries the method for establishing an

age-scale on a sedimentary record is to use a comparable well-dated signal as a reference

signal and then to optimize some measurement of the similarity between the two series,

while changing the depth scale of the first one to the age-scale of the second (Labeyrie

and Yiou, 1996). Analyseries then generates a pointer file, which allows plotting of the

isotope data from the two patterns on the same scale. The oxygen isotope values versus

distance from the origin were used to generate the pointer files and the carbon isotope

data (otherwise not discussed in this paper) was used as a check for the quality of the

match of the oxygen isotope data.









Results and Discussion

Fossil and Recent Shark Centra Mineralization and Diagenesis

FT-IR spectra indicate differences in shape and crystallinity indices between

modern shark centra and Eocene 0. obliquus centra (Fig. 2-3 and Table 2-1). Modern

shark centra (UF47943 and UF31648, FLMNH Environmental Collection) are

characterized by low CI (2.79 -2.84), low C/P values (0.32 0.34), and the absence of the

1096 cm-1 peak. The 0. obliquus centra (UF162732) have high CI (4.44 4.83), low

carbonate content (0.15 0.20), and a pronounced 1096 cm-1 peak, which indicates an

increase in crystallinity, a decrease in amount of carbonate, and the formation of a new

mineral phase after burial (francolite). There was no significant variation between the

samples treated with acetic acid compared to those samples not treated with acetic acid.

Fourier transform infrared spectroscopy allows for the evaluation of the mineral

characteristics of modern and fossil shark centra and enables detection of any diagenetic

changes to the mineralogy. The differences between the crystallinity indices of modern

shark centra and fossil 0. obliquus (Table 2-1) suggest that the three 0. obliquus centra

have been recrystallized, presumably due to diagenetic processes involving the growth of

larger crystals at the expense of smaller ones. The FT-IR spectra of the 0. obliquus

centra are indicative of two chemical changes occurring during recrystallization: (1) type

B carbonate substitution for P04-2 decreases with increasing crystallinity, which is seen in

reduced C/P values and increased CI (Fig. 2-4); and (2) an increase in fluorine content

with increasing crystallinity, which is indicated by a distinct peak at 1096 cm-1 (Fig. 2-3).

These changes in the FT-IR spectra signify a transformation from dahllite (carbonate










Table 2-1. Comparison of crystallinity index (CI) and carbonate content (C/P) from FT-
IR spectrum for samples treated with acetic acid and samples that were not
treated with acetic acid and from Eocene and modern samples.

Sample # CI C/P

Eocene 0. obliquus, UF 162732A

j102-51 4.62 0.18

j102-52 4.67 0.18

j102-53 4.62 0.19

Eocene O. obliquus, UF 162732B

j102-54 4.83 0.16

j102-56 4.51 0.20

j102-26* 4.58 0.17

j102-47* 4.53 0.15

Eocene O. obliquus, UF 162732C

j102-57 4.55 0.17

j102-58 4.57 0.17

j102-94* 4.44 0.15

j102-106* 4.45 0.16

Modem C. carcharias, UF 47943

j102-60 2.82 0.34

Modem C. carcharias, UF 211351

j102-317 2.80 0.32

Modem/. oxyrhinchus, UF 31648

j102-318 2.79 0.34

Modems. paucus, UF 211353

j102-319 2.84 0.33

*Samples that have been treated with acetic acid.












P043-
1.4
1.3- UF162732C
1.2-
1.1 -


10-
O 0.9'-
-0B
< 0.7 P043-
0.6-
0.5-
0.4- CO32-
0.3-
0.2-
0.1-
1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400
Wavenumber (cm-1)

P043-
1.2
1.-UF211353
1.1-
1.0- P043-
S0.9-
-0 0.8-
SOrganics
m 0.7- CO32-
0.6-
CO32-
0.5-

0.4-

0.3-
0.2-

1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400
Wavenumber (cm-1)


Figure 2-3. Fourier transform infrared spectra of (a) Otodus obliquus centrum
(UF162732C, sample j102-58) (scale on y-axis 0 to 1.4) and (b) Isurus paucus
centrum (UF211353, sample j102-319) (scale on y-axis 0 to 1.2). The arrow
shows peak 1096 cm-1, which is diagnostic of francolite (F-apatite)and is only
present in the fossil (UF 162732C) specimen and not the modern specimen
(UF211353).


hydroxyapatite) to francolite (carbonate fluorapatite). The lack of significant differences


in the FT-IR spectrum of the 0. obliquus samples treated with acetic acid and those that


were not treated suggest that these samples were not diagenetically enriched in CO3-2 and


no secondary carbonate minerals were present. The absence of a 710 cm-1 peak on the


FT-IR spectrum, which represents the presence of calcite, indicates that no secondary










carbonate minerals were present. Comparison of the FT-IR spectra from various

locations along the growth axis of a single 0. obliquus centra and between the three

centra show insignificant variation, which indicates that the chemical changes that occur

during diagenesis are uniform along the growth axis and between the three centra.


0.4

0.35 UF162732A
o3 o UF162732B
0.3
A UF162732B (T)
0.25 UF162732C
(0.2 UF162732C (T)
O U o Modern
0.15 -

0.1

0.05

0 --------------------
2 2.5 3 3.5 4 4.5 5
CI

Figure 2-4. Graph representing decrease in the amount of carbonate with an increase in
crystallinity.

Physical Increments and Variation

Physical growth couplets annulii) are evident in the gross morphology and x-

radiographs of each of the fossil Otodus obliquus centra. Contact prints of x-rays of the

three 0. obliquus centra (Fig. 2-5) were used to enhance the visibility of the growth rings.

The counts on the three contact prints indicate a total of 19 growth couplets for each

centrum. A growth couplet is defined as a band pair, composed of one opaque (darker)

band and one lighter band. The location of the 19 growth couplets is consistent among

the three centra and fit a characteristic growth function (von Bertalanffy, 1938; and von

Bertalanffy, 1960), i.e., with the most rapid growth during early ontogeny and










incrementally decreasing growth rate during later ontogeny. Given the fact that all three

centra come from the same individual, the observation of 19 equivalent band couplets is

both expected and corroborates these as growth related phenomena. Assuming one

growth couplet is deposited annually as proposed for other lamnids (Cailliet at al. 1983;

Campana et al. 2002; Labs-Hochstein, submitted) this shark has a minimum age of 19

years.

UF162732A IJF162'328














uF 162732C













cm




Figure 2-5. Contact prints of the three Otodus obliquus (UF 162732) x-rays. The "*"
symbols indicate dark growth bands on centra.









Stable Isotope (6SO ) Signal Archived in Eocene Otodus obliquus centra

A series of microsamples taken along the growth axis of three centra (UF

162732A, UF162732B, and UF162732C) of Otodus obliquus reveals a systematic pattern

of change in 6180 values (Fig. 2-6; Table 2-2). There appears to be a systematic,

sinusoidal, variation of 6180 values representing at least eight isotopic cycles. The

extremes of these cycles are interpreted to represent warm seasonal signals, with lower

6180 values between approximately -3.5 to -4.0%o and cold seasonal signal with higher

6180 values between about -2.0 to -3.3%o.

There are several points of discussion concerning these data. Firstly, two adjacent

microsamples were taken, so far as possible, to correspond with dark-light band couplets

within an annulus. The results indicate that more positive 6180 values correspond to the

darker bands, confirming the prediction that these represent a colder signal. Conversely,

the more negative 6180 values were taken in the lighter band, indicating a warmer signal.

This correspondence, plus the non-random pattern of isotopic variation therefore suggests

that these isotopic data are archiving a record of paleoclimate cycles (although perhaps

not the actual amplitude of paleotemperature change, see below).

Secondly, the oxygen isotopic data of all three centra have eight annual growth

band couplets. However, there is no consistency in the amplitude of the bands. In

different centra the same growth bands have different isotopic values, and the extremes of

each annual cycle are not recorded uniformly in each of the three centra. This may be

due to several reasons, such as; (1) the growth band is not sampled in the exact same

place on each of the three centra; (2) there may be an ossification order such that the

centra closer to the head ossify before the centra towards the tail or vice versa









(Ridewood, 1921); (3) the seasonal variation may not have been as strong at various

times during the sharks life; and/or (4) possible dampening/amplification of the original

signal by diagenesis.

Finally, there are fewer annuli indicated from the isotopes (8) than those observed

from the physical growth couplets (19). Otodus obliquus could have been

homoeothermic (maintain a body temperature above its surroundings). All modern day

lamnids have the ability to be homoeothermic (Campango, 2002) and it is not known

when lamnids evolved this characteristic. However since porebeagles, makos, and great

whites are all homoeothermic it suggest that the common ancestor may also have been

homoeothermic. Using molecular clocks calibrated for sharks Martin (1996)

demonstrated that all three genera of lamnids diverged at nearly identical times during the

Paleocene or early Eocene. Another possibility is that all modern lamnids independently

evolved homeothermy. Only seven out of the more than 360 known modern species of

sharks are known to be homoeothermic, the five species of modem lamnids and two

species of modem thresher sharks (Campango, 2002). Therefore homeothermy is rare in

modern sharks and most likely evolved in lamnids through a common ancestor. The

possibility exists that the oxygen isotopic data is showing the shifts in body temperature

in 0. obliquus as it came in contact with varying temperatures of water and not the actual

water temperature. The oxygen isotopic signal gives an age of half what the growth ring

counts estimates suggesting that this shark may not have been migrating into waters with

enough variation in the oxygen isotopic signal on an annual basis or it was not encounter

waters that changed its body temperature on an annual basis if 0. obliquus was

homoeothermic.







19








I cm
15





-4.5


-4.0


-3.5


-3.0


-2.5


-4.0








-2.5


-2.0


-4.0


-3.5



UF162732A
"--UF162732B
-2.5 \ I --UF162732C


10 15 20 25 30 35 40 45 50 55
Distance from Center (mm)



Figure 2-6. Centrum of Otodus obliquus, UF 162732A, from the early Eocene of
Morocco showing exact sampling locations (grooves, top) and plot of
variation in 6180c (bottom), the three plots represent the correlation between
each of the centra produced by using AnalyseriesTM. The gray lines connect
the dark growth bands that correspond to the possible annual signal.










Table 2-2. Stable isotopic data for three vertebral centra of Otodus obliquus, UF 162732,
from the Early Eocene of Morocco.


mm 618~ 0/oo mm 618O 0/oo mm 68Ce /oo


UF162732A


11.60
13.44
14.54
15.8
16.67
17.60
19.06
20.10
21.13
22.63
23.95
24.81
26.57
27.50
28.93
29.81
31.27
32.41
33.98
34.74
35.87
36.83
37.90
39.20
39.94
41.70
42.58
43.10
44.31
45.21
46.36
47.34
48.45
49.33
50.89
51.73
53.47


-6.2
-6.0
-5.9
-6.1
-5.9
-5.9
-5.7
-5.8
-5.8
-5.7
-5.6
-5.4
-6.1
-5.8
-6.1
-6.1
-6.3
-6.3
-6.3
-6.2
-6.4
-6.4
-6.2
-6.3
-6.2
-6.6
-6.6
-6.6
-6.3
-6.5
-6.4
-6.5
-6.5
-6.7
-7.0
-6.7
-6.7


UF162732B


2.81
14.94
16.61
17.89
19.77
23.08
24.56
26.32
27.62
21.50
30.06
31.58
32.78
33.99
35.69
36.78
38.04
39.30
40.65
42.16
43.30
44.70
46.27
46.77
47.81
49.41
50.35
51.13
52.50


-5.4
-5.7
-6.3
-6.0
-5.4
-5.8
-5.9
-5.3
-5.7
-5.8
-5.7
-5.8
-5.8
-5.9
-6.1
-6.1
-6.3
-6.1
-5.9
-5.9
-6.3
-6.3
-6.2
-6.8
-6.9
-6.8
-6.4
-6.8
-7.0


UF162732C


10.81
12.94
14.93
16.84
17.95
19.42
20.56
22.04
24.27
25.35
27.25
28.78
30.87
32.25
34.60
36.31
36.66
38.16
39.60
40.07
42.17
42.93
45.42
46.01
47.25
48.72
49.70
51.43
52.92


-6.4
-6.0
-5.9
-5.7
-5.4
-5.7
-5.8
-5.5
-5.5
-5.8
-5.0
-5.6
-5.6
-5.7
-5.7
-5.9
-6.0
-6.0
-5.8
-6.2
-6.0
-6.0
-6.4
-6.4
-6.1
-6.3
-6.7
-6.4
-6.6









Conclusions

Because of their cartilaginous skeletons, which characteristically do not fossilize,

the secondarily calcified centra provide a unique opportunity to assess incremental

growth and age determination in fossil sharks. The FT-IR spectra indicate that the three

0. obliquus centra have undergone diagenesis. Modem skeletal tissue is composed of

carbonate hydroxyapatite while according to the FT-IR spectra the 0. obliquus centra

have francolite (carbonate fluorapatite) and have lost carbonate and organic relative to

modern shark centra. All three centra have undergone similar diagenesis indicating that

in this case the diagenesis was uniform throughout this specimen. There was no

difference between the acetic acid treated samples and those that were not treated,

indicating that these specimens did not have any secondary carbonate present and the

acetic acid does not affect the structural carbonate.

Despite this degree of alteration, two important conclusions derived from this study

are that: (1) the incremental growth banding, i.e., annuli, retain their original physical

structure; and (2) the non-random pattern of oxygen isotopic variation therefore suggests

that these isotopic data are archiving a record of paleoclimate cycles. With regard to the

latter observation, I caution that the oxygen isotopic variation may represent a signal

damped/amplified by diagenesis or the possibility exists that 0. obliquus was

homoeothermic and therefore the shifts in oxygen isotopes represent changes in body

temperature as it came in contact water of different temperatures. Accordingly, I do not

advocate using these data to attempt calculations of paleotemperatures in the early

Eocene seas. The oxygen isotopic signal gives an age of about half of what the growth

ring counts estimates suggest (assuming one growth ring pair per year). One explanation

of this observation is that this shark may not have been migrating into waters with









enough oxygen isotopic variation on an annual basis. Another explanation, if 0. obliquus

was warm-bodied, is it was not encountering waters that changed its body temperature on

an annual. These findings are similar to modern day lamnids, where by larger sharks

oxygen isotope estimates yield ages equal to approximately 1/2 of the ring counts (Labs-

Hochstein, submitted).

The analysis of Otodus obliquus centra from the early Eocene of Morocco

potentially have broad ramifications for understanding the evolution of developmental

strategies in fossil sharks. This application can potentially answer some unresolved

questions about the developmental mechanisms that resulted in huge body size in fossil

lamnid sharks (Gottfried et al., 1996) such as Miocene Carcharodon megalodon, which is

a close relative of 0. obliquus. In all such future studies, however, analytical techniques

that assess diagenesis, such as FT-IR, should be used in combination with isotopic studies

to produce the most insightful analysis of fossil shark paleobiology.














CHAPTER 3
QUANTIFICATION OF DIAGENESIS IN CENOZOIC SHARKS: ELEMENTAL AND
MINERALOGICAL CHANGES

Introduction

Fossilized vertebrate skeletal tissues, including teeth and bone, have recently

received considerable attention as geochemical archives of paleoecological and

paleoenvironmental information (Piper, 1974; Kolodny et al., 1983; Elderfield and

Pagett, 1986; Kolodny and Luz, 1991; Lecuyer et al., 1993; Picard et al., 1998; Shields

and Stille, 2001; Picard et al., 2002; MacFadden et al., 2004; Puceat et al., 2004). In these

studies, fossil tooth enamel has been the preferred material for analysis because it is

compact, relatively non-porous mineral and consists of >95% hydroxyapatite. However,

isotopic data have been used to eludicate paleobiological information (e.g., to reconstruct

dinosaur physiology; Barrick and Showers, 1994). These studies have come under close

scrutiny (Kolodny et al., 1996) because porous bone is more prone to diagenesis than

teeth (Wang and Cerling, 1994).

There are certain situations in which fossil bone is either the only skeletal material

available for study (e.g., in those vertebrates that lack teeth, such as most birds), or is

preferred because certain skeletal elements archive incremental growth. One example of

an archive of incremental growth is shark vertebral centra. Although shark skeletons are

initially cartilaginous (i.e., composed of soft supporting tissue that does not fossilize), the

cartilage is replaced in the vertebral centra by carbonate hydroxyapatite during the

growth of the individual. This growth is periodic and incremental rings are called annuli









because of their presumed annular cyclicity (although this is not always the case

Branstetter et al., 1987). These growth rings are easily observed in both modern and

fossilized shark centra. During a related research project investigating stable isotopic

signatures archived in fossil shark centra (MacFadden et al. 2004), we became interested

in the extent of diagenesis and how it may have affected the geochemistry of fossil bone.

The purpose of this study is to quantify diagenesis of shark bone through analysis

of a suite of physical and chemical characters including crystallinty index, carbonate

content, and major, minor, trace elemental concentrations. The sharks are all from the

group known as the superfamily Lamnoidea (Capetta 1987), that includes the modern

great white (Carcharodon carcharias), and six closely related, extinct species that range

in geologic age from the Cretaceous to the Pliestocene. The modern shark species are

included in this study to provide an unaltered "end-member" in which initial physical

parameters and elemental concentrations can be determined.

Bone Chemistry and Diagenesis

Stable isotopes and rare earth elements (REE) of biogenic apatites have been used

for paleoclimate reconstruction, to trace ocean currents and water masses, to quantify

redox conditions, for incremental growth studies, and to reconstruct diet (Piper, 1974;

Kolodny et al., 1983; Elderfield and Pagett, 1986; Kolodny and Luz, 1991; Lecuyer et al.,

1993; Picard et al., 1998; Shields and Stille, 2001; Picard et al., 2002; MacFadden et al.,

2004; Puceat et al., 2004). Partial or complete dissolution, precipitation, recrystallization,

and ion uptake by adsorption and diffusion may lead to changes in chemical composition

and lattice structure of the biogenic apatite (Reiche et al., 2003); therefore, the original

chemical signatures of biogenic apatites may be modified through diagenesis, resulting in

the interpretation of erroneous biological signals (Puceat et al., 2004).









Modem bone is composed of carbonate hydroxyapatite, Calo(PO4)6(CO3)x(OH)2-

2x, that has small crystallites, large surface area (200 m2/g; Weiner and Price, 1986), and

high organic content (- 35%, principally collagen and water; Williams, 1989; Carlson,

1990; Koch et al., 1992). The high reactivity of biogenic apatite is due to small

crystallite size and high surface area of the bone hydroxyapatite (Trueman, 1999). Many

substitutions are possible for both the anions and cations in biogenic hydroxyapatite

(Table 3-1; Nathan, 1981). In modern biogenic apatites, carbonate (C032-) can substitute

for either OH- (A site) or P043- (B site), but primarily substitutes for the latter (Shemesh,

1990; Lee-Thorp and van der Merwe, 1991; Rink and Schwarcz, 1995). Substitution of

carbonate for phosphate distorts the crystal lattice increasing the solubility of biogenic

apatite (Nelson, 1981; Nelson et al. 1983).

During fossilization, the biogenic apatite alters to a more stable, less reactive form

of apatite (carbonate fluorapatite some times referred to as "francolite") by losing

carbonate and hydroxyl ions and gaining fluoride (Nathan and Sass, 1983; Newesely,

1989; Greene et al., 2004). The loss of carbonate decreases the defect densities within

the hydroxyapatite lattice resulting in an increase in crystallite size and order and

decreaed solubility relative to carbonate hydroxyapatite (Greene et al., 2004).

Table 3-1. Some possible substitutions in the apatite crystal structure.
Constituent ion Substituting ion

Ca Na K Sr Mn Mg Zn ,Ba Sc3,
Y3+, REEs, U4
P043- CO32-, SO42-, Cr042-, CO3sF3-,C03 OH4-, SiO44-

OH- F-, C1-, Br, 02-









Through the processes of diagenesis, trace element concentrations can either

increase or decrease relative to unaltered bone concentration. This phenomenon is well-

documented and described in the literature (Elderfield and Pagett, 1986; Wright et al.,

1987; Williams, 1988; Grandjean and Albarede, 1989; Koeppekastrop and DeCarlo,

1992; Grandjean-Lecuyer et al., 1993; Denys et al., 1996; Hubert et al., 1996; Laenen et

al., 1997; Reynard et al., 1999; Trueman, 1999; Staron et al., 2001). Trace elements are

most likely incorporated into bone apatite during early diagenesis through the process of

substitution. Trace element signatures acquired during the initial stages of diagenesis

appear to be stable and resistant to later modification (Bernat, 1975; Grandjean and

Albarede, 1989; Grandjean-Lecuyer et al., 1993). Because of similar ionic size, REE3

ions are easily substituted into the Ca2+ site by means of coupled substitution (Whittacker

and Munts, 1970). Adsoprtion of trace elements onto the surface may also be a

qunatatively significant mode of uptakeof biogenic apatite (Reynard et al., 1999).

Surface adsorbed species are typically only weakly bound to the mieral surfaces, and

therefore ions adsorbed are susceptible to exchange as long as the crystal surface remains

exposed (Reynard et al., 1999). However, if during diagenesis the inter-crystalline

porosity is closed, then individual crystallite surfaces will be protected from further

exchange. Ultimately, the final trace element composition of the biogenic apatite is

controlled by the concentration of trace elements in the system, apatite-fluid partition

coefficienst, chemistry of the burial microenvironment, bone microstructure, and length

of exposure (Trueman, 1999).

Rare Earth Elements

Although the REE typically exist in the 3+ oxidation state two exceptions are

Cerium and Europium. Cerium can undergo oxidation in seawater from the solvated 3+









state to the relatively insoluble 4+ state (de Baar et al., 1985a). Under oxic conditions,

Ce4+ is readily removed from seawater onto particle surface coatings or into authigenic

minerals (Sholkovitz et al., 1994; Koeppenkastrop and De Carlo, 1992) and under

reducing conditions Ce3+ may be released back into the water column or pore waters.

Therefore when Ce is depleted (i.e., under oxic conditions) in the water column a

negative Ce anomaly (Ce anom.) is present and vice versa (German and Elderfield, 1990).

Europium may undergo oxidation from the Eu3+ to Eu2, which is significant in the

oceans because of its preferential ability of Eu2+ to substitute for Ca2+ in apatites

(Elderfield, 1988). de Barr et al. (1985a) illustrate that in both the Atlantic and Pacific

Oceans all REE, with the exception of Ce, increase with water depth. Concentrations of

Ce decrease with water depth and therefore the negative Ce anomaly observed increases

with depth (de Baar et al., 1985a). Thus, when the seawater signal is preserved in

biogenic apatites the Ce anomaly may be a useful relative indicator of water depth at

which fossils have been deposited.

Fossil biogenic apatites contain several tens to several hundreds parts per million

(ppm) of REE, whereas REE maximum concentration in pore water and seawater are in

the range of parts per billion (ppb) and part per trillion (ppt), respectively (Elderfield and

Greaves, 1982; Elderfield and Sholkovitz, 1987). Bernat (1975) reported high REE

concentrations in ichthyoliths from the upper-most 4 cm of sediment of ocean cores.

These ichthyoliths exhibit bulk REE patterns similar to overlying waters. Furthermore,

Bernat (1975) analyzed ichthyoliths from 4 to 600 cm of the sediments, and showed that

their REE compositions were similar to the ichthyoliths from the upper-most 4 cm of the

same cores. These results suggest that in this case ichthyoliths inherit a REE composition









directly from seawater at the sediment/seawater interface during early diagenesis, with

little or no fractionation. Direct uptake of REE in biogenic apatites from pore waters

and/or seawater raises serious problems. Assuming that REE are directly taken up

through advection of pore waters, about a ton of pore water would be needed to give the

biogenic apatite enough REE to fit observed concentrations (several tens to several

hundreds parts per million, ppm). Such a water/rock ratio would require an exceedingly

high flux of pore water through the sedimentary column, and reasonably this cannot be

considered for the cause of the enrichment of biogenic phosphates (Grandjean et al.,

1987; Grandjean and Albarede, 1989; Grandjean-Lecuyer et al., 1993). Grandjean et al.

(1987) proposed quantitative uptake of non-detrital REE locally released at the

sediment/seawater interface to explain biogenic apatite enrichment. Abundant debris

with large specific surfaces, which easily absorb large amounts of REE from seawater,

are dispersed in the oceans and are known to settle to the ocean floor. Such a rain of REE

rich carriers has been identified in sediment traps (Murphy and Dymond, 1984) and

comprises a variety of inorganic (detrital minerals, oxy-hydroxide flocs) and organic

(pellets, organic debris) phases. The decay of the REE rich carriers at the

sediment/seawater interface, associated with that of biogenic apatite, and the resulting

reducing conditions eventually cause the dissolution of Fe-Mn oxy-hydroxides and favor

transfer of REE from large volumes of seawater to recrystallized biogenic apatite in a

rather short period of time (Grandjean et al., 1989). This extension of Bernat's (1975)

model implies that both that the ultimate source of phosphatic REE is seawater, and there

is an early diagenetic transfer to the phosphate through a short-lived phase made of

oxihydroxides and organic detritus. Upon completion of the early diagenetic processes









and once most oxihydroxides have been dissolved, phosphate remains the major non-

detrital REE repository in sediments, so its REE concentrations must reflect to a large

extent the flux of seawater derived REE exclusive of the detrital particulate accumulation

(Grandjean et al., 1989).

Variations in host lithology of marine biogenic apatites may influence the REE

contents due to differences in permeability, the flux of REE from diagenetic fluids

expelled from sediments, and organic and oxy-hydroxide contents (Grandjean-Lecuyer et

al., 1993; Lecuyer et al., 2004). Terrestrially derived sediments have shale-normalized

REE patterns that carry a shale-like signal (i.e., flatten REE pattern) and no Ceanom. is

expected from common fine-grained detrital material (Grandjean et al., 1987). REE in

pore waters are derived from the surrounding sediment particles and development of

large pore water concentration gradients will allow fluxes of REE from sediments to

seawater (i.e., diagenetic fluids expelled from sediments into seawater; Elderfield and

Sholkovitz, 1987). Therefore, REE contents of biogenic apatites deposited in terrestrial

derived sediments (clays and sands) may have flattened shale-normalized REE patterns

that are intermediate between those of seawater and those of shale (Grandjean et al.,

1988; Elderfield et al., 1990). Sediments that precipitate directly from seawater

carbonatess and phosphorites) with little to no terrestrial input have diagenetic fluids

reflecting the composition of the overlying water column. Biogenic apatite deposited in

marine precipitated sediments should show a seawater REE pattern since the diagenetic

signature would be the same as the overlying water column (Lecuyer et al., 2004).

Therefore, the REE signature in fossil biogenic apatites results from a mass balance

between the flux of REE from decaying organic and oxy-hydroxides (primary carriers









with seawater signature), the flux of REE from diagenetic fluids expelled from sediments

(diagenetic signature),and the flux of REE from rivers (detrital signature) (Grandjean and

Albarede, 1989).

Materials and Methods

This study analyzes the composition of nine shark centra (Fig. 3-1), two modern

great white, Carcharodon carcharias, and seven fossil specimens ranging in age from

Cretaceous to Pliocene (Table 3-2). These specimens were selected because they all are

within the monophyletic superfamily Lamnoidea, the group that includes great white

sharks, makos (Isurus), and their close fossil relatives. Likewise, these sharks were

selected because they span an age range from Cretaceous to Recent and are widely

distributed geographically. A broad geographic distribution of fossils should illuminate

the effects of different extents and environments of diagenetic alteration. The vertebral

centra were chosen because they are the primary ossified skeletal tissue that fossilizes in

sharks (i.e., other than teeth).

Two analytical techniques were used to determine the chemical and mineralogical

properties of the nine shark centra: (1) Fourier Transform Infrared Spectroscopy (FT-IR).

FT-IR, which is used here to determine crystallinity. FT-IR has advantages over x-ray

diffraction (XRD), because only a small amount of sample is required (<1 mg),

preparation is easier and produces more accurate results; and additionally, carbonate

content can be assessed from FT-IR. (2) Inductively Coupled Plasma Mass Spectrometry

(ICPMS), which allows for the determination of the major, minor, and trace elements in

modern and fossil shark centra. ICPMS is a comprehensive technique that is extremely

sensitive (detection limits in the ppb range for many elements in aqueous solution). The










high level of relative accuracy (1 to 2%) coupled with sensitivity allows analysis at

concentrations ranging over more than nine orders of magnitude (Montaser, 1998).

Table 3-2. Lamnid shark specimens used in this study.
ecie M I Lcait Age Sediment & Depositional
Species Museum ID Locality Eim
Environment
Carcharodon Recent
Carchar n BT0433 E coast, South Africa
carcharias
Carcharodon Recent
Carcharon UF211351 Islamorada, Florida
carcharias
Pliocene Shallow bay sandstone
Isurus hastalis UF211358 Pisco Fm., Peru ene a a a
(Brand et al., 2004)
Carcharodon 120A Saitama Prefecture, Miocene Nearshore sandy siltstone
megalodon Japan (Hayashi et al., 2003)
Carcharodon OU22261 Kokoamu Greensand, Oligocene Shelf glauconitic sand
angustidens New Zealand (Ayress, 1993)
Carcharodon Brussels Sand, Oligocene Near shore shelf sandstone
au s EF809A Belm (Hooyberghs, 1990; and
SHerman et al. 2000)
Eocene Shelf phosphorite (Lancelot
Otodus obliquus UF162732B Oued Zem, Morocco E e Sf p
and Seibold, 1978)
Eocene Shelf phosphorite (Lancelot
Otodus obliquus UF162732D Oued Zem, Morocco and Seibo, 1978)
and Seibold, 1978)
Cretaceous Shallow epicontinental sea
reotxyrina UF211357 Niobrara Fm., Kansas outer shelf chalky shale
mantelli (Hattin, 1981)


Fourier Transform Infrared Spectroscopy (FT-IR)

Three -1 mg samples were drilled with a low-speed Foredom drill from each of

the nine centra. Samples were taken along the growth axes, i.e., one from the center, one

from the middle, and one from the edge of each centrum. Potassium Bromide (KBr)

pellets were prepared using the method discussed in MacFadden et al. (2004). Infrared

spectra were obtained between 4000 and 400 cm'1 on a FT-IR Nicolet 20 SXB Bench

housed at the Major Analytical Instrument Center in the UF Material Science and

Engineering Department. Interferences from KBr were cancelled by subtracting a

standard KBr spectrum from the sample spectra. The size distribution of crystallites and

the degree of substitution order-disorder within the crystal lattice of biogenic apatite can

be determined by calculating the crystallinity index (CI) from the extent of phosphate

peak splitting at 565-605 cm-1 in an FT-IR spectrum. Apatites with larger, more ordered









crystals show greater separation of these peaks and a higher CI (Shemesh, 1990; Wright

and Schwarcz, 1996). An estimate of the carbonate content is given by the absorption

ratio of the height of the carbonate peak at 1428 cm-1 to the height of the phosphate peak

at 1042 cm-1 of the FT-IR spectrum (Featherstone et al., 1984; Lee-Thorp and van der

Merwe, 1991; Stuart-Williams et al., 1996; Wright and Schwarcz, 1996).


Figure 3-1. Nine vertebral centra used in this study. A. Carcharodon carcharias
(BT0433) B. Carcharodon carcharias (UF211351) C. Isurus hastalis D.
Carcharodon megalodon E. Carcharodon angustidens F. Carcharodon
auriculatus G. Otodus obliquus H. Otodus obliquus I. Creotxyrhina
mantelli.









Elemental Analysis (ICPMS)

Approximately 6 mg of bulk sample was drilled with a slow speed Foredom drill

from each of the nine centra. 5 mg of each sample were weighed out and placed into 3

mL Savillex vials, dissolved in 1 mL of 3M HNO3, and heated overnight. Samples were

allowed to cool and then dried. Next 2 mL of 1% HNO3 was added, heated overnight, and

allowed to cool. Samples were analyzed on an Element 2 High Resolution Inductively

Coupled Plasma Mass Spectrometer (HR-ICP-MS) at the Center for Trace Element

Analysis at the University of Southern Mississippi. All samples were corrected by

subtracting the blank, corrected for instrumental drift based on internal machine standards

that were analyzed during the run (initial quantification based on comparing the corrected

ion counts of the samples with ion count for the standards), and corrected ion counts to a

constant response to the known amount.

All REE values were shale-normalized to PAAS (Post-Archean Australian Shale

Standard) in order to clearly illuminate enrichment-depletion trends relative to average

crust (e.g., Grandjean et al., 1988; Elderfield et al., 1990; Grandjean-Lecuyer et al., 1993;

Reynard et al., 1999; Trueman and Tuross, 2001). The Ce anomaly (Ceanom) was

calculated from Ceanom=Log[3CeN/(2LaN+NdN)] (Elderfield and Greaves, 1982).

Results

Mineralogical Changes

FT-IR spectra of the modern and fossil shark centra are shown in Fig. 3-2. Both

the modem and fossil samples have the same characteristic absorption bands as the FT-IR

spectra of synthetic apatites containing CO32- at both A- and B- sites (Bonel, 1972). The

FT-IR spectra for modern specimens are characterized by large H20 bands (which

usually mask the OH- band at 3567 cm-1) and the presence of organic represented by the









three amide group bands amidee I 1660 cm-1, amide II 1550 cm-1, and amide III 1236 cm

1, mean values). The FT-IR spectra for fossil specimen are characterized by reduced H20

bands, lack of OH- band, and absence of one or more of the amide group bands (Fig.3-2).

There are three intense phosphate (P043-) absorption bands: the main absorbance peak is

recorded at 1041 cm-1 and a doublet at 605 cm-1 and ~ 568 cm-1, consistent with

previous studies (Shemesh, 1990). In the modem specimens, the 605 cm-1 absorption

band has a smaller intensity than the 568 cm-1 band and but the reverse holds for the

fossil specimens (Fig. 3-3). The modern specimens have an average crystallinity index

(CI) of 2.83, while in the fossil specimens CI is increased to 3.19-5.39 (Table 3-3; Fig. 3-

4). B-type carbonate substitution (replacement of P043- by CO32-; Shemesh, 1990; Dahm

and Risnes, 1999) is represented by a set of absorption bands at 1460 cm-1, 1428 cm-1,

and 870 cm-1 (average values). Carbonate content (C/P) is much greater in modern

specimens (0.35 and 0.43) than fossil specimens (range from 0.10 to 0.29) (Table 3-3;

Fig. 3-4). The lack of the 713 cm-1 absorbance band in all samples indicates that there is

no authigenic calcite present, and the 1092 cm-1 band (average value), which is found

only in the fossil specimens, demonstrates the presence of fluorine (Fig. 3-2).

Elemental Concentration

The effects of diagenesis on elemental concentrations can be assessed by

comparing the modem unaltered centra with the altered fossil centra. This is illustrated

by the isocon plots (Grant, 1982) in Fig. 3-5. The linear trends (labeled isocon in Fig. 3-

5) are the average of the two modem shark centra elemental compositions and represent

no elemental loss or gain during diagenesis. In modem sharks the major and minor

elements vary (Table 3-3), therefore in the isocon plot of the major and minor elements






































Wavenumber (cm-1)


Figure 3-2. FT-IR spectra of all nine shark vertebral centra illustrating the differences
from modern (C. carcharias) to fossil biogenic apatites.


568 cm-1
605 cm-1 A


800 750 700 650 600 550 500 450 400
Wavenumber (cm-1)


Figure 3-3. FT-IR spectra from 400 to 850 cm-1, illustrating the y4 P043- band
differences between modem (solid line) to fossil (dashed line) shark centra.














































2.50 3.00 3.50 4.00
Crystalllnlty Index (CI)


* O. obliquus
1 O. obliquus
A C. carcharias
SC. carcharias
+ C. mantelli
X I. hastalis
A C auriculatus
o C. angustidens
o C. megalodon


4.50 5.00 5.50 600


Figure 3-4. Carbonate content (C/P) vs. crystallinity index (CI) of the nine shark vertebral

centra.


Table 3-3. Elemental and mineralogical data of nine shark vertebral centra. Elemental

concentration are in ppm. Crystallinity index (CI) and carbonate content

(C/P) were calculated from FT-IR spectra.
Specimen Ca P Mg Na Zn Fe K Sr Al Si Mn Ba Pb B
C. carcharias 732027 59982.3 1752.9 4053.4 904.8 177.2 1133.6 352.5 266.0 28.8 4.8 1.5 9.1 9.1
(BT0433)
C. carcharias 131144.2 1156974 4239.8 3446.9 2318.8 523.9 314.3 644.8 189.6 1663.7 12.4 1.9 47.0 23.8
(UF211351)
I hastials 179681.8 132370.8 5234.1 18385.2 449.6 3408.4 2242.6 1031.2 2999.2 118.2 494.6 30.6 13.7 46.9
C. megalodon 129279.7 78068.0 1521.4 1540.9 575.5 2790.4 239.2 1716.6 2592.8 543.9 667.9 444.4 9.9 49.8
C. auriculatus 88019.48 61402.8 2027.8 1879.8 1417.0 957.9 183,9 519.9 373.3 953.7 31.7 26.2 29.7 24.5
C angustidens 101257.9 61242.3 489.8 1600.8 18.8 883.8 588.5 512.9 568,7 117.6 11.0 24.6 2.6 17.9
O. obliquus 159115.7 116988.6 1029.3 3260.4 942.4 133.0 98.9 653.5 149.5 0.0 3.8 62.9 8.1 17.5
(UF162732B)
O. obliquus 186700.9 130473.9 1149.3 37458.4 853.6 113.1 86.0 791.9 106.4 0.0 3.2 76.9 7.9 21.6
(UF162732D)
C. mantelli 89909.63 58346.8 648.6 1151.9 516.8 1109.5 145.9 1326.5 553.6 0.0 10.2 283.7 4.7 19.9

Specimen Y La Ce Nd Sm Gd Dy Yb EREE U La/Yb Ca/P Ce I CI C/P
C carcharias 0.56 0.23 0.09 0.14 0.02 0.04 0.04 0.03 1.15 0.28 7.7 1.2 -0.73 2.82 0.43
(BT0433)
C. carcharias 0.62 0.30 0.57 0.34 0.07 0.07 0.09 0.04 2.11 0.27 7.7 1.1 -0.11 2.83 0.34
(UF211351)
I hastials 9.94 15,04 22.65 9.93 1.85 1.55 1.31 0.97 63.2 6.66 15.5 1.4 -0.09 4.30 0.14
C. megalodon 16.14 23.78 41.26 16.22 3.37 3.29 2.59 1.03 107.7 1.59 23.1 1.7 -0.05 5.39 0.10
C. ariculatus 75.95 104.39 128.85 75.73 13.37 14.71 10.25 3.8 427.1 24.84 27.5 1.4 -0.21 3.41 0.24
C. angustidens 21.11 12.46 13.29 10.85 2.16 2.36 2.19 1.01 65.4 32.9 12.3 1.7 -0.18 3.60 0.30
O. obliquus 31.64 14.19 3.79 6.57 1,26 1.92 2.07 1,86 63.3 48.05 7.7 1.4 -0,85 4.55 0.17
(UF162732B)
O. obliquus 32.89 3.76 6.88 1.29 1.98 2.19 2.00 66.4 57.92 7.6 1.4 -0.85 4.55 0 17
(UF162732D)
C. mantelli 98.90 66.39 91.75 39.43 7.40 8.63 9.99 6.34 328.8 27.26 10.5 1.5 -0.14 4.02 0.21


0,45


0.40-


036 -


030 -
-"

0.25-


0.20-
e-





0.15-
01-


0.10-


0.05-


fnmA II









(Fig. 3-5A) the elemental concentrations range in modern sharks for each element is

indicated by a vertical line. Most fossil sharks have comparable Ca, P, Zn, Si, Pb, and B

concentrations to the modem sharks (Fig. 3-5A). The minor elements Mg, Na, K, Sr, and

Mn are more variable, with some fossil specimens exhibiting concentrations similar to

modern sharks while Fe, Al, and Ba have no fossils with similar concentrations as

modern sharks (Fig. 3-5A). All fossil specimens are enriched in Ba relative to modern,

while most fossils are enriched relative to modern in B, Mn, Al, Fe, and Sr (Fig. 3-5A).

All fossil sharks are depleted relative to modem in Pb, while most fossils are depleted

relative to modern in K, Si, Zn, Mg, and Na (Fig. 3-5A). It can be seen in Fig. 3-5B that

Y, U, and REE's are enriched in fossil bone relative to modern. Fig. 3-5C shows the

Ba/Ca ratio for the fossils are higher than in modern sharks, because diagenesis has

significantly enriched Ba (one to two orders of magnitude greater in fossils than modem

shark centra) in all the fossil centra used in this study. The fossil centra Ca/P ratios are

slightly higher (0.2-0.5 higher) than modern sharks (Fig. 3-5C).

The shale-normalized REE concentrations of the shark centra are enriched relative

to seawater by about 105-107 (Fig. 3-6). The modem specimen C. carcharias

(UF211351), which was caught off the east coast of Florida, has a shale-normalized REE

pattern similar to seawater but is slightly flattened and depleted in Yb, a small negative

Ceanom. (-0.11), a higher concentration of Ce than the other C. carcharias (BT0433), and

(La/Sm)N and (La/Yb)N ratios that overlap those found in coastal waters (Fig. 3-6 and

Fig. 3-7). C. carcharias (BT0433), which was caught off the east coast of South Africa,

has a seawater-like shale-normalized REE pattern (enriched with HREE), has depleted Ce

concentrations, a much larger Ceanom. (-0.73) than the other C. carcharias (UF211351),













A. 1000000

100000
U1
C
0
I 10000


o iooo
o 1000.


o 100.


10.
U,
0
I.
1.



E 1000
0.
A
U,
100


I 10
0
0


1 1
O
W 0.1

0
U-


0.1

0
0 0.01

CI


c
S0.001


= 0.0001

n tnnLL


p Ca


Na


AIFe

Ba rn sr Z

+ Mg
B \ 0






1 10 100 1000 10000 100000 100t
Modem Shark Centra Concentrations (ppm)



LaCe y
Nd & GdU$
+ o"
Sm Lx
A Yb 8S
+ + x x
0 A o 0









0.01 0.1
Modem Shark Centra Concentrations (ppm)


Ca/P






Sr/Ca

Ba/Ca
>P


* 0. obliquus
o 0. obliquus
+ C. mantelli
x hastalis
A C. auriculatus
* C. angustidens
oC. megalodon


0.00001 U0.001 0.001 U.01 U.1 1 1U
Modem Shark Centra Concentrations (ppm)

. Isocon plots (Grant, 1982) depicting variations in fossil elemental

concentrations and ratios due to diagenesis. The isocon lines are the average

of the elemental concentrations in the two modern shark centra. A. Isocon

plot of the major and minor elements, with the red vertical lines represent the

variation between the elemental concentrations in the two modern shark

centra. B. Isocon plot of Y and REE. C. Isocon plot of Ba/Ca, Sr/ Ca, and

Ca/P ratios.


Figure 3-5


U.UUUU ... .... .. .. -.


I










and (La/Sm)N and (La/Yb)N ratios that overlap with oceanic waters (Fig. 3-6 and Fig. 3-

7).

Fossil specimens (Fig. 3-6) may be divided into four groups based on their REE

composition. The first group, which includes the two 0. obliquus specimens, have shale-

normalized REE patterns similar to the modern C. carcharias (BT0433) and seawater

(HREE enriched) (Fig. 3-6A), show a minimum at Sm (disregarding Ce),


La Ce Nd Sm Gd Dy Yb


La Ce Nd Sm Gd Dy Yb


Figure 3-6. PAAS normalized REE of the nine vertebral centra divided into four
diagenetic groups. A. First group has seawater-like REEN pattern. B. Second
and third groups have mixed shale- and seawater-like REEN patterns. C.
Fourth group has shale-like REEN patterns. Seawater is average seawater
(Elderfield and Greaves, 1982) and multiplied by 106 and modern C.
carcharias, UF211351 and BT0433, are multiplied by 101.


B
C. auiculatus

mantelli
C. angustidens
Seawater

0.1 C. carcharias(UF2 1351)



001 C N S
La Ce Nd Sm Gd Dy Yb












*


*
^


VI r j (r Coastal waters
0.1- AA* A A A
@ 00 :00


o0


Oceanic waters
Continental waters w


0.1
0.1 1 10
(LalSm)N

Figure 3-7. Compilation of observed (La/Yb)N vs. (La/Sm)N in biogenic apatites of
various ages and types, and fresh and oceanic waters (based on Reynard et al.,
1999). *, Jurassic fish and reptile teeth (Picard et al., 2002); A, Tertiary and
Mesozoic fish teeth (Grandjean et al., 1988; Grandjean and Albarede, 1989);
Cretaceous reptile and dinosaur bones (Samoilov and Benjamini, 1996) and
Jurassic coprolites (Kemp and Trueman, 2002); 0, North Atlantic seawater
from 0-4000 m depth (Elderfield and Greaves, 1982), Southern Ocean from 0-
4000 m depth (German et al., 1990 ) and North Pacific surface and deepwater
(Piepgrass and Jacobsen ,1992); *, bottom waters (German et al., 1991); 0,
coastal waters (Elderfield and Sholkovitz, 1987) and coastal waters (Hoyle et
al., 1984); -, Scotland river waters (Hoyle et al., 1984); @, anoxic waters
(Elderfield and Sholkovitz, 1987); and current research, A and 0, C.
carcharias, X, I. hastalis, 0, C. angustidens, A, C. auriculatus, o, C.
megalodon, 1 and 0, O. obliquus, and +, C. mantelli. Oceanic, Coastal, and
Continental boxes are based on values given by Reynard et al. (1999). Blue
circles indicate samples with seawater-like REEN patterns, green circles
indicate samples with shale-like REEN patterns, and red circles indicate
samples with mixed seawater- and shale-like REEN patterns.









large Ceanom. (greater than -0.3), and an (La/Yb)N ratio that falls within coastal waters. 0.

obliquus is the only sample that has a (La/Sm)N ratio greater than oceanic waters. The

second group, which includes C. angustidens and C. mantelli, have shale-normalized

REE patterns similar to the modern shark pattern of C. carcharias (UF211351) and close

to seawater but with some flattening (Fig. 3-6B), show a minimum at Nd (disregarding

Ce), (La/Yb)N and (La/Sm)N ratios that fall within or just outside of coastal waters (Fig.

3-7), and small negative Ceanom. (less than -0.3). The third group, which includes C.

auriculatus, shows a minimum in HREE, shale-normalized REE pattern between

seawater and the modern C. carcharias (UF211351) pattern (Fig. 3-6B), (La/Yb)N ratio

above marine and continental waters, (La/Sm)N ratio within coastal and oceanic waters

(Fig. 3-7), and a small negative Ceanom. (less than -0.3). The fourth group, which includes

C. megalodon and I. hastalis, show a minimum at Yb, a flat shale-normalized pattern or a

maximum in the heavy-middle REE (Fig3- 6C), a high (La/Yb)N ratios (above seawater

and continental waters), and (La/Sm)N ratio within coastal and oceanic waters (Fig. 3-7).

Discussion

Mineralogical Characterization of Centra

Fossil specimens have lost most, if not all, of their organic content through

diagenetic processes and contain less absorbed (3430 cm-1 band) and structural H20

(3330 cm-1 band; Holcomb and Young, 1980; Michel et al., 1995) than modern

specimens. The weak intensity of the absorption band near 1660 cm-1, corresponding to

vCONH of the amide group amidee I), and the absence of the two other amide bands

amidee II and III) signify a significant loss of organic (Reiche et al., 2003) in the fossils.

In contrast, the FT-IR spectra of the modern specimens show intense amide I, II, and III

bands and thus indicate the presence of organic matter (Reiche at al., 2003).









When comparing the two modem specimens, the CIs are similar, but the carbonate

content differs, with the South African C. carcharias shark (BT0433) having a higher

carbonate content than the one from Florida. This may be due to due to natural

variability within a shark species or overlapping absorption peaks that have both

carbonate substitution in the A-site for OH- and B-site for PO43- (i.e., peaks 1460 cm-1

and 870 cm-1) are not included in the estimation of carbonate content. Since, the

carbonate content estimation is only normalized by a phosphate peak, the use of A-site

substituted carbonate would not be an accurate representation of carbonate content.

Usually in modern specimens the OH- band at 3567 cm-1 is masked by the water bands

and is completely absent in fossil specimens (Fig. 3-2), making it difficult for

normalization. Lower C/P and higher CI in fossil specimens (Fig. 3-4) indicate

diagenetic loss of carbonate during recrystallization and possibly dissolution of the

mineral phase.

Implications for Diagenetic and Biological Signal Reconstruction

The shale normalized REE patterns of the two modern shark specimens (Fig. 3-6)

have similar patterns to seawater, however, C. carcharias (BT0433) from South Africa

has a more negative Ceanom. and greater HREE enrichment than UF211351 from Florida.

The larger negative Ceanom. in C. carcharias (BT0433) may indicate that sharks from the

coast of Africa live and/or spent most of their time at greater depths than sharks from the

East coast of North America. Alternatively, larger great white sharks may feed and/or

spend a majority of their time at shallower depths than smaller great whites (UF211351

was 12'9" in length when captured and BT0433 was 6'9" in length when captured).

Further study of modem great white shark behavior is needed to fully understand the bulk

chemistry variations within this species.









The first fossil group, which includes the two 0. obliquus specimens, have

seawater-like patterns indicating that REE enrichment involved quantitative uptake of

REE without fractionation. In other words, there was no absorption taking place, no

indications of a terrestrially derived diagenetic signature (flattening of the shale-

normalized REE patterns; Grandjean et al., 1987), or no fractionation of REE (i.e. no

MREE enrichment or bell shape pattern; Reynard et al., 1999). The La/Yb ratio of about

7 and a larger negative Ceanom. points to deposition in an environment with a deepwater

influence (Grandjean, 1987; Grandjean et al., 1988; German and Elderfield, 1990;

Pipegras and Jacobsen, 1992). Results from DSDP Leg 41 off the Moroccan coast have

identified the onset of abundant chert deposition during the early Tertiary reflecting the

input of cold bottom water (Lancelot and Seibold, 1978). Cappetta (1981) showed that

Ypresian fish associations from the Ouled Abdoun basin (Oued Zem, location of O.

obliquus, is located in the eastern part of the Ouled Abdoun basin) were indicative of

greater depth than previous periods. All this evidence indicates either upwelling of deep

water onto the continental shelf or progressive deepening of the troughs and channels in

which the phosphorite was deposited (Grandjean, 1987) and suggests an influence of

deepwater during the diagenesis of 0. obliquus. The original REE signal of the 0.

obliquus centra have been replaced during diagenesis at/or near the sediment/seawater

interface with the seawater signal present at time of deposition of the centra. Therefore,

the two 0. obliquus centra preserved a seawater signal at or near the seawater/sediment

boundary and will be useful in reconstructing paleoceanographic environments.

The second diagenetic grouping, which includes C. angustidens and C. mantelli,

have shale-normalized REE patterns similar to the modem C. carcharias (UF211351)









and close to seawater but with some flattening (Fig. 3-6B). C. angustidens was deposited

in glauconitic shelf sand (Kokoamu Greensand) at a water depth of 50-100 m. The

presence of glauconite indicates slow sedimentation and low inputs of detritus from land.

New Zealand, as a whole, was low-lying and almost fully submerged at this time and

therefore the terrestrial input was minor (Ayrees, 1993). Minor flattening of the C.

angustidens shale-normalized REE pattern relative to seawater supports that the

diagenetic signature, which is produced by large pore water concentration gradients that

allow fluxes of REE from sediments to seawater, has had a small influence from

terrestrial derived sediments. C. mantelli was deposited in the Smoky Hill Chalk

Member of the Niobrara Formation in a water depth between 30-180 m (Hattin, 1981).

The sediments of the Smoky Hill Chalk Member are from the mid to outer shelf of the

Cretaceous epicontinental seaway. Once again, slight flattening of C. mantelli shale-

normalized REE pattern relative to seawater supports a small influence from a

terrestrially derived diagenetic signature. Neither C. angustidens nor C. mantelli have

any indications of REE fractionation ("bell-shaped" shale-normalized REE pattern). C.

angustidens and C. mantelli centra have the potential to be used for a general

paleoceanographic and paleoenvironmental reconstruction, but with caution, because they

do have a mixture of seawater signal and diagenetic signature, which will decrease the

Ceanom. as well as flatten the shale normalized REE pattern.

The third group, which includes C. auriculatus, has a shale-normalized REE

pattern between seawater and the modern C. carcharias (UF211351) (Fig. 3-6B). The C.

auriculatus centrum was deposited in the Brussels Sand in a shelf/nearshore environment

(Hooyberghs, 1990; Herman et al., 2000). The shale-normalized REE pattern, unlike the









second fossil group and C. carcharias (UF211351), has a high at Gd and is more depleted

in Dy and Yb. The flattening of the shale-normalized REE pattern, the (La/Yb)N ratio

greater than seawater, and the depletion of Dy and Yb in the C. auriculatus centrum

indicates a diagenetic signature that is more strongly derived from terrestrial sediments

than that of the second fossil group. The C. auriculatus centrum has the potential to be

used for a general paleoenvironmental reconstruction, but with caution, because of the

terrestrially influenced diagenetic signature, which will decrease the Ceanom. and flatten

the shale-normalized REE pattern.

The fourth group, which includes C. megalodon and I. hastalis have a flat shale-

normalized pattern or a maximum in the heavy-middle REE (Fig3- 6C). The C.

megalodon centrum was deposited in sandy siltstone near the Kanto Mountains in Japan

(Hayashi et al., 2003). The shale-normalized pattern of C. megalodon is almost

completely flat indicating a diagenetic signature that is extensively derived from

terrestrial sediments. The I. hastilas centrum was deposited in a shallow bay sandstone

and has a flat shale-normalized REE pattern, which indicates a diagenetic signature

derived primarily from terrestrial sediments and/or major influence from river water.

This is supported by fresh water diatoms in the sediments of the Pisco Formation

suggesting the influx of river water entering the basin (Brand et al., 2004). The high

(La/Yb)N ratios found in C. megalodon and I. hastalis can be explained by the extensive

terrigenous influence (Grandjean et al., 1987). C. megalodon and I. hastalis do not show

any indications of late diagenesis ("bell-shaped" shale-normalized REE pattern). These

two centra indicate the quantitative intake of REEs during early diagenesis with a strong

influence of continental sedimentary supply in a near-shore environment; therefore they









are not useful in paleoceanographic reconstruction (global controls) but the record of the

diagenetic signature indicates the sedimentary environment (local controls) can be

reconstructed.

For the fossil shark centra used in this study the major control of the shale-

normalized REE pattern would be the diagenetic signature and how the terrestrially

derived sediments generate concentration gradients between pore waters and seawater.

The more prominent the terrestrial sediments the flatter and more shale-like the shale-

normalized REE signal become. Each depositional environment must be assessed in

order to determine which fossils can be used in paleoceanographic studies versus

depositional environment reconstructions.

Conclusions

While certain variables independently provide information about diagenesis, the

simultaneous use of FT-IR and elemental concentrations gives a much better picture of

depositional environment and the extent of diagenesis. There is no doubt that diagenetic

alteration has affected the REE composition of these seven fossil shark centra. Through

the processes of diagenesis, the centra have been imprinted with an REE seawater and

diagenetic REE pattern at the sediment/seawater interface. The REE seawater signature

was incorporated into the biogenic apatite via a transfer from a short-lived phase made of

oxy-hydroxides and organic detritus (Grandjean et al., 1987). The diagenetic signature is

caused by the development of pore water concentration gradients, which allow fluxes of

REE from sediments to seawater. The amount of terrigenous input and therefore the REE

composition of diagenetic signature controls whether the shale-normalized REE patterns

and Ce anomalies are representative of the original seawater signal for these seven fossil

shark centra. This is clearly seen for I. hastalis, and C. megalodon, where these two









specimens have a strong terrestrially influence diagenetic signature, which disturbs the

oceanic signal and Ceanom.. In contrast, the two 0. obliquus centra preserve a REE

seawater signal at the time of deposition and have no indication of a diagenetic signature

derived from terrestrial sediments. The remaining three centra (C. angustidens, C.

megalodon, and C. auriculatus) have diagenetic signatures that have some influence from

terrestrial sediments, which is evident by the slightly flattened shale-normalized REE

patterns. These later kinds of samples would require extreme care when interpreting Ce

anomalies because it is difficult to determine how much the Ceanom. has been reduce by

the diagenetic signature. Hence, even if there is no negative Ceanom. that does not

necessarily indicate an anoxic environment but may represent a strong continental

influence in the depositional environment. In summary, geochemical data from biogenic

apatite of fossil marine vertebrates, like lamnid sharks, have the potential to be used to

understand diagenesis, depositional environments (local controls), and/or

paleoceanography (global controls).














CHAPTER 4
OXYGEN ISOTOPIC AND RARE EARTH ELEMENTAL ANALYSIS OF MODERN
LAMNID SHARKS: DETERMINATION OF LIFE HISTORY?

Introduction

Lamnid sharks, Family Lamnidae (great white sharks and their relatives), are of

great interest not only to the scientific community but the public as well. Scientists have

spent a great deal of time trying to study and understand the life history of great whites

(Carcharodon carcharias) and their relatives. Because great whites do not survive well

in captivity, tagging and recapture studies and captured sharks from fishermen have been

the main source of study.

Sharks deposit light and dark bands on their vertebral centra throughout their lives

(Fig. 4-1). It is known that in most sharks the darker, denser portions are deposited

during slower growth times (e.g., winter) and lighter portions are deposited during more

rapid growth (e.g., summer). The problem is that the growth rate is affected by the

physical environment (including temperature and water depth), food availability, and

stress (Branstetter et al., 1987). Therefore, it cannot be assumed that a band pair (one

light and one dark band) reflects a single year (called annulus).

Wintner and Cliff (1999) estimated ages of great white sharks from the East coast

of South Africa by counting growth rings in the centra. The vertebrae of 61 females and

53 males were x-rayed and counts were made from the x-rays. X-rays enhance the

visibility of growth rings in shark centra and have been used successfully to accurately

determine ontogenic age of several species (Cailliet et al., 1983; Yudin and Cailliet,









1990; Ferreira and Vooren, 1991). Of particular interest from Wintner and Cliffs (1999)

study was the one shark that was injected with oxytetracycline (OTC) on October 10,

1994, and was recaptured on May 27, 1997 (specimen BTO433). BTO433 was tagged at

140 cm and grew 69 cm within that two year, seven month and 27 day period. The OTC

indicated annual growth ring deposition in most of the centra from BTO433; however,

this could not be confirmed from growth ring counts of the entire sample (Wintner and

Cliff, 1999).



Dark growth band
Birth Mark
OTC Mark:10/30/94
Captured: 5/27/97














Figure 4-1. Scanned contact print of BTO433 centra. (Top) Dark growth bands, OTC
mark and birth mark have all been indicated. Dark growth rings on the
contact print show up as white bands, which can be seen in the lower image of
the centra.

Bomb Carbon and Oxygen Isotopes

Atmospheric testing of atomic bombs in the 1950s and 1960s resulted in a rapid

increase in radiocarbon (14C) in the world's oceans (Druffel and Linick, 1978). The

period of radiocarbon increase was almost synchronous in marine carbonates such as









corals, bivalves, and fish otoliths around the world (Kalish, 1993; Campana, 1997; Baker

and Wilson, 2001), providing a date marker in calcified structures exhibiting incremental

growth. More recently, analysis of bomb radiocarbon has been used to validate age

estimates derived from vertebral centra of sharks (because sharks do not contain otoliths

that grow incrementally; Campana et al., 2002). Campana et al. (2002) used bomb

carbon dating as an age validation method for long-lived sharks. They compared

radiocarbon assays in young, known-age porbeagles (Lamna) collected in the 1960s with

corresponding growth bands in old porbeagles collected later. With this method Campana

et al. (2002) confirmed the validity of porbeagle vertebral growth band counts as accurate

annual indicators.

Because shark vertebrae grow incrementally, the oxygen isotopic signal preserved

should reflect the seawater conditions at the time of formation. Oxygen isotopes vary

with temperature and salinity (Hoefs, 1988), consequently variations preserved in the

vertebral centra could indicate migration into various water bodies and/or water depths.

Also, it might be possible to determine the frequency of the migration and, if annual, the

oxygen isotopic signals could be used to estimate ontogenic age of the individual.

Rare Earth Elements

The rare earth elements (REE) consist of fifteen elements which form a series from

the lightest REE, lanthanum (La), to the heaviest, lutecium (Lu). With the exception of

multiple oxidation states for Ce and Eu, the other REEs have trivalent oxidation state in

most natural waters. Cerium may undergo oxidation in seawater from the solvated Ce3+

state to insoluble Ce4 consequently Ce fractionates relative to other REE (German and

Elderfield, 1990). Europium may undergo reduction from the Eu3+ to Eu2 which

substitutes readily for Ca2+ inCa- bearing minerals such as apatites (Elderfield, 1988).









The residence time of REE's in seawater is 102-103 years and is therefore shorter than the

mixing time of the oceans (1600 years) making these elements useful tracers of

oceanographic events and processes (Elderfield and Greaves, 1982; Bertram and

Elderfield, 1993; Nozaki et al., 1999; Lacan and Jeandel, 2001). Because most REE

concentrations increase with water depth (Elderfield and Greaves, 1982; deBaar et al.,

1985a; deBaar et al, 1985b; Dubinin, 2004) they may act as a proxy to indicate the

relative water depth at which these individual sharks are living

This study tests whether the chemistry of vertebral centra can be used to improve

our understanding of the life history of sharks. The possibility exists to elucidate shark

migration, the relative depth of habitat, and determination of individual age using rare

earth elemental compositions, bomb carbon, and oxygen isotopes.

Background

Five species of lamnid sharks live today, Carcharodon carcharias (great white),

Isurus paucus (longfin mako), Isurus oxyrinchus (shortfin mako), Lamna ditropis

(salmon shark), and Lamna nasus (porbeagle). This study focuses only on the largest

three members of Lamnidae, the great white, longfin mako, and shortfin mako (Table 4-

1), due to ease in sampling larger vertebral centra.

Great White (Carcharodon carcharias)

The great white is the largest extant lamnid shark and has one of the broadest

distributions of all modern sharks. The great white is cosmopolitan in cold temperate to

tropical seas, living primarily in coastal and offshore habitats of continental and insular

shelves. However large individuals have been recorded off oceanic islands. The known

depth range of great whites is from the surface to at least 6,150 ft (1.875 km). Great

whites maintain a body temperature up to 270F (150C) warmer than surrounding waters









by an adaptation to its circulatory system that does not allow the heat generated within

the body to escape through the gills (Compango, 2002). Great whites prefer waters with

a sea surface temperature between 59-720F (15-220C). Most individuals are 12-16 ft

(3.7-4.9 m) long with a maximum length of 20 ft (6.1 m). At birth a great white is

between 3'11" to 4'3" in length. Juvenile sharks feed on bottom-dwelling teleost fish,

small sharks, and rays, while adult sharks feed on sharks, rays, teleost fish, seals, sea

lions, dolphins, whale blubber (scavenged), squid, seabirds, marine turtles, crabs, and

snails (Campagno, 2002).

Longfin Mako (Isurus paucus)

The longfin mako was first described in 1966 and is one of the least-known

lamnids. It is also the second largest member of Lamnidae, after the great white.

Longfin makos have an appearance similar to shortfin makos but have a slimmer body,

larger eyes, and larger pectoral fins. Most specimens are about 7 ft (2.2 m) long and the

maximum known length is 14 ft (4.3 m), which is based on a male specimen taken from

15 mi (24 km) off Pompano Beach, FL, in February 1984 (Compango, 2002). At birth a

longfin mako is between 3' to 3' 11" (92 to 120 cm) in length. Longfin makos are widely

distributed in tropical to warm temperate seas. They are fairly common in the western

Atlantic (Gulf Stream waters, northern Cuba to southeast Florida) and possibly in the

central Pacific (near Phoenix Island and north of Hawaii), although rare, longfin makos

have been recorded off northwestern Africa and the Iberian Peninsula, from the northern

Gulf of Mexico to the Grand Banks, Bahamas and off New South Wales Australia

(Compango, 2002). Most specimens of longfin makos are caught on long-lines in deep

tropical waters, from depths of 360-720 ft (110-220 m). The long broad pectoral fins

suggest that longfin makos are slower and less active than shortfin makos. This inference









is also supported by the fact that longfin makos have the same heat-retaining

modifications to the circulatory system as other lamnids; however longfin makos are

unique among the members of its family in that this species is not warm bodied. The diet

of a longfin mako consists of schooling fish and pelagic squid (Compagno, 2002).

Table 4-1. Lamnid specimens used in this study.
Number Body
Taxon Specimen Location of Capture of Length
Centra (ft)
Carcharodon
Cn BTO433 Capetown, S. Africa 1 6'11"
carcharias
Carcharodon
Cr UF211351 Islamorada, Florida 1 12'9"
carcharias
Carcharodon
Cn UF211352 Marathon, Florida 1 12'5 /4"
carcharias
Carcharodon .
Cr UF31648 Florida 1 N/A
carcharias
Isuruspaucus UF211355 Miami, Florida 3 8

Pompano Beach, 8
Isuruspaucus UF211354 o o 3 84"
Florida
Isuruspaucus UF211353 o ao a 1 14
Florida
Isurus
Isus UF47943 Florida 1 N/A
oxyrhincus

Shortfin Mako (Isurus oxyrinchus)

The shortfin mako is the fastest swimming shark and has a global distribution in

tropical and temperate waters. Shortfin makos are common in coastal and oceanic

regions of tropical and temperate seas but seldom occur in waters less than 610F (160C).

They range from California to Chile in the Pacific Ocean and from the Grand Banks of

the Bahamas to Brazil, including the Gulf of Mexico and the Caribbean Sea in the

Atlantic Ocean. In the eastern Atlantic, shortfin makos range from Norway to South

Africa, including the Mediterranean, and is found throughout the Indian Ocean from

South Africa to Australia. In the western Pacific it can be found from Japan to New









Zealand and in the central Pacific it occurs from the Aleutian Islands to the Society

Islands (Compango, 2002). The known depth range is from the surface down to at least

1,300 ft (400 m). Shortfin makos tend to follow movements of warm water in extreme

northern and southern parts of its range. Tagging studies off the northeastern US show a

seasonal pattern of abundance along the western margin of the Gulf Stream, moving

inshore and into higher latitude waters as the stream shifts northward from April-October,

possibly wintering in the Sargasso sea from November to March (Compango, 2002). At

birth a shortfin mako is 2 to 2'3" long with most individuals 6-8 ft (1.8-2.5 m) long and a

maximum recorded length of 12.8 ft (3.9 m). Shortfin makos maintain body temperatures

12.5-180F (7-100C) warmer than the ambient water and are capable of rapid acceleration

and bursts of speed when hooked or in pursuit of prey. Adults have been clocked at 31

mph (50 km/hr) (Compango, 2002).

Materials and Methods

X-radiograph Analyses

X-radiographs were used to enhance the visibility of the growth rings. X-

radiographs of whole centra were taken at the C.A. Pound Human Identification

Laboratory at the University of Florida. The x-rays are set at 78 kV for 2 minutes. The

x-rays are used to make contact prints, which are a reversed pattern of the x-ray (i.e., dark

lines on the x-ray are the light lines on the contact print). The dark and light alternating

growth rings are easily seen on the contact prints (Fig. 4-1). These contact prints were

digitally scanned and Adobe PhotoshopT was used to enhance the images. Growth ring

counts were made from the scanned contact prints and interpretation of the vertebral

growth bands was made using published criteria for porbeagles and Pacific shortfin

makos. Campana et al. (2002) has proven with the use of bomb carbon, annual growth









band pairs form in porbeagles, and Cailliet at al. (1983) demonstrated that a single band

pair formed each year in Pacific shortfin mako based on growth ring counts.

Oxygen Isotopic Preparation and Analyses

Oxygen isotopes can be used for the interpretation of temperature. For each of the

centra, about 1 mg was drilled with a foredoom slow speed drill approximately every 1

mm across the growth axis starting from the birthmark and ending at the external margin.

An interval of 1 mm insured that each primary growth band was sampled. Sample

powders were treated with standard isotope preparation techniques (e.g., MacFadden et

al., 1999). At least 0.75 mg of each treated sample powder was then measured into

individual metal boats and placed in the carousel of the isocarb device for introduction

into the VG Prism mass spectrometer in the Stable Isotope Laboratory in the UF

Department of Geological Sciences. The sample runs were calibrated to internal

laboratory and NBS 19 standards. The oxygen isotopic results are reported in the

standard "6" convention: 6 (parts per mil, %o) = (Rsampe/Rstandard)-I) x 1,000), where R =

13/13C or 180/160, and the standard is VPDB. Ontogenic ages based on the oxygen

isotopic data were estimated by counting the number of peaks or valleys present long the

growth axis of the centra.

Bomb Carbon Dating Preparation and Analysis

Atmospheric testing of atomic bombs in the 1950's and 1960's produced a time-

specific radiocarbon marker, which allows for material formed between the 1950's and

the present to be dated (Kalish, 1993; Weidman and Jones, 1993). To determine if bomb

carbon dating would work on great white centra, only one shark centra, BTO433, was

chosen for dating. BTO433 was chosen because the capture date is known and this shark









was injected with oxytetracycline (OTC) on October 30th 1994. The OTC mark gives a

second reference for the accuracy of the bomb carbon dates generated.

About 60 mg of sample was extracted from BTO433 first formed growth band

(corresponding to the first year of growth) and the last growth band. The external surface

of the centrum was removed in order to minimize surface contamination. The sample was

weighed to the nearest 0.01 mg in preparation for assay with Accelerator Mass

Spectrometry (AMS). The sample was assayed at the Keck Carbon Cycle AMS

Laboratory, UC Irvine for 613C (to determine the carbon source) and A14C (measure of

radiocarbon), with A14C calculated per Stuiver and Polach (1977).

To assign dates of formation to an unknown sample, it is necessary that the A14C of

the unknown sample be compared with a A14C of a known-aged material. BTO433 A14C

data were compared to the A14C values of Pagrus auratus otolith collected off the East

coast of North Island, New Zealand (Kalish, 1993). An age for BTO433 based off the

Campana et al. (2002) reference curve was assigned by correcting the 1997 A14C value of

BTO433 to the1997 porbeagle reference curve value generated by Campana et al. (2002),

then the same correction was applied to the first growth band A14C value and that value

was compared to the porbeagle curve to determine a year for deposition of the first

growth band (Fig. 4-2). This was done to determine if the second method would produce

similar results to the first, because reference curves are not always available for the area

in which the specimen was recovered and this would allow for the use of previously

produced reference curves. It is important to have a local reference curve since the

carbon was not uniformly distributed at the time of atomic bomb testing, especially










between the Northern and Southern Hemisphere as most of the bomb testing was done in

the Northern Hemisphere.






50 *BT0433B

-" o

.50

-100

-150
1952 1956 1960 1964 1968 1972 1976 1980 1984 1988 1992 1996 2000 2004
Year

Figure 4-2. BT0433 bomb carbon data plotted vs. two reference curves (a) fish otolith
from New Zealand (Kalish, 1983) and (b) porbeagle (Campana, 2002).
BT0433 is plotted on the fish otolith curve and BT0433B is plotted on the
porbeagle curve.

Inductively Coupled Plasma Mass Spectroscopy (ICPMS)

Inductively coupled plasma mass spectroscopy (ICPMS) allows for the

quantification of elemental abundances within the shark centra. Approximately 5-10 mg

of bulk sample was drilled from each of the twelve centra. 5 mg of each sample were

weighed into 3 mL Savillex vials and dissolved in 1 mL of 3M HNO3 and heated

overnight. Samples were allowed to cool and then dried. Then 2 mL of 1% HNO3 was

added, heated overnight, and allowed to cool. Samples were analyzed on an Element 2

High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS) at the

UF Department of Geological Sciences. All samples were corrected by subtracting the

blank, corrected for instrumental drift based on internal machine standards that were

analyzed during the run, and correcting ion counts to a constant response to the known

amount.









All REE values were shale-normalized to PAAS (Post-Archean Shale Standard) in

order to eliminate the odd-even effect of the natural abundances of the REE. The Ce

anomaly (Ceanom) was calculated with the Elderfield and Greaves (1982) formula,

Ceanom=Log[3 CeN/(2LaN+NdN)].

Results and Discussion

Ontogenic Age Determinations

The ontogenic ages determined from oxygen isotopic cyclicity, growth ring counts

and the A14C of the eight sharks are presented in Table 4-2. The ontogenic ages

estimated based on the oxygen isotopic cyclicity (Fig. 4-3) give ages that are too young

with the exception of UF31648 and BTO433 (Table 4-2) when compared to growth ring

counts. For UF211351, UF211352, and UF47943 the oxygen isotopic signal gave an

ontogenic ages of 5+ years, 7+ years, and 4+ years, respectively, which are all several

years too young compared to growth ring counts. For specimens UF211354 and

UF211353 the oxygen isotopic data gave ages of 6+ and 8+, respectively, which are

about half of the ages estimated from the growth ring counts. The three UF211355 have

age estimates of 3+, which is 8+ years too young according to the growth ring counts.

One problem with the oxygen isotopic data is that sampling every growth ring is

challenging. As sharks get older, their growth rate decreases and therefore the size of the

growth rings become narrower making it difficult to sample only one growth ring at a

time. Also, the location of the centra in the sharks vertebral column (i.e., towards the

head or towards the tail) will affect the age estimate for both oxygen isotopic analysis and

growth ring counts. Sampling the exact same location for multiple centra within an

individual is difficult to accomplish because not all the vertebrae grow at the exact same

time. This accounts for the offset of the isotopic data in sharks UF211354 and UF211355









Table 4-2. Ontogenic age estimates based growth ring counts (GR), oxygen isotopic
(6180) cyclicity, and bomb carbon (A13C). Ages estimated from growth ring
counts should be considered minimum ages since the last growth band may
not have been formed completely at time of capture.
Taxon Specimen GR counts 610 age A14C age
Carcharodon carcharias BTO433 3+ 3+ 3
Carcharodon carcharias UF211352 9+ 7+
Carcharodon carcharias UF211351 7+ 5+
Carcharodon carcharias UF47943 4+ 3+
Isurus paucus UF211355A 11+ 3+
Isurus paucus UF211355B 11+ 3+
Isurus paucus UF211355C 11+ 4+
Isurus paucus UF211354C 11+ 4+
Isurus paucus UF211354B 13+ 5+
Isurus paucus UF211354A 12+ 5+
Isurus paucus UF211353 16+ 8+
Isurus oxyrhincus UF31648 3+ 3+

of the three centra analyzed (Fig. 4-3). Choosing the largest centra and/or centra that are

closer to the head than the tail would probably alleviate some of these effects. Another

possibility is that the sharks studied did not migrate into bodies of water with enough

temperature variation to alter the oxygen isotopic signal. Also, great whites and shortfin

makos are warm-bodied and maintain a body temperature above the surrounding water

temperature, which will affect the oxygen isotopic signal, (i.e., the isotopic variation

might not reflect the actual water temperature change, but the changes in body

temperature as the sharks encounter colder or warmer waters). It has been shown that

muscle temperature of lamnid sharks changes in response to changes in ambient water

temperature (Carey et al., 1982; Tricas and McCosker, 1984; and Carey et al. 1985).

Oxygen isotopic signals may reflect changes in body temperature; these changes may

accentuate or dampen the oxygen isotopic signal preserved in the centra. The shark

centra analyzed in this study do not seem to have a dampened oxygen isotopic signal (i.e.,

oxygen isotopic values along the growth axis have similar absolute values) indicating that









great whites and shortfin makos do not maintain a constant body temperature (Fig. 4-3).

Unlike the shortfin mako and great white, the longfin mako is not warm-bodied and

therefore does not maintain a body temperature above that of its surroundings and

consequently the oxygen isotopic signal should reflect the temperature of the surrounding

water.

BT0433 age is estimated by the A14C values, which are plotted against the two

reference curves in Fig. 4-2. BT0433 A14C values were plotted against the first reference

curve, otoliths from fish off the coast of New Zealand (Kalish, 1993), and no correction

was necessary because the 1997 age plotted on the curve. The second method entailed

subtracting the 1997 A14C reference curve value generated from the porbeagle centra

(Campana, 2002) from the BT0433 1997 A14C value and then subtracting that same

amount from the BT0433 first growth ring A14C value. Both methods produce the same

ontogenic age of 3 years. More work needs to be done in order to determine if

subtracting the difference of the A14C values of the known age of the specimen to the

reference curve works for sharks that are older. However, the fact that both

methods(growth ring counting and bomb carbon dating) produced an ontogenic age of 3

years for BT0433 indicates that at least at young ages great whites deposit growth bands

on an annual basis. Also, if two growth band pairs form per year, this shark would only

be 11/2 years old and that is impossible since 2.6 years passed before recapture of BT0433

after the OTC injection (Wintner and Cliff, 1999). However one individual cannot be

used to provide a definitive growth rate for great whites, further radiocarbon assays of

lamnid shark vertebrae is needed to accomplish this goal.


























8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
mm

-7.5
-7 UF211351
-6.5 Great White


-5
-4.5
-4
-3.5

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
mm


1.351


7 8 9 10 11 12 13 14 15 16 17 18 19
mm

0
BT0433
1 Great White I


9 10 11 12 13 14 15 16 17 18 19 20
mm


5 6 7 8 9 10 11 12 13 14 15
mm


5 6 7 8 9 10 11 12 13 14
mm


.3
UF211353
.5 Longfin Mako
.7
.9


.3
.5
.7
5678910111213141516171819202122232425


5 6 7 8 9 1011 1213141516171819202122232425
mm

Figure 4-3. Oxygen isotopic data (VPDB) for the shark centra analyzed. UF211354 and

UF31155 both have three centra per specimen.




Rare Earth Elements


The elemental concentrations are given in Table 4-3 and the shale-normalized REE


patterns are shown in Fig. 4-4. The shale-normalized REE concentrations are enriched


relative to seawater by about 104 -105. For all the sharks captured off the coast of


Florida, Eu is enriched relative to seawater, Eu was not analyzed for the specimen caught


UF31648
Great White


UF211352
Great White


0.2
0.4
06E
0.8

1.

1.6
1.6


0.



0.





1
0.

o0.



1
1
1
1.


0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
mm


UF211355
Longfin Mako
B


0.2
0.4
0.6
00.8

1.2
1.4
1.6
1.8


n7e









off the coast of South Africa. Eu is known to replace Ca in apatite more readily than

other REE since Eu2+ has the same oxidation state and a similar ionic radius to Ca2+

(Elderfield, 1988), therefore expectations are for marine biogenic apatite to be enriched in

Eu relative to seawater. Specimens UF211353, UF31648, UF47943, and UF211352 all

have depletion in Gd relative to seawater. The degree of enrichment of Eu and depletion

of Gd varies between specimens. BT0433 and UF211352 have shale normalized REE

patterns similar to average seawater, ignoring the Eu enrichment (Fig. 4-4). UF211351,

UF211354, and UF211355 have shale-normalized REE patterns between seawater and

coastal waters (Fig. 4-4), ignoring the Eu enrichment. UF211353, UF31648, and

UF47943 have shale normalized REE patterns similar to coastal waters (Fig. 4-4),

ignoring the Eu enrichment.

The REE patterns differ with location along the Florida coast, indicating that REE

shale-normalized patterns may serve as a provence tool (i.e., location determination). In

this study the two centra, UF31648 and UF47943, belong to sharks without capture

location data but exhibit REE distributions similar to that of UF211353. To use bulk

samples as a provenace tool numerous sharks from an area should be analyzed, which is

evident from the differences between UF211354 and UF211353. Both of these sharks

were caught off the coast of Pompano Beach, Florida but they show different shale-

normalized REE patterns, because they probably spent time in different waters/depths

throughout their lives. The REE pattern throughout life will be averaged when bulk

samples are used; therefore changes in the REE shale-normalized patterns along the

growth axis of the centra (i.e., migration patterns) should be shown when microsampling

is utilized.









The REE content variation in seawater correlates to the circumcontinental zonality

(Dubinin and Rozanov, 2001; Tachikawa et al., 1999) due to an exchange between the

dissolved REE and absorbed complex of terrigenous suspended matter. The decrease in

terrigenous particulate content toward the pelagic area leads to the increase of the

dissolved REE concentration (Dubinin, 2004). Dissolved REE content increases with

water depth by several times in the Atlantic Ocean (de Barr et al., 1985; Sholkovitz,

1994; Dubinin, 2004), therefore REE concentrations could be a proxy for relative water

depth for these shark centra. The plot of La+Sm+Yb (ppm) versus (Sm/Yb)N (Fig. 4-5)

indicates that (a) UF211351, UF211353,UF211355, UF47943, and UF31648 lived at

similar depth closest to the surface; and (b) BTO433, UF211352, and UF211354 also

lived at similar depth, but deeper than the previously mentioned group. UF211353,

UF211354, and UF211355 should plot at similar depths (i.e., have similar REE

concentrations) since it is known that most longfin makos live and feed between 360-720

ft and expectations are that the REE values would not vary much within this depth range

since it is only a difference of 360 ft. Shortfin makos have a larger depth range in which

they feed and inhabit than longfin makos, however according to these data UF47943

lived or ate at a fairly shallow depth relative to the other sharks analyzed.

Conclusions

This is the first study to analyze the chemical and light stable isotopic composition of

lamnid shark centra. Because lamnid sharks cannot be kept in captivity, REE

concentrations, oxygen isotopes, and bomb carbon are new approaches to gaining

knowledge about these animals. Bomb carbon dating is an effective way to accurately

date shark centra, and this has been tested with an individual with a known capture date.
















Table 4-3. Elemental data (in ppm), and oxygen isotopic and bomb carbon dating ages. N/A represents elements that were not
analyzed and (-) indicates that the concentrations were below detection limits of the ICPMS.
Taxon Specimen Na Mg Al Mn Fe Ni Cu Zn Sr Y Ba Pb U P
266.0
C. carcharas BT0433 4053.43 1752.92 1 4.80 177.22 N/A N/A 904.81 352.49 0.56 1.47 9.11 0.28 59982.29
C. carcharas UF211352 8391.58 3196.68 82.32 6.95 35.19 1.54 2.13 255.64 1428.63 0.05 3.20 1.04 2.42 144448.89
C. carcharas UF211351 6559.68 2678.65 30.78 6.43 408.77 7.91 6.65 29.51 1120.88 0.03 1.76 0.54 0.14 116058.87
C. carcharas UF47943 5012.73 2822.44 75.03 4.82 19.52 5.72 8.62 36.04 1181.54 0.05 3.47 2.47 0.11 119935.33
I. paucus UF211355A 6012.29 3053.64 88.06 9.80 42.78 0.66 1.15 52.40 1541.16 0.06 6.62 0.19 0.10 131921.99
I. paucus UF211355B 5001.04 1868.17 52.46 10.60 16.28 0.75 0.93 31.45 1545.61 0.04 8.27 0.15 0.11 135493.72
I. paucus UF211355C 5992.55 3050.59 83.15 9.25 14.71 0.86 1.81 46.09 1478.26 0.04 6.67 0.27 0.09 130841.17
I. paucus UF211354C 4915.74 2651.50 46.06 8.94 79.74 0.84 1.09 35.57 1417.09 0.08 6.78 0.64 0.21 131198.65
I. paucus UF211354B 4548.74 2327.36 21.16 7.01 41.85 0.38 0.60 28.17 1226.32 0.04 6.05 0.32 0.18 117790.59
I. paucus UF211354A 4782.93 2627.17 49.99 7.77 54.53 0.76 0.95 32.09 1337.93 0.08 7.43 0.67 0.21 125275.09
I. paucus UF211353 7522.55 2775.05 11.37 10.52 16.66 0.45 1.05 54.17 1596.44 0.01 4.52 24.26 0.17 137821.84
I. oxyrhincus UF31648 5869.75 3483.01 20.78 7.62 14.73 1.61 5.87 54.54 1563.24 0.09 5.87 0.57 0.21 142857.41


Ce
Taxon Specimen K Ca La Ce Pr Nd Sm Eu Gd Dy Yb Lu anom.
C. carcharas BT0433 1133.63 73202.7 0.23 0.09 N/A 0.14 0.02 N/A 0.04 0.04 0.03 N/A -0.68
C. carcharas UF211352 322.38 302628.1 0.168 0.028 0.033 0.135 0.025 0.014 0.007 0.024 0.016 0.003 -1.08
C. carcharas UF211351 261.62 246712.3 0.044 0.068 0.01 0.035 0.009 0.006 0.005 0.008 0.005 0.002 -0.11
C. carcharas UF47943 185.32 250650.4 0.055 0.138 0.011 0.044 0.011 0.013 0.008 0.005 0.001 0.1
I. paucus UF211355A 187.57 280707.6 0.081 0.097 0.018 0.078 0.014 0.022 0.029 0.014 0.008 0.003 -0.25
279298.9
I. paucus UF211355B 187.16 8 0.086 0.132 0.021 0.087 0.018 0.023 0.012 0.016 0.009 0.002 -0.15
I. paucus UF211355C 217.55 277130.3 0.185 0.17 0.041 0.16 0.029 0.026 0.018 0.027 0.009 0.002 -0.35
I. paucus UF211354C 131.12 280654.2 0.476 1.45 0.13 0.62 0.145 0.047 0.156 0.183 0.103 0.019 0.1
I. paucus UF211354B 105.88 252319.5 0.2223 0.444 0.057 0.245 0.051 0.024 0.036 0.055 0.034 0.007 -0.05
I. paucus UF211354A 109.88 270683.3 0.271 0.773 0.073 0.336 0.07 0.033 0.1 0.089 0.058 0.009 0.08
I. paucus UF211353 639.50 291824.4 0.05 0.071 0.012 0.047 0.01 0.017 0.01 0.006 0.001 -0.18
I. oxyrhincus UF31648 192.68 30000.6 0.055 0.131 0.013 0.051 0.013 0.018 0.007 0.006 0.001 0.05

















0.01


0.001:





0.0001


0.1





0.01

z
LU

0.001





0.0001


0.1:





0.01
z
LU
LU
cc

0.001:


* BT0433 Caught off coast of S. Africa
-F UF211352 Caught off coast of Marathon, FL
- UF211351 Caught off coast of Islamorada, FL .-...""


A.,
X" ...... .....


A. ..---w---^""







---- Seawater (x 06)
---- Connecticut Coastal Waters (x104)

La Ce Nd Sm Eu Gd Dy Yb Lu


Caught off coast of Miami, FL

.. ... .... -.... .....







X- .---x
x... .

-.-UF211355A
-+-UF211355B --a-Seawater (x10s)
--UF211355C *x-- Connecticut Coastal Waters (x10)


La Ce Nd Sm Eu Gd Dy Yb Lu


0.0001 I.
La Ce Nd Sm Eu Gd Dy Yb Lu

Figure 4-4. Post Archean Australian Shale normalized rare earth element plots for the
eight sharks analyzed compared with average seawater (Elderfield and
Sholkovitz, 1987) and Connecticut coastal waters (Elderfield et al., 1990).


r-*-UF211354C
Caught off coast of J --UF211354B
Pompano Beach, FL -o.UF21135A
S- f-UF31648
Unkown capture location --_UF47943












A m BT0433
UF211352
A E UF211351
E A 0UF47943
C A AUF211355
S\ AUF211354
"\ AUF211353
+E0.1 A *UF31648

+ \
-I-
S Increasing water depth



0.01 .. ...
0.1 1 10
(Sm/Yb)
Figure 4-5. Depth estimates for the eight lamnid sharks. Arrow indicates direction of
increasing water depth.

Both methods of bomb carbon age estimation (correcting to a known curve and plotting

against a known curve) seem to work for at least the one great white analyzed here.

BTO433 has annual growth ring deposition giving it an age of 3+ years, which coincides

with the growth ring counts from the contact prints of the centra.

Oxygen isotopes give an indication of changes in body temperature for shortfin

makos and great white sharks, and water temperature for longfin makos throughout their

lives. Ontogenic age estimated based on the oxygen isotopic data did not give similar

ages for most sharks when compared to the growth ring counts. Either the eight sharks

studied were not encountering waters that shifted their body temperature enough to

change the oxygen isotopic signal seasonally, or during sampling some growth bands

were missed and/or more than one band was sampled averaging the oxygen isotopic data.

Annual growth deposition in lamnid sharks is supported by bomb carbon dating from

Camapana (2002) and from the great white BTO433 from this study. Also, UF211353 a









14' longfin mako, which is twice the usual size, would only be 8 years old if two growth

band pairs were deposited annually, indicating that the longfin mako, which is the slow

moving non-warm bodied lamnid, would grow at a faster rate than the warm-bodied fast

moving shortfin mako.

REE shale normalized patterns have the potential to be utilized for assessing the

general location of sharks that have unknown capture locations. Elemental analysis was

done on bulk samples, which would average the REE values along the growth axis.

Sharks with similar REE patterns most likely lived in similar waters. UF31648 and

UF47943 were captured off the coast of Florida and no other location data were given.

When compared to the other centra analyzed from the coast of Florida UF31648 and

UF47943 REE shale-normalized patterns are almost identical to UF211353, indicating

that these sharks may have spent a significant amount of time in similar waters. Also

with microsampling the changes of the shale-normalized REE patterns along the growth

axis of the centra will allow for the determination of migration timing. The use of

La+Sm+Yb vs. (La/Sm)N demonstrates the possibility of estimating relative water depth

for sharks. According to this interpretation (Fig. 4-5), the great whites in this study have

a large depth distribution and longfin makos in this study have a fairly restricted depth

distribution, which is supported by that fact that great whites have been seen from the

surface down to 6,150 ft and longfin makos are usually captured depths between 360-720

ft. More shark centra must be analyzed in order to determine whether REE in shark

centra accurately represent water depth. This study demonstrates the potential of using

elemental and isotopic analysis to learn more about the life histories of sharks from the

chemical make-up of their vertebral centra.














CHAPTER 5
SUMMARY AND CONCLUSIONS

Diagenesis is pervasive in fossil bones and ancient sedimentary environments even

though in paleontology this process is all too often ignored. Understanding how

diagenesis affects the biological signal preserved in fossils is imperative to correctly

interpret analytical findings. The study presented here gives several procedures

(including FT-IR, ICMPS, and stable isotope analysis) to help unravel the diagenetic

story. A modem analog must always be understood before the fossil data can be

interpreted. Therefore, if the end memebr composition of unaltered specimens is not

known there is no way to interpret the signal preserved in the fossil (all fossil undergo

diagenesis just to varying degrees).

Otodus obliquus centra from the Eocene of Morocco demonstrate that a biological

oxygen isotopic signal remains preserved along the growth axis even though diagenesis

has taken place. The oxygen isotopic signal does not seem to be annual in this case,

because if annual growth ring deposition is assumed as in other lamnids, then the oxygen

isotopic age is eight years too young. Given that the oxygen isotopes vary with

temperature, this distribution relative to the growth rings may indicate that these sharks

were not migrating annually into waters with temperature shifts detectable by the oxygen

isotopes. Another possibility is that Otodus obliquus, like modern lamnids, may be

warm-bodied. If this were the case, then the oxygen isotopic data represent body

temperature and not water temperature and 0. obliquus may be migrating on an annual

basis but it may not be encountering waters that raise or lower its body temperature. The









oxygen isotopic analysis of Otodus obliquus centra have prospective broad ramifications

for understanding the evolution of growth rates and developmental strategies in fossil

sharks; however, analytical techniques that assess diagenesis should be used in

combination with isotopic studies in order to produce the most insightful analysis of

fossil shark paleobiology.

Diagenesis can be quantified by the use of multiple variable analyses. The use of

ICPMS and FT-IR data provide a clearer picture of the depositional environment and the

extent of diagenesis. Seven fossil lamnid shark centra from all over the world were

analyzed for elemental and mineralogical composition. All seven fossil centra are

diagenetically altered, which is evident from the FT-IR spectra indicating the presence of

fluorine and the decrease in carbonate content, and ICPMS data which show an

enrichment in REE, Y, and U. However, through diagenesis the centra have been

imprinted with the seawater signal at/near the sediment/water interface. The type of

diagenetic fluids expelled from sediments into the water column determines how

representative the shale-normalized REE patterns and Ce anomalies are to the original

seawater signal. The more terrigenous sediments present in the depositional environment

the flatter and more shale-like the normalized REE patterns of the centra. Therefore

samples that contain a terrestrial influence would require extreme care when interpreting

Ce anomalies because it is difficult to determine how much the Ce anomaly has been

reduced by continental input. In these cases, the chemistry of depositional environment

is determined rather than the chemistry of the seawater. These seven shark centra

illustrate that geochemical data from biogenic apatites of fossil marine vertebrates have









the potential to be used to understand diagenesis, depositional environments, and/or

paleoceanography.

Modem lamnid sharks, which include great whites, longfin makos, and shortfin

makos, have vertebral centra composed of carbonate hydroxyapatite similar to other

vertebrates. The oxygen isotopic signals preserved along the growth axis of great whites

and shortfin makos represent changes in body temperature as they encounter varying

water temperatures, while in the longfin mako the actual water temperature is

represented. This is because great whites and shortfin makos are warm-bodied and

longfin makos are cold-bodied (Campana, 2002). Unfortunately, the oxygen isotopic

signal recorded in the eight modem sharks studied did not demonstrate annual cyclicity.

Growth ring counts made on all specimens did indicate annual growth, but the oxygen

isotopic data usually gave an age of about half of the actual age. Annual growth ring

deposition in lamnid sharks was supported by bomb carbon dating of lamnid sharks

(BT0433) and a comparison of growth rates for three different lamnid species. Rare

earth elemental data illustrate that shale-normalized rare earth elements can be used as to

determine habitat for sharks with an unknown capture site off the coast of Florida and can

be used for relative depth estimation of the eight sharks in this study.

These studies will potentially serve as a general model for other researchers

interested in assessing the extent of diagenesis of their fossils in a particular study area.

These studies therefore have broad applicability to paleobiologists, paleoclimatologists,

paleoceanographers, and archaeologists.

Plans to continue this study include geochemical modeling of the diagenetic system

using thermodynamics and kinetics to better understand the stability of biogenic apatites









and chemical pathways for diagenesis. Also a comparison of the accuracy of laser

ablation techniques with conventional dissolution and dilution methods currently used to

quantify the elemental concentrations on the ICPMS. The precision of laser ablation will

allow for fine scale resolution along the growth axis of the shark's centra and will

minimize destruction to the specimen. Future work will include applying the techniques

presented in this dissertation to the terrestrial system in order to determine how

diagenesis affects the orthodentine of Edentates (sloths and armadillos). Edentates,

unlike other mammals, do not have enamel on their teeth and are therefore more prone to

diagenesis. Finally, future work will be to expand the techniques used here to other

groups of fishes (including modem and fossil) and possibly mososaurs and plesiosaurs

(which preserve incremental growth in their vertebrae).















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BIOGRAPHICAL SKETCH

Joann Labs Hochstein was born in Rockville Center, NY, on January 13, 1977.

She graduated from Commack High School on Long Island in June 1995. In August

1995 she attended South Dakota School of Mines and Technology in Rapid City, SD,

where her research was focused on vertebrate paleontology. In 1999 she graduated with

honors with her Bachelor of Science degree in geology. After, Joann attended the

University of Florida, focusing her research on the high resolution study of the variations

in paleointensity of the Earth's magnetic field. In May 2001 she received her Master of

Science degree in geological sciences. In August 2001 Joann entered the Ph.D. program

at the University of Florida. Her research focused on understanding how diagenesis

affects the chemistry of fossils. Joann will complete her Ph.D. at the University of

Florida in 2005.




Full Text

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QUANTIFICATION OF THE EXTENT OF DIAGENESIS IN BIOGENIC APATITE OF CENOZOIC SHARK CENTRA By JOANN LABS HOCHSTEIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Joann Labs Hochstein

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This dissertation is dedicated to my parent s, Robert and Denise, and my Husband, Jason for all their love and support.

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ACKNOWLEDGMENTS Firstly, I would like to thank my advisor, Bruce MacFadden, for his insights, expert guidance, support, patience, and continual enthusiasm for this project. I also wish to thank my committee members, Ellen Martin, Phil Neuhoff, Neil Opdyke, and John Krigbaum, for their patience and support throughout this project. George Kamenov, Jason Curtis, and Penny Higgins gave invaluable insight during sample preparation and analysis. I am grateful for Clifford Jeremiah for providing the inspiration of this project with the donation of Otodus obliquus specimens analyzed in this study. I would also like to thank Michael Gottfried, Gordon Hubbell, Dirk Nolf, O. Sakamoto, and Sabine Wintner for allowing me to borrow and sample specimens. I would like to thank all my friends, especially Helen Evans and Steve Volpe, for their continual support and comic relief. The staff of the Geological Sciences Department, Ron Ozbun, Jody Gordon, and Mary Ploch, has been very helpful during my study at the University of Florida. I would like to thank my parents and sister for their constant support and patience. Finally, I would like to thank my husband, Jason, for the encouragement to accomplish my dreams and his unconditional love. This research was supported by Geological Society of America Grant number 2009018 and National Science Foundation grant EAR 0418042. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 DIAGENSIS AND VARIATION OF THE OXYGEN ISOTOPIC SIGNATURES IN VERTEBRAL CENTRA FROM Otodus obliquus.................................................3 Introduction...................................................................................................................3 Materials and Methods.................................................................................................8 Gross X-ray Analyses............................................................................................9 Fourier Transform Infrared Spectroscopy Preparation and Analysis....................9 Stable Isotope Analyses.......................................................................................10 Results and Discussion...............................................................................................12 Fossil and Recent Shark Centra Mineralization and Diagenesis.........................12 Physical Increments and Variation......................................................................15 Stable Isotope ( 18 O ) Signal Archived in Eocene Otodus obliquus centra........17 Conclusions.................................................................................................................21 3 QUANTIFICATION OF DIAGENESIS IN CENOZOIC SHARKS: ELEMENTAL AND MINERALOGICAL CHANGES............................................23 Introduction.................................................................................................................23 Bone Chemistry and Diagenesis..........................................................................24 Rare Earth Elements............................................................................................26 Materials and Methods...............................................................................................30 Fourier Transform Infrared Spectroscopy (FT-IR).............................................31 Elemental Analysis (ICPMS)..............................................................................33 Results.........................................................................................................................33 Mineralogical Changes........................................................................................33 Elemental Concentration.....................................................................................34 v

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Discussion...................................................................................................................41 Mineralogical Characterization of Centra...........................................................41 Implications for Diagenetic and Biological Signal Reconstruction....................42 Conclusions.................................................................................................................46 4 OXYGEN ISOTOPIC AND RARE EARTH ELEMENTAL ANALYSIS OF MODERN LAMNID SHARKs: DETERMINATION OF LIFE HISTORY?...........48 Introduction.................................................................................................................48 Rare Earth Elements............................................................................................50 Background.................................................................................................................51 Great White (Carcharodon carcharias)..............................................................51 Longfin Mako (Isurus paucus)............................................................................52 Shortfin Mako (Isurus oxyrinchus).....................................................................53 Materials and Methods...............................................................................................54 X-radiograph Analyses........................................................................................54 Oxygen Isotopic Preparation and Analyses.........................................................55 Bomb Carbon Dating Preparation and Analysis.................................................55 Inductively Coupled Plasma Mass Spectroscopy (ICPMS)................................57 Results and Discussion...............................................................................................58 Ontogenic Age Determinations...........................................................................58 Rare Earth Elements............................................................................................61 Conclusions.................................................................................................................63 5 SUMMARY AND CONCLUSIONS.........................................................................68 LIST OF REFERENCES...................................................................................................72 BIOGRAPHICAL SKETCH.............................................................................................82 vi

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LIST OF TABLES Table page 2-1. Comparison of crystallinity index (CI) and carbonate content (C/P) from FT-IR spectrum for samples treated with acetic acid and samples that were not treated with acetic acid and from Eocene and modern samples...........................................13 2-2. Stable isotopic data for three vertebral centra of Otodus obliquus, UF 162732, from the Early Eocene of Morocco..........................................................................20 3-1. Some possible substitutions in the apatite crystal structure.......................................25 3-2. Lamnid shark specimens used in this study................................................................31 3-3. Elemental and mineralogical data of nine shark vertebral centra. Elemental concentration are in ppm..........................................................................................36 4-1. Lamnid specimens used in this study........................................................................53 4-2. Ontogenic age estimates based growth ring counts (GR), oxygen isotopic ( 18 O) cyclicity, and bomb carbon ( 13 C)...........................................................................59 4-3. Elemental data (in ppm), and oxygen isotopic and bomb carbon dating ages...........64 vii

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LIST OF FIGURES Figure page 2-1. Modern white shark, Carcharodon carcharias (UF211352, left), and Eocene Otodus obliquus (UF162732, right) showing well defined growth increments.........5 2-2. Infrared spectrum of O. obliquus (UF162732A, sample jl02-53) between 500 and 700 cm1....................................................................................................................7 2-3. Fourier transform infrared spectra of (a) Otodus obliquus centrum (UF162732C, sample jl02-58) (scale on y-axis 0 to 1.4) and (b) Isurus paucus centrum (UF211353, sample jl02-319) (scale on y-axis 0 to 1.2)..........................................14 2-4. Graph representing decrease in the amount of carbonate with an increase in crystallinity...............................................................................................................15 2-5. Contact prints of the three Otodus obliquus (UF162732) x-rays. The symbols indicate dark growth bands on centra.......................................................................16 2-6. Centrum of Otodus obliquus, UF 162732A, from the early Eocene of Morocco showing exact sampling locations (grooves, top) and plot of variation in 18Oc (bottom)....................................................................................................................19 3-1. Nine vertebral centra used in this study......................................................................32 3-2. FT-IR spectra of all nine shark vertebral centra illustrating the differences from modern (C. carcharias) to fossil biogenic apatites..................................................35 3-3. FT-IR spectra from 400 to 850 cm-1, illustrating the 4 PO 4 3band differences between modern (solid line) to fossil (dashed line) shark centra.............................35 3-4. Carbonate content (C/P) vs. crystallinity index (CI) of the nine shark vertebral centra........................................................................................................................36 3-5. Isocon plots (Grant, 1982) depicting variations in fossil elemental concentrations and ratios due to diagenesis......................................................................................38 3-6. PAAS normalized REE of the nine vertebral centra divided into four diagenetic groups.......................................................................................................................39 viii

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3-7. Compilation of observed (La/Yb) N vs. (La/Sm) N in biogenic apatites of various ages and types...........................................................................................................40 4-1. Scanned contact print of BTO433 centra...................................................................49 4-2. BTO433 bomb carbon data plotted vs. two reference curves.....................................57 4-3. Oxygen isotopic data (VPDB) for the shark centra analyzed.....................................61 4-4. Post Archean Australian Shale normalized rare earth element plots for the eight sharks analyzed........................................................................................................65 4-5. Depth estimates for the eight lamnid sharks. Arrow indicates direction of increasing water depth..............................................................................................66 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy QUANTIFICATION OF THE EXTENT OF DIAGENESIS IN BIOGENIC APATITE OF CENOZOIC SHARK CENTRA By Joann Labs Hochstein December 2005 Chair: Bruce J. MacFadden Major Department: Geological Sciences Diagenesis of bone in the fossil record is pervasive; however, the extent of this process varies with depositional environment. Diagenesis is any chemical or physical change that occurs below 200C. This study quantifies the extent of diagenesis in shark vertebral centra through analysis of a suite of physical and chemical properties including crystallinity index, carbonate content, isotopes, and major, minor, and trace elemental concentrations. The sharks used in this study (Family Lamnidae) range in geographic location and geological age from the Cretaceous to Recent. Although shark skeletons are initially cartilaginous, the cartilage of the vertebral centra is replaced with carbonate hydroxyapatite during growth of the individual. Understanding chemical changes to biogenic apatite informs of the extent diagenesis has altered the biological signal preserved in vertebrate bones. Modern lamnid vertebral centra establish a modern analog for comparison to fossil lamnid sharks. Rare earth element (REE) compositions, 14 C, and 18 O, give indications x

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of timing of when these sharks migrate, changes in their eating habits, changes in water depth, and determination of ontogenic age and growth rates. Fossil shark centra used in this study have undergone diagenesis; therefore how have the processes of diagenesis affected the original signal recorded in these centra? Shale-normalized REE patterns indicate that diagenesis has erased the original signal; however because diagenesis occurred at or near the seawater/sediment interface, a seawater REE signal may still be preserved in lamnid shark centra (related to the time of deposition and location). Therefore, with caution, geochemical data from biogenic apatite of fossil marine vertebrates, such as lamnid sharks, may be used to understand paleoceanography and paleoenvironment. Also, the centra from Otodus obliquus demonstrate that the biological oxygen isotopic signal is not completely erased by diagenesis. Therefore, biological signals and diagenetic signals of fossil lamnid sharks can be utilized to understand paleobiology, paleoclimatology, and paleoceanography. xi

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CHAPTER 1 INTRODUCTION Diagenesis is a fundamental and pervasive aspect of the fossil record. Diagenesis is defined here as all physical and chemical changes that occur to vertebrate fossils (bones and teeth) after deposition and below 200C. For vertebrate bones, rapid physical and chemical changes are known to occur within relatively short geological timescales. For example, Trueman (2004) demonstrated that after death and decay of a modern animal, their bones will uptake rare earth elements (REE) within 25 years. The changes that occur in bones during diagenesis can affect crystallinity, organic content, mineralogy, density, major, minor, and trace element concentrations, and stable isotopic compositions. Any scientist interested in interpreting the fossil record must understand how diagenesis has affected the evidence. Despite the importance of understanding how diagenesis affects biogenic apatite, this process is poorly understood and frequently considered to be an impediment to progress. In many cases the effects of diagenesis are explained as being of minor importance, or insignificant, to the interpretation of geochemical data archived in the fossil record. In this context, the fundamental questions that I seek to answer in this research project include: How does diagenesis affect the geochemistry of fossil bone? How can these changes be quantified? Can life histories of sharks (e.g., age determination and ecology) be determined by the chemical signal preserved in modern and fossil shark vertebral centra? 1

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2 Although initially cartilaginous, sharks deposit carbonate hydroxyapatite in their vertebral centra throughout their life, and therefore their centra are prone to fossilization (except for the ubiquitous teeth, the rest of the skeleton remains cartilaginous and usually does not fossilize). Lamnid shark centra were chosen to understand the affects of diagenesis on fossil bone for the following reasons: 1. Vertebral centra in sharks are incrementally calcified during an individuals lifetime. Calcified bands deposited in concentric rings are either, dark and compact representing periods of slow growth whereas light, less dense bands correspond to periods of faster growth. According to Ridewood (1921) and Moss (1977), there is no resorption or redeposition of bone during life as is the case in other vertebrates, e.g., the limb bones of reptiles (e.g., Francillon-Vieillot et al. 1990). 2. Lamnid sharks (Family Lamnidae, sensu Gottfried et al., 1996) are widely distributed in space, time, and different marine sedimentary (phosphate, carbonate, and clastic) environments throughout the Cenozoic. 3. Lamnid sharks are large, including the largest shark ever to have lived, the Mio-Pliocene Carcharodon megalodon (Gottfried et al. 1996). Consequently, their vertebral centra are physically large and therefore can be more easily sampled for higher resolution. 4. Modern lamnids such as, Carcharodon carcharias (the Great White Shark) and Isurus (makos), are available for analysis of an unaltered end-member. This dissertation will discuss the use of oxygen isotopic data as a tool for incremental growth studies in fossil sharks, and the chemical and mineralogical alteration of lamnid shark centra caused by diagenesis. Also, discussed is the use of growth ring counts, bomb carbon, and oxygen isotopic data in modern analogs (i.e., modern great white and mako centra) as ontogenic age determination tools.

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CHAPTER 2 DIAGENSIS AND VARIATION OF THE OXYGEN ISOTOPIC SIGNATURES IN VERTEBRAL CENTRA FROM Otodus obliquus Introduction Sharks have been an abundant part of the marine fossil record since they first appeared in the Devonian. Sharks are usually represented in the fossil record by their durable teeth; most of the remainder of the skeletal tissue is composed of hyaline cartilage, and thus not prone to fossilization. However, vertebral centra, and in rare instances the jaws, both undergo secondary calcification during ontogeny resulting in more durable skeletal elements composed of carbonate hydroxyapatite that are frequently preserved in the fossil record (Ridewood, 1921; Goodrich, 1930; Applegate, 1967; Moss, 1977; Compango, 1999). Shark centra grow incrementally, laying down a dark band that represents time of slow growth and a light band that represents times of faster growth. Most shark species deposit a set of bands (one dark, which represents winter and one light, which represents summer) annually (called annuli), but this may vary depending on the species, physical environment (including temperature and water depth), food availability, and stress (Branstetter et al., 1987). Clearly, the periodicity of the growth bands cannot be established in modern or fossil sharks just by counting the growth rings. Therefore, the challenge for paleobiological interpretation is how to interpret the periodicity of the growth bands. Stable isotopic analysis provides the potential to independently determine whether growth band increments represent annual growth, and therefore the ontogenetic age of an individual. In most modern fish 18 O in body fluids is 3

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4 close to that of ambient water, and since PO 4 3and CO 3 2are cogenetic oxygen-bearing phases in isotopic equilibrium with the same oxygen reservoir at the same temperature, a linear correlation should exist between the oxygen isotopic composition of the phosphate and carbonate, 18 O p and 18 O c respectively (Iacumin et al., 1996). The results below show that isotopic 18 O c are archived in mineralized bone of fossil shark centra, even when it is diagenetically altered. These data may not represent the actual amplitude of temperature change, but demonstrate seasonal cycles that can be used to corroborate age determinations based on counting physical growth bands. The preservation of incremental growth layers in fossil vertebrate skeletal tissues provides the opportunity to assess growth rates of individual species and the evolution of developmental strategies in ancestral and descendant species. In addition to the physical archives of bone growth preserved in the fossil record, recent studies have also applied stable isotope analyses to understand periodic growth and related parameters of diet and seasonality preserved in fossil bone and teeth (Longinelli and Nuti, 1973; Kolodny et al. 1983; Cerling and Sharp 1996; Bocherens et al., 1996; MacFadden et al., 1999; and Vennemann et al., 2001). Results from associated vertebral centra of the lamnid shark Otodus obliquus from the early Eocene (Ypresian) of Morocco (Fig. 2-1) are presented in this chapter. This is a cosmopolitan species and is well represented in the highly fossiliferous phosphate mines of the Oued Zem, central Morocco (Arambourg, 1952). The current study was undertaken to determine: If 50-million-year-old shark centra preserve an archive of incremental growth and isotopic data that can be interpreted in a meaningful ontogenetic and phylogenetic context.

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5 The extent to which secondary calcification during ontogeny and/or diagenesis after death obscures or removes the biologically significant incremental growth and stable isotopic variation preserved in the centra. If there is intravertebral variation in the physical incremental growth preserved in the centra. Figure 2-1. Modern white shark, Carcharodon carcharias (UF211352, left), and Eocene Otodus obliquus (UF162732, right) showing well defined growth increments. The Eocene shark Otodus obliquus was chosen for this study for several reasons. This species is represented by excellent specimens of intact, fossilized portions of the vertebral column in association with teeth. Otodus obliquus is conservatively classified within the Lamnidea, the family that includes the modern mako (Isurus oxyrinchus), white (Carcharodon carcharias), and extinct shark species, including the Carcharodon megalodon (Gottfried and Fordyce, 2002). Therefore, this study is a necessary foundation for further studies of the evolution of development and body size in extinct lamnid sharks.

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6 Isotopic studies of bone are rare due to the potential for diagenetic alteration as a result of large surface area and small crystal size. During fossilization, replacement and recrystallization occurs within the crystal lattice, which changes the original composition of carbonate hydroxyapatite to carbonate fluorapatite (sometimes referred to as francolite) and eventually to fluorapatite. Transformation of carbonate hydroxyapatite to fluorapatite occurs with the loss of CO 2 and OH and addition of F which causes an increase in crystallinity (Barrick, 1998; Wang and Cerling, 1994; Shemesh et al., 1983). Fourier transform infrared (FT-IR) spectroscopy has been effectively used to evaluate mineral characteristics of fossils. Infrared spectroscopy measures the absorption of infrared radiation by the sample at the vibrational frequencies of its component molecular bonds, allowing characterization of its structural sites. In addition, the magnitude of IR absorption is proportional to the concentration of a molecular species in the sample (Sibilia et al., 1988). The crystalline structure of bone can be determined by calculating the crystallinity index (CI) from the extent of phosphate peak splitting at 565-605 cm -1 in an FT-IR spectrum (Figure 2-2). The 605 cm -1 peak intensity increases with respect to the 565 cm -1 peak intensity with an increasing degree of fluorination. Ulatimately, the CI is influenced by the size distribution of crystallites and the degree of the substitutional order-disorder within the crystal lattice (Shemesh, 1990). Apatites with larger, more ordered crystals show greater separation of these peaks and a higher CI, while in poorly crystallized apatites the peaks are closer together and therefore have a lower CI (Weiner and Bar-Yosef, 1990; Wright and Schwarcz, 1996).

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7 In the apatite lattice carbonate can substitute in two sites, OH -1 and PO 4 -2 (Shemesh, 1990; Lee-Thorp and van der Merwe, 1991; Rink and Schwarcz, 1995), indicated by the superscripts A and B in the formula: Ca 5 [(PO 4 ) 3 -B(CO 3 ) B ][(OH) 1 -A(CO 3 ) A ] which results in two sets of absorption bands in an FT-IR spectrum, corresponding to A(1545-1450-890 cm -1 ) and B (1465-1412-873 cm -1 ). Carbonate content can be estimated from the ratio of the absorbance of the CO 3 and PO 4 peaks (C/P) in the FT-IR spectrum (Shemesh, 1990; and Wright and Schwarcz, 1996). The amount of carbonate present in apatite affects the CI, due to type B carbonate substitution for PO 4 which produces smaller crystals with greater strain; therefore, highly carbonated apatites show little peak splitting and have lower crystal indices. Fourier transform infrared spectrum will indicate the presence of non-apatite mineral structures, such as fluorine. Francolite (carbonate fluorapatite) has a characteristic peak at 1096 cm -1 therefore the presence of fluorine can be determined (Wright and Schwarcz, 1996). Figure 2-2. Infrared spectrum of O. obliquus (UF162732A, sample jl02-53) between 500 and 700 cm1. The crystallinity index, (CI) is calculated by (A + B)/C, where A, B, and C represent the peak height from the baseline.

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8 It has been found that recrystallization during diagenesis does not necessarily affect the isotopic composition (Barrick, 1998). When bone recrystallizes in a closed system, there is no alteration of the isotopic value (Stuart-Williams et al. 1996). Studies have shown (see Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996) that when a sample is treated with buffered 1M acetic acid any apatite that has been diagenetically enriched in CO 3 2and secondary carbonate minerals can be removed (Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996). It is known that when CO 3 -2 substitution increases apatite solubility, rendering it more susceptible to diagenesis (Krueger, 1991; Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996). As a consequence, 18 O c values may not be a reliable paleothermometer in fossil bone. However, fossils that have been affected by diagenesis the oxygen isotope composition may have some biological information preserved, as will be discussed below. Materials and Methods Three associated fossil shark precaudal centra were analyzed from a single individual catalogued in the Vertebrate Paleontology Collection, Florida Museum of Natural History (FLMNH), University of Florida (UF) 162732. These centra are identified as Otodus obliquus based on association with a diagnostic dentition. This specimen was collected from the Early Eocene (Ypresian) unit within the phosphate mines at Oued Zem, central Morocco. For comparison with the fossil shark, four modern shark centra representing Carcharodon carcharias (great white, UF Environmental Archaeology specimen 31648 and UF Vertebrate Paleontology specimen 211351), Isurus oxyrinchus (shortfin mako, UF Environmental Archaeology specimen 47943), and Isurus paucus (longfin mako, UF Vertebrate Paleontology specimen 211353) were analyzed.

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9 Gross X-ray Analyses Traditional x-radiographic (x-rays) photography can reveal physical differences in bone density, such as those representing incremental growth bands. X-rays of the centra were taken at the C.A. Pound Human Identification Laboratory at UF. The x-rays are set at 78 kV for 2 minutes. The x-rays are used to make contact prints, which are a reversed pattern of the x-ray (i.e. dark lines on the x-ray are the light lines on the contact print). The dark and light alternating growth rings are easily seen on the contact prints and are marked to indicate the location of the samples used for oxygen isotope analysis. These contact prints were scanned digitally and modified for presentation using Adobe Photoshop TM Fourier Transform Infrared Spectroscopy Preparation and Analysis For the intended study presented here, Fourier transform infrared spectroscopy has its advantages over x-ray diffraction (XRD), including: (1) only a small amount of sample is required (<1 mg); (2) preparation is easier and produces more accurate results; and (3) carbonate content can be assessed from FT-IR. Four ~2 mg microsamples were drilled with a low speed Foredom drill from each of the three centra. For the FT-IR analyses, two samples came from the center, and two from the edge of each centrum. One-half of the samples were treated with the same procedures as those analyzed for oxygen isotope composition (Table 2-1) to compare with the results from samples not treated with acetic acid and hydrogen peroxide. The other half of the samples were left untreated to serve as a control. The samples were weighed out to 0.8 mg, and combined with 150 mg of spectral grade KBr, and ground together in a ball mill. The KBr dye was put under vacuum for 5 minutes and compressed (under vacuum) at 20,000 psi for another 8 minutes. The vacuum was

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10 removed and the KBr dye remained under 20,000 psi for another 2 minutes, which generated a 13 mm pellet. Infrared spectra were obtained between 4000 and 400 cm -1 on a FT-IR Nicolet 20 SXB Bench in the Major Analytical Instrument Center in the UF Material Science and Engineering Department. Interferences from KBr were cancelled by subtracting a standard KBr spectra from the sample spectra. The crystallinity index, (CI) measures the degree of PO 4 3band splitting and is defined by: CI= (A 605 + A 565 )/(A 595 ) where Ax is the absorbance at wave number x (Shemesh, 1990), assuming a straight baseline between 700 and 500 cm -1 (Fig. 2-2). An estimate of the carbonate content is given by the absorption ratio of the height of the carbonate peak at 1428 cm -1 to the height of the phosphate peak at 1042 cm -1 of the FT-IR spectrum (Featherstone et al., 1984; Lee-Thorp and van der Merwe, 1991; Wright and Schwarcz, 1996; Stuart-Williams et al., 1996), that is: C/P=A 1428 /A 1042 Stable Isotope Analyses For each of the three centra, from 24 to 28 microsamples of ~5 mg each were drilled with a low speed Foredom drill across the growth axis starting from the center and ending at the external margin. As far as practicable, the goal was to sample each annulus twice, i.e., once in the dark portion and another in the light portion of the mineralized bone. Sample powders were treated with standard isotope preparation techniques (e.g., MacFadden et al., 1999) used to analyze teeth. This included first washing with H 2 O 2 overnight to remove organic contaminants and then with weak (0.1 N) acetic acid overnight to remove mineral (principally CaCO 3 ) contaminants, and then dried using methanol. About 2 mg of each treated sample powder were then measured into

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11 individual vials and placed in the automated Multiprep device for introduction into the VG Prism mass spectrometer in the Stable Isotope Laboratory in the UF Department of Geological Sciences. The sample runs were calibrated to internal laboratory and NBS 19 standards. The carbon and oxygen isotopic results are reported in the standard convention: value = [(R sample /R standard )-1] x 1,000 (parts per mil, ), where R = 13 C/ 12 C or 18 O/ 16 O, and standard is VPDB (Vienna Pee Dee Belemnite). After the isotopic data were run and plotted against distance from origin of the centra, they were the entered into a time series analysis program, Analyseries TM version 1.2. This is necessary because each of the three centra are of slightly different sizes and the growth lines do not match exactly, i.e., as measured by the distance from center. Analyseries is traditionally used in paleoclimatological interpretation to construct age-depth relations for sedimentary records. In Analyseries the method for establishing an age-scale on a sedimentary record is to use a comparable well-dated signal as a reference signal and then to optimize some measurement of the similarity between the two series, while changing the depth scale of the first one to the age-scale of the second (Labeyrie and Yiou, 1996). Analyseries then generates a pointer file, which allows plotting of the isotope data from the two patterns on the same scale. The oxygen isotope values versus distance from the origin were used to generate the pointer files and the carbon isotope data (otherwise not discussed in this paper) was used as a check for the quality of the match of the oxygen isotope data.

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12 Results and Discussion Fossil and Recent Shark Centra Mineralization and Diagenesis FT-IR spectra indicate differences in shape and crystallinity indices between modern shark centra and Eocene O. obliquus centra (Fig. 2-3 and Table 2-1). Modern shark centra (UF47943 and UF31648, FLMNH Environmental Collection) are characterized by low CI (2.79 -2.84), low C/P values (0.32 0.34), and the absence of the 1096 cm -1 peak. The O. obliquus centra (UF162732) have high CI (4.44 4.83), low carbonate content (0.15 0.20), and a pronounced 1096 cm -1 peak, which indicates an increase in crystallinity, a decrease in amount of carbonate, and the formation of a new mineral phase after burial (francolite). There was no significant variation between the samples treated with acetic acid compared to those samples not treated with acetic acid. Fourier transform infrared spectroscopy allows for the evaluation of the mineral characteristics of modern and fossil shark centra and enables detection of any diagenetic changes to the mineralogy. The differences between the crystallinity indices of modern shark centra and fossil O. obliquus (Table 2-1) suggest that the three O. obliquus centra have been recrystallized, presumably due to diagenetic processes involving the growth of larger crystals at the expense of smaller ones. The FT-IR spectra of the O. obliquus centra are indicative of two chemical changes occurring during recrystallization: (1) type B carbonate substitution for PO 4 -2 decreases with increasing crystallinity, which is seen in reduced C/P values and increased CI (Fig. 2-4); and (2) an increase in fluorine content with increasing crystallinity, which is indicated by a distinct peak at 1096 cm -1 (Fig. 2-3). These changes in the FT-IR spectra signify a transformation from dahllite (carbonate

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13 Table 2-1. Comparison of crystallinity index (CI) and carbonate content (C/P) from FT-IR spectrum for samples treated with acetic acid and samples that were not treated with acetic acid and from Eocene and modern samples. __________________________________________ Sample # CI C/P _______________________________________ Eocene O. obliquus, UF 162732A jl02-51 4.62 0.18 jl02-52 4.67 0.18 jl02-53 4.62 0.19 Eocene O. obliquus, UF 162732B jl02-54 4.83 0.16 jl02-56 4.51 0.20 jl02-26* 4.58 0.17 jl02-47* 4.53 0.15 Eocene O. obliquus, UF 162732C jl02-57 4.55 0.17 jl02-58 4.57 0.17 jl02-94* 4.44 0.15 jl02-106* 4.45 0.16 Modern C. carcharias, UF 47943 jl02-60 2.82 0.34 Modern C. carcharias, UF 211351 jl02-317 2.80 0.32 Modern I. oxyrhinchus, UF 31648 jl02-318 2.79 0.34 Modern I. paucus, UF 211353 jl02-319 2.84 0.33 __________________________________________ *Samples that have been treated with acetic acid.

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14 Figure 2-3. Fourier transform infrared spectra of (a) Otodus obliquus centrum (UF162732C, sample jl02-58) (scale on y-axis 0 to 1.4) and (b) Isurus paucus centrum (UF211353, sample jl02-319) (scale on y-axis 0 to 1.2). The arrow shows peak 1096 cm-1, which is diagnostic of francolite (F-apatite)and is only present in the fossil (UF 162732C) specimen and not the modern specimen (UF211353). hydroxyapatite) to francolite (carbonate fluorapatite). The lack of significant differences in the FT-IR spectrum of the O. obliquus samples treated with acetic acid and those that were not treated suggest that these samples were not diagenetically enriched in CO 3 -2 and no secondary carbonate minerals were present. The absence of a 710 cm -1 peak on the FT-IR spectrum, which represents the presence of calcite, indicates that no secondary

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15 carbonate minerals were present. Comparison of the FT-IR spectra from various locations along the growth axis of a single O. obliquus centra and between the three centra show insignificant variation, which indicates that the chemical changes that occur during diagenesis are uniform along the growth axis and between the three centra. 00.050.10.150.20.250.30.350.422.533.544.55CIC/P UF162732A UF162732B UF162732B (T) UF162732C UF162732C (T) Modern Figure 2-4. Graph representing decrease in the amount of carbonate with an increase in crystallinity. Physical Increments and Variation Physical growth couplets (annuli) are evident in the gross morphology and x-radiographs of each of the fossil Otodus obliquus centra. Contact prints of x-rays of the three O. obliquus centra (Fig. 2-5) were used to enhance the visibility of the growth rings. The counts on the three contact prints indicate a total of 19 growth couplets for each centrum. A growth couplet is defined as a band pair, composed of one opaque (darker) band and one lighter band. The location of the 19 growth couplets is consistent among the three centra and fit a characteristic growth function (von Bertalanffy, 1938; and von Bertalanffy, 1960), i.e., with the most rapid growth during early ontogeny and

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16 incrementally decreasing growth rate during later ontogeny. Given the fact that all three centra come from the same individual, the observation of 19 equivalent band couplets is both expected and corroborates these as growth related phenomena. Assuming one growth couplet is deposited annually as proposed for other lamnids (Cailliet at al. 1983; Campana et al. 2002; Labs-Hochstein, submitted) this shark has a minimum age of 19 years. Figure 2-5. Contact prints of the three Otodus obliquus (UF162732) x-rays. The symbols indicate dark growth bands on centra.

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17 Stable Isotope ( 18 O ) Signal Archived in Eocene Otodus obliquus centra A series of microsamples taken along the growth axis of three centra (UF 162732A, UF162732B, and UF162732C) of Otodus obliquus reveals a systematic pattern of change in 18 O values (Fig. 2-6; Table 2-2). There appears to be a systematic, sinusoidal, variation of 18 O values representing at least eight isotopic cycles. The extremes of these cycles are interpreted to represent warm seasonal signals, with lower 18 O values between approximately -3.5 to -4.0 and cold seasonal signal with higher 18 O values between about -2.0 to .3. There are several points of discussion concerning these data. Firstly, two adjacent microsamples were taken, so far as possible, to correspond with dark-light band couplets within an annulus. The results indicate that more positive 18 O values correspond to the darker bands, confirming the prediction that these represent a colder signal. Conversely, the more negative 18 O values were taken in the lighter band, indicating a warmer signal. This correspondence, plus the non-random pattern of isotopic variation therefore suggests that these isotopic data are archiving a record of paleoclimate cycles (although perhaps not the actual amplitude of paleotemperature change, see below). Secondly, the oxygen isotopic data of all three centra have eight annual growth band couplets. However, there is no consistency in the amplitude of the bands. In different centra the same growth bands have different isotopic values, and the extremes of each annual cycle are not recorded uniformly in each of the three centra. This may be due to several reasons, such as; (1) the growth band is not sampled in the exact same place on each of the three centra; (2) there may be an ossification order such that the centra closer to the head ossify before the centra towards the tail or vice versa

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18 (Ridewood, 1921); (3) the seasonal variation may not have been as strong at various times during the sharks life; and/or (4) possible dampening/amplification of the original signal by diagenesis. Finally, there are fewer annuli indicated from the isotopes (8) than those observed from the physical growth couplets (19). Otodus obliquus could have been homoeothermic (maintain a body temperature above its surroundings). All modern day lamnids have the ability to be homoeothermic (Campango, 2002) and it is not known when lamnids evolved this characteristic. However since porebeagles, makos, and great whites are all homoeothermic it suggest that the common ancestor may also have been homoeothermic. Using molecular clocks calibrated for sharks Martin (1996) demonstrated that all three genera of lamnids diverged at nearly identical times during the Paleocene or early Eocene. Another possibility is that all modern lamnids independently evolved homeothermy. Only seven out of the more than 360 known modern species of sharks are known to be homoeothermic, the five species of modern lamnids and two species of modern thresher sharks (Campango, 2002). Therefore homeothermy is rare in modern sharks and most likely evolved in lamnids through a common ancestor. The possibility exists that the oxygen isotopic data is showing the shifts in body temperature in O. obliquus as it came in contact with varying temperatures of water and not the actual water temperature. The oxygen isotopic signal gives an age of half what the growth ring counts estimates suggesting that this shark may not have been migrating into waters with enough variation in the oxygen isotopic signal on an annual basis or it was not encounter waters that changed its body temperature on an annual basis if O. obliquus was homoeothermic.

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19 Figure 2-6. Centrum of Otodus obliquus, UF 162732A, from the early Eocene of Morocco showing exact sampling locations (grooves, top) and plot of variation in 18Oc (bottom), the three plots represent the correlation between each of the centra produced by using AnalyseriesTM. The gray lines connect the dark growth bands that correspond to the possible annual signal.

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20 Table 2-2. Stable isotopic data for three vertebral centra of Otodus obliquus, UF 162732, from the Early Eocene of Morocco. mm 18 O c o / oo mm 18 O c o / oo mm 18 O c o / oo UF162732A UF162732B UF162732C 11.60 13.44 14.54 15.8 16.67 17.60 19.06 20.10 21.13 22.63 23.95 24.81 26.57 27.50 28.93 29.81 31.27 32.41 33.98 34.74 35.87 36.83 37.90 39.20 39.94 41.70 42.58 43.10 44.31 45.21 46.36 47.34 48.45 49.33 50.89 51.73 53.47 -6.2 -6.0 -5.9 -6.1 -5.9 -5.9 -5.7 -5.8 -5.8 -5.7 -5.6 -5.4 -6.1 -5.8 -6.1 -6.1 -6.3 -6.3 -6.3 -6.2 -6.4 -6.4 -6.2 -6.3 -6.2 -6.6 -6.6 -6.6 -6.3 -6.5 -6.4 -6.5 -6.5 -6.7 -7.0 -6.7 -6.7 2.81 14.94 16.61 17.89 19.77 23.08 24.56 26.32 27.62 21.50 30.06 31.58 32.78 33.99 35.69 36.78 38.04 39.30 40.65 42.16 43.30 44.70 46.27 46.77 47.81 49.41 50.35 51.13 52.50 -5.4 -5.7 -6.3 -6.0 -5.4 -5.8 -5.9 -5.3 -5.7 -5.8 -5.7 -5.8 -5.8 -5.9 -6.1 -6.1 -6.3 -6.1 -5.9 -5.9 -6.3 -6.3 -6.2 -6.8 -6.9 -6.8 -6.4 -6.8 -7.0 10.81 12.94 14.93 16.84 17.95 19.42 20.56 22.04 24.27 25.35 27.25 28.78 30.87 32.25 34.60 36.31 36.66 38.16 39.60 40.07 42.17 42.93 45.42 46.01 47.25 48.72 49.70 51.43 52.92 -6.4 -6.0 -5.9 -5.7 -5.4 -5.7 -5.8 -5.5 -5.5 -5.8 -5.0 -5.6 -5.6 -5.7 -5.7 -5.9 -6.0 -6.0 -5.8 -6.2 -6.0 -6.0 -6.4 -6.4 -6.1 -6.3 -6.7 -6.4 -6.6

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21 Conclusions Because of their cartilaginous skeletons, which characteristically do not fossilize, the secondarily calcified centra provide a unique opportunity to assess incremental growth and age determination in fossil sharks. The FT-IR spectra indicate that the three O. obliquus centra have undergone diagenesis. Modern skeletal tissue is composed of carbonate hydroxyapatite while according to the FT-IR spectra the O. obliquus centra have francolite (carbonate fluorapatite) and have lost carbonate and organics relative to modern shark centra. All three centra have undergone similar diagenesis indicating that in this case the diagenesis was uniform throughout this specimen. There was no difference between the acetic acid treated samples and those that were not treated, indicating that these specimens did not have any secondary carbonate present and the acetic acid does not affect the structural carbonate. Despite this degree of alteration, two important conclusions derived from this study are that: (1) the incremental growth banding, i.e., annuli, retain their original physical structure; and (2) the non-random pattern of oxygen isotopic variation therefore suggests that these isotopic data are archiving a record of paleoclimate cycles. With regard to the latter observation, I caution that the oxygen isotopic variation may represent a signal damped/amplified by diagenesis or the possibility exists that O. obliquus was homoeothermic and therefore the shifts in oxygen isotopes represent changes in body temperature as it came in contact water of different temperatures. Accordingly, I do not advocate using these data to attempt calculations of paleotemperatures in the early Eocene seas. The oxygen isotopic signal gives an age of about half of what the growth ring counts estimates suggest (assuming one growth ring pair per year). One explanation of this observation is that this shark may not have been migrating into waters with

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22 enough oxygen isotopic variation on an annual basis. Another explanation, if O. obliquus was warm-bodied, is it was not encountering waters that changed its body temperature on an annual. These findings are similar to modern day lamnids, where by larger sharks oxygen isotope estimates yield ages equal to approximately 1/2 of the ring counts (Labs-Hochstein, submitted). The analysis of Otodus obliquus centra from the early Eocene of Morocco potentially have broad ramifications for understanding the evolution of developmental strategies in fossil sharks. This application can potentially answer some unresolved questions about the developmental mechanisms that resulted in huge body size in fossil lamnid sharks (Gottfried et al., 1996) such as Miocene Carcharodon megalodon, which is a close relative of O. obliquus. In all such future studies, however, analytical techniques that assess diagenesis, such as FT-IR, should be used in combination with isotopic studies to produce the most insightful analysis of fossil shark paleobiology.

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CHAPTER 3 QUANTIFICATION OF DIAGENESIS IN CENOZOIC SHARKS: ELEMENTAL AND MINERALOGICAL CHANGES Introduction Fossilized vertebrate skeletal tissues, including teeth and bone, have recently received considerable attention as geochemical archives of paleoecological and paleoenvironmental information (Piper, 1974; Kolodny et al., 1983; Elderfield and Pagett, 1986; Kolodny and Luz, 1991; Lcuyer et al., 1993; Picard et al., 1998; Shields and Stille, 2001; Picard et al., 2002; MacFadden et al., 2004; Pucat et al., 2004). In these studies, fossil tooth enamel has been the preferred material for analysis because it is compact, relatively non-porous mineral and consists of >95% hydroxyapatite. However, isotopic data have been used to eludicate paleobiological information (e.g., to reconstruct dinosaur physiology; Barrick and Showers, 1994). These studies have come under close scrutiny (Kolodny et al., 1996) because porous bone is more prone to diagenesis than teeth (Wang and Cerling, 1994). There are certain situations in which fossil bone is either the only skeletal material available for study (e.g., in those vertebrates that lack teeth, such as most birds), or is preferred because certain skeletal elements archive incremental growth. One example of an archive of incremental growth is shark vertebral centra. Although shark skeletons are initially cartilaginous (i.e., composed of soft supporting tissue that does not fossilize), the cartilage is replaced in the vertebral centra by carbonate hydroxyapatite during the growth of the individual. This growth is periodic and incremental rings are called annuli 23

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24 because of their presumed annular cyclicity (although this is not always the case Branstetter et al., 1987). These growth rings are easily observed in both modern and fossilized shark centra. During a related research project investigating stable isotopic signatures archived in fossil shark centra (MacFadden et al. 2004), we became interested in the extent of diagenesis and how it may have affected the geochemistry of fossil bone. The purpose of this study is to quantify diagenesis of shark bone through analysis of a suite of physical and chemical characters including crystallinty index, carbonate content, and major, minor, trace elemental concentrations. The sharks are all from the group known as the superfamily Lamnoidea (Capetta 1987), that includes the modern great white (Carcharodon carcharias), and six closely related, extinct species that range in geologic age from the Cretaceous to the Pliestocene. The modern shark species are included in this study to provide an unaltered end-member in which initial physical parameters and elemental concentrations can be determined. Bone Chemistry and Diagenesis Stable isotopes and rare earth elements (REE) of biogenic apatites have been used for paleoclimate reconstruction, to trace ocean currents and water masses, to quantify redox conditions, for incremental growth studies, and to reconstruct diet (Piper, 1974; Kolodny et al., 1983; Elderfield and Pagett, 1986; Kolodny and Luz, 1991; Lcuyer et al., 1993; Picard et al., 1998; Shields and Stille, 2001; Picard et al., 2002; MacFadden et al., 2004; Pucat et al., 2004). Partial or complete dissolution, precipitation, recrystallization, and ion uptake by adsorption and diffusion may lead to changes in chemical composition and lattice structure of the biogenic apatite (Reiche et al., 2003); therefore, the original chemical signatures of biogenic apatites may be modified through diagenesis, resulting in the interpretation of erroneous biological signals (Pucat et al., 2004).

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25 Modern bone is composed of carbonate hydroxyapatite, Ca 10 (PO 4 ) 6 (CO 3 ) x (OH) 2-2x that has small crystallites, large surface area (200 m 2 /g; Weiner and Price, 1986), and high organic content (~ 35%, principally collagen and water; Williams, 1989; Carlson, 1990; Koch et al., 1992). The high reactivity of biogenic apatite is due to small crystallite size and high surface area of the bone hydroxyapatite (Trueman, 1999). Many substitutions are possible for both the anions and cations in biogenic hydroxyapatite (Table 3-1; Nathan, 1981). In modern biogenic apatites, carbonate (CO 3 2) can substitute for either OH (A site) or PO 4 3(B site), but primarily substitutes for the latter (Shemesh, 1990; Lee-Thorp and van der Merwe, 1991; Rink and Schwarcz, 1995). Substitution of carbonate for phosphate distorts the crystal lattice increasing the solubility of biogenic apatite (Nelson, 1981; Nelson et al. 1983). During fossilization, the biogenic apatite alters to a more stable, less reactive form of apatite (carbonate fluorapatite some times referred to as francolite) by losing carbonate and hydroxyl ions and gaining fluoride (Nathan and Sass, 1983; Newesely, 1989; Greene et al., 2004). The loss of carbonate decreases the defect densities within the hydroxyapatite lattice resulting in an increase in crystallite size and order and decreaed solubility relative to carbonate hydroxyapatite (Greene et al., 2004). Table 3-1. Some possible substitutions in the apatite crystal structure. Constituent ion Substituting ion Ca 2+ Na 2+ K 2+ Sr 2+ Mn 2+ Mg 2+ Zn 2+ ,Ba 2+ Sc 3+ Y 3+ REEs, U 4+ PO 4 3CO 3 2, SO 4 2, CrO 4 2, CO 3 F 3,CO 3 OH 4, SiO 4 4OH F Cl Br O 2

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26 Through the processes of diagenesis, trace element concentrations can either increase or decrease relative to unaltered bone concentration. This phenomenon is well-documented and described in the literature (Elderfield and Pagett, 1986; Wright et al., 1987; Williams, 1988; Grandjean and Albarde, 1989; Koeppekastrop and DeCarlo, 1992; Grandjean-Lcuyer et al., 1993; Denys et al., 1996; Hubert et al., 1996; Laenen et al., 1997; Reynard et al., 1999; Trueman, 1999; Staron et al., 2001). Trace elements are most likely incorporated into bone apatite during early diagenesis through the process of substitution. Trace element signatures acquired during the intial stages of diagenesis appear to be stable and resistant to later modification (Bernat, 1975; Grandjean and Albarde, 1989; Grandjean-Lcuyer et al., 1993). Because of similar ionic size, REE 3+ ions are easily substituted into the Ca 2+ site by means of coupled substitution (Whittacker and Munts, 1970). Adsoprtion of trace elements onto the surface may also be a qunatatively significant mode of uptakeof biogenic apatite (Reynard et al., 1999). Surface adsorbed species are typically only weakly bound to the mieral surfaces, and therefore ions adsorbed are susceptible to exchange as long as the crystal surface remains exposed (Reynard et al., 1999). However, if during diagenesis the inter-crystalline porosity is closed, then individual crystallite surfaces will be protected from further exchange. Ultimately, the final trace element composition of the biogenic apatite is controlled by the concentration of trace elements in the system, apatite-fluid partition coefficienst, chemistry of the burial microenvironment, bone microstructure, and length of exposure (Trueman, 1999). Rare Earth Elements Although the REE typically exist in the 3+ oxidation state two exceptions are Cerium and Europium. Cerium can undergo oxidation in seawater from the solvated 3+

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27 state to the relatively insoluble 4+ state (de Baar et al., 1985a). Under oxic conditions, Ce 4+ is readily removed from seawater onto particle surface coatings or into authigenic minerals (Sholkovitz et al., 1994; Koeppenkastrop and De Carlo, 1992) and under reducing conditions Ce 3+ may be released back into the water column or pore waters. Therefore when Ce is depleted (i.e., under oxic conditions) in the water column a negative Ce anomaly (Ce anom. ) is present and vice versa (German and Elderfield, 1990). Europium may undergo oxidation from the Eu 3+ to Eu 2+ which is significant in the oceans because of its preferential ability of Eu 2+ to substitute for Ca 2+ in apatites (Elderfield, 1988). de Barr et al. (1985a) illustrate that in both the Atlantic and Pacific Oceans all REE, with the exception of Ce, increase with water depth. Concentrations of Ce decrease with water depth and therefore the negative Ce anomaly observed increases with depth (de Baar et al., 1985a). Thus, when the seawater signal is preserved in biogenic apatites the Ce anomaly may be a useful relative indicator of water depth at which fossils have been deposited. Fossil biogenic apatites contain several tens to several hundreds parts per million (ppm) of REE, whereas REE maximum concentration in pore water and seawater are in the range of parts per billion (ppb) and part per trillion (ppt), respectively (Elderfield and Greaves, 1982; Elderfield and Sholkovitz, 1987). Bernat (1975) reported high REE concentrations in ichthyoliths from the upper-most 4 cm of sediment of ocean cores. These ichthyoliths exhibit bulk REE patterns similar to overlying waters. Furthermore, Bernat (1975) analyzed ichthyoliths from 4 to 600 cm of the sediments, and showed that their REE compositions were similar to the ichthyoliths from the upper-most 4 cm of the same cores. These results suggest that in this case ichthyoliths inherit a REE composition

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28 directly from seawater at the sediment/seawater interface during early diagenesis, with little or no fractionation. Direct uptake of REE in biogenic apatites from pore waters and/or seawater raises serious problems. Assuming that REE are directly taken up through advection of pore waters, about a ton of pore water would be needed to give the biogenic apatite enough REE to fit observed concentrations (several tens to several hundreds parts per million, ppm). Such a water/rock ratio would require an exceedingly high flux of pore water through the sedimentary column, and reasonably this cannot be considered for the cause of the enrichment of biogenic phosphates (Grandjean et al., 1987; Grandjean and Albarde, 1989; Grandjean-Lcuyer et al., 1993). Grandjean et al. (1987) proposed quantitative uptake of non-detrital REE locally released at the sediment/seawater interface to explain biogenic apatite enrichment. Abundant debris with large specific surfaces, which easily absorb large amounts of REE from seawater, are dispersed in the oceans and are known to settle to the ocean floor. Such a rain of REE rich carriers has been identified in sediment traps (Murphy and Dymond, 1984) and comprises a variety of inorganic (detrital minerals, oxy-hydroxide flocs) and organic (pellets, organic debris) phases. The decay of the REE rich carriers at the sediment/seawater interface, associated with that of biogenic apatite, and the resulting reducing conditions eventually cause the dissolution of Fe-Mn oxy-hydroxides and favor transfer of REE from large volumes of seawater to recrystallized biogenic apatite in a rather short period of time (Grandjean et al., 1989). This extension of Bernats (1975) model implies that both that the ultimate source of phosphatic REE is seawater, and there is an early diagenetic transfer to the phosphate through a short-lived phase made of oxihydroxides and organic detritus. Upon completion of the early diagenetic processes

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29 and once most oxihydroxides have been dissolved, phosphate remains the major non-detrital REE repository in sediments, so its REE concentrations must reflect to a large extent the flux of seawater derived REE exclusive of the detrital particulate accumulation (Grandjean et al., 1989). Variations in host lithology of marine biogenic apatites may influence the REE contents due to differences in permeability, the flux of REE from diagenetic fluids expelled from sediments, and organic and oxy-hydroxide contents (Grandjean-Lcuyer et al., 1993; Lcuyer et al., 2004). Terrestrially derived sediments have shale-normalized REE patterns that carry a shale-like signal (i.e., flatten REE pattern) and no Ce anom. is expected from common fine-grained detrital material (Grandjean et al., 1987). REE in pore waters are derived from the surrounding sediment particles and development of large pore water concentration gradients will allow fluxes of REE from sediments to seawater (i.e., diagenetic fluids expelled from sediments into seawater; Elderfield and Sholkovitz, 1987). Therefore, REE contents of biogenic apatites deposited in terrestrial derived sediments (clays and sands) may have flattened shale-normalized REE patterns that are intermediate between those of seawater and those of shale (Grandjean et al., 1988; Elderfield et al., 1990). Sediments that precipitate directly from seawater (carbonates and phosphorites) with little to no terrestrial input have diagenetic fluids reflecting the composition of the overlying water column. Biogenic apatite deposited in marine precipitated sediments should show a seawater REE pattern since the diagenetic signature would be the same as the overlying water column (Lcuyer et al., 2004). Therefore, the REE signature in fossil biogenic apatites results from a mass balance between the flux of REE from decaying organic and oxy-hydroxides (primary carriers

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30 with seawater signature), the flux of REE from diagenetic fluids expelled from sediments (diagenetic signature),and the flux of REE from rivers (detrital signature) (Grandjean and Albarde, 1989). Materials and Methods This study analyzes the composition of nine shark centra (Fig. 3-1), two modern great white, Carcharodon carcharias, and seven fossil specimens ranging in age from Cretaceous to Pliocene (Table 3-2). These specimens were selected because they all are within the monophyletic superfamily Lamnoidea, the group that includes great white sharks, makos (Isurus), and their close fossil relatives. Likewise, these sharks were selected because they span an age range from Cretaceous to Recent and are widely distributed geographically. A broad geographic distribution of fossils should illuminate the effects of different extents and environments of diagenetic alteration. The vertebral centra were chosen because they are the primary ossified skeletal tissue that fossilizes in sharks (i.e., other than teeth). Two analytical techniques were used to determine the chemical and mineralogical properties of the nine shark centra: (1) Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR, which is used here to determine crystallinity. FT-IR has advantages over x-ray diffraction (XRD), because only a small amount of sample is required (<1 mg), preparation is easier and produces more accurate results; and additionally, carbonate content can be assessed from FT-IR. (2) Inductively Coupled Plasma Mass Spectrometry (ICPMS), which allows for the determination of the major, minor, and trace elements in modern and fossil shark centra. ICPMS is a comprehensive technique that is extremely sensitive (detection limits in the ppb range for many elements in aqueous solution). The

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31 high level of relative accuracy (1 to 2%) coupled with sensitivity allows analysis at concentrations ranging over more than nine orders of magnitude (Montaser, 1998). Table 3-2. Lamnid shark specimens used in this study. Species Museum ID Locality Age Sediment & Depositional Environment Carcharodon carcharias BTO433 E coast, South Africa Recent Carcharodon carcharias UF211351 Islamorada, Florida Recent Isurus hastalis UF211358 Pisco Fm., Peru Pliocene Shallow bay sandstone (Brand et al., 2004) Carcharodon megalodon 120A Saitama Prefecture, Japan Miocene Nearshore sandy siltstone (Hayashi et al., 2003) Carcharodon angustidens OU22261 Kokoamu Greensand, New Zealand Oligocene Shelf glauconitic sand (Ayress, 1993) Carcharodon auriculatus EF809A Brussels Sand, Belgium Oligocene Near shore shelf sandstone (Hooyberghs, 1990; and Herman et al. 2000) Otodus obliquus UF162732B Oued Zem, Morocco Eocene Shelf phosphorite (Lancelot and Seibold, 1978) Otodus obliquus UF162732D Oued Zem, Morocco Eocene Shelf phosphorite (Lancelot and Seibold, 1978) Creotxyrhina mantelli UF211357 Niobrara Fm., Kansas Cretaceous Shallow epicontinental sea outer shelf chalky shale (Hattin, 1981) Fourier Transform Infrared Spectroscopy (FT-IR) Three ~1 mg samples were drilled with a low-speed Foredom drill from each of the nine centra. Samples were taken along the growth axes, i.e., one from the center, one from the middle, and one from the edge of each centrum. Potassium Bromide (KBr) pellets were prepared using the method discussed in MacFadden et al. (2004). Infrared spectra were obtained between 4000 and 400 cm -1 on a FT-IR Nicolet 20 SXB Bench housed at the Major Analytical Instrument Center in the UF Material Science and Engineering Department. Interferences from KBr were cancelled by subtracting a standard KBr spectrum from the sample spectra. The size distribution of crystallites and the degree of substitution order-disorder within the crystal lattice of biogenic apatite can be determined by calculating the crystallinity index (CI) from the extent of phosphate peak splitting at 565-605 cm-1 in an FT-IR spectrum. Apatites with larger, more ordered

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32 crystals show greater separation of these peaks and a higher CI (Shemesh, 1990; Wright and Schwarcz, 1996). An estimate of the carbonate content is given by the absorption ratio of the height of the carbonate peak at 1428 cm -1 to the height of the phosphate peak at 1042 cm -1 of the FT-IR spectrum (Featherstone et al., 1984; Lee-Thorp and van der Merwe, 1991; Stuart-Williams et al., 1996; Wright and Schwarcz, 1996). Figure 3-1. Nine vertebral centra used in this study. A. Carcharodon carcharias (BTO433) B. Carcharodon carcharias (UF211351) C. Isurus hastalis D. Carcharodon megalodon E. Carcharodon angustidens F. Carcharodon auriculatus G. Otodus obliquus H. Otodus obliquus I. Creotxyrhina mantelli.

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33 Elemental Analysis (ICPMS) Approximately 6 mg of bulk sample was drilled with a slow speed Foredom drill from each of the nine centra. 5 mg of each sample were weighed out and placed into 3 mL Savillex vials, dissolved in 1 mL of 3M HNO 3 and heated overnight. Samples were allowed to cool and then dried. Next 2 mL of 1% HNO 3 was added, heated overnight, and allowed to cool. Samples were analyzed on an Element 2 High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS) at the Center for Trace Element Analysis at the University of Southern Mississippi. All samples were corrected by subtracting the blank, corrected for instrumental drift based on internal machine standards that were analyzed during the run (initial quantification based on comparing the corrected ion counts of the samples with ion count for the standards), and corrected ion counts to a constant response to the known amount. All REE values were shale-normalized to PAAS (Post-Archean Australian Shale Standard) in order to clearly illuminate enrichment-depletion trends relative to average crust (e.g., Grandjean et al., 1988; Elderfield et al., 1990; Grandjean-Lcuyer et al., 1993; Reynard et al., 1999; Trueman and Tuross, 2001). The Ce anomaly (Ce anom ) was calculated from Ce anom =Log[3Ce N /(2La N +Nd N )] (Elderfield and Greaves, 1982). Results Mineralogical Changes FT-IR spectra of the modern and fossil shark centra are shown in Fig. 3-2. Both the modern and fossil samples have the same characteristic absorption bands as the FT-IR spectra of synthetic apatites containing CO 3 2at both Aand Bsites (Bonel, 1972). The FT-IR spectra for modern specimens are characterized by large H 2 O bands (which usually mask the OH band at 3567 cm -1 ) and the presence of organics represented by the

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34 three amide group bands (amide I 1660 cm -1 amide II 1550 cm -1 and amide III 1236 cm -1 mean values). The FT-IR spectra for fossil specimen are characterized by reduced H 2 O bands, lack of OH band, and absence of one or more of the amide group bands (Fig.3-2). There are three intense phosphate (PO 4 3) absorption bands: the main absorbance peak is recorded at ~ 1041 cm -1 and a doublet at ~ 605 cm -1 and ~ 568 cm -1 consistent with previous studies (Shemesh, 1990). In the modern specimens, the 605 cm -1 absorption band has a smaller intensity than the 568 cm -1 band and but the reverse holds for the fossil specimens (Fig. 3-3). The modern specimens have an average crystallinity index (CI) of 2.83, while in the fossil specimens CI is increased to 3.19-5.39 (Table 3-3; Fig. 3-4). B-type carbonate substitution (replacement of PO4 3by CO 3 2; Shemesh, 1990; Dahm and Risnes, 1999) is represented by a set of absorption bands at 1460 cm -1 1428 cm -1 and 870 cm -1 (average values). Carbonate content (C/P) is much greater in modern specimens (0.35 and 0.43) than fossil specimens (range from 0.10 to 0.29) (Table 3-3; Fig. 3-4). The lack of the 713 cm -1 absorbance band in all samples indicates that there is no authigenic calcite present, and the 1092 cm -1 band (average value), which is found only in the fossil specimens, demonstrates the presence of fluorine (Fig. 3-2). Elemental Concentration The effects of diagenesis on elemental concentrations can be assessed by comparing the modern unaltered centra with the altered fossil centra. This is illustrated by the isocon plots (Grant, 1982) in Fig. 3-5. The linear trends (labeled isocon in Fig. 3-5) are the average of the two modern shark centra elemental compositions and represent no elemental loss or gain during diagenesis. In modern sharks the major and minor elements vary (Table 3-3), therefore in the isocon plot of the major and minor elements

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35 Figure 3-2. FT-IR spectra of all nine shark vertebral centra illustrating the differences from modern (C. carcharias) to fossil biogenic apatites. Figure 3-3. FT-IR spectra from 400 to 850 cm-1, illustrating the 4 PO 4 3band differences between modern (solid line) to fossil (dashed line) shark centra.

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36 Figure 3-4. Carbonate content (C/P) vs. crystallinity index (CI) of the nine shark vertebral centra. Table 3-3. Elemental and mineralogical data of nine shark vertebral centra. Elemental concentration are in ppm. Crystallinity index (CI) and carbonate content (C/P) were calculated from FT-IR spectra.

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37 (Fig. 3-5A) the elemental concentrations range in modern sharks for each element is indicated by a vertical line. Most fossil sharks have comparable Ca, P, Zn, Si, Pb, and B concentrations to the modern sharks (Fig. 3-5A). The minor elements Mg, Na, K, Sr, and Mn are more variable, with some fossil specimens exhibiting concentrations similar to modern sharks while Fe, Al, and Ba have no fossils with similar concentrations as modern sharks (Fig. 3-5A). All fossil specimens are enriched in Ba relative to modern, while most fossils are enriched relative to modern in B, Mn, Al, Fe, and Sr (Fig. 3-5A). All fossil sharks are depleted relative to modern in Pb, while most fossils are depleted relative to modern in K, Si, Zn, Mg, and Na (Fig. 3-5A). It can be seen in Fig. 3-5B that Y, U, and REEs are enriched in fossil bone relative to modern. Fig. 3-5C shows the Ba/Ca ratio for the fossils are higher than in modern sharks, because diagenesis has significantly enriched Ba (one to two orders of magnitude greater in fossils than modern shark centra) in all the fossil centra used in this study. The fossil centra Ca/P ratios are slightly higher (0.2-0.5 higher) than modern sharks (Fig. 3-5C). The shale-normalized REE concentrations of the shark centra are enriched relative to seawater by about 10 5 -10 7 (Fig. 3-6). The modern specimen C. carcharias (UF211351), which was caught off the east coast of Florida, has a shale-normalized REE pattern similar to seawater but is slightly flattened and depleted in Yb, a small negative Ce anom. (-0.11), a higher concentration of Ce than the other C. carcharias (BTO433), and (La/Sm) N and (La/Yb) N ratios that overlap those found in coastal waters (Fig. 3-6 and Fig. 3-7). C. carcharias (BTO433), which was caught off the east coast of South Africa, has a seawater-like shale-normalized REE pattern (enriched with HREE), has depleted Ce concentrations, a much larger Ce anom (-0.73) than the other C. carcharias (UF211351),

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38 A. B. C. Figure 3-5. Isocon plots (Grant, 1982) depicting variations in fossil elemental concentrations and ratios due to diagenesis. The isocon lines are the average of the elemental concentrations in the two modern shark centra. A. Isocon plot of the major and minor elements, with the red vertical lines represent the variation between the elemental concentrations in the two modern shark centra. B. Isocon plot of Y and REE. C. Isocon plot of Ba/Ca, Sr/ Ca, and Ca/P ratios.

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39 and (La/Sm) N and (La/Yb) N ratios that overlap with oceanic waters (Fig. 3-6 and Fig. 3-7). Fossil specimens (Fig. 3-6) may be divided into four groups based on their REE composition. The first group, which includes the two O. obliquus specimens, have shale-normalized REE patterns similar to the modern C. carcharias (BTO433) and seawater (HREE enriched) (Fig. 3-6A), show a minimum at Sm (disregarding Ce), Figure 3-6. PAAS normalized REE of the nine vertebral centra divided into four diagenetic groups. A. First group has seawater-like REE N pattern. B. Second and third groups have mixed shaleand seawater-like REE N patterns. C. Fourth group has shale-like REE N patterns. Seawater is average seawater (Elderfield and Greaves, 1982) and multiplied by 10 6 and modern C. carcharias, UF211351 and BTO433, are multiplied by 10 1

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40 Figure 3-7. Compilation of observed (La/Yb) N vs. (La/Sm) N in biogenic apatites of various ages and types, and fresh and oceanic waters (based on Reynard et al., 1999). Jurassic fish and reptile teeth (Picard et al., 2002); Tertiary and Mesozoic fish teeth (Grandjean et al., 1988; Grandjean and Albarde, 1989); Cretaceous reptile and dinosaur bones (Samoilov and Benjamini, 1996) and Jurassic coprolites (Kemp and Trueman, 2002); North Atlantic seawater from 0-4000 m depth (Elderfield and Greaves, 1982), Southern Ocean from 0-4000 m depth (German et al., 1990 ) and North Pacific surface and deepwater (Piepgrass and Jacobsen ,1992); bottom waters (German et al., 1991); coastal waters (Elderfield and Sholkovitz, 1987) and coastal waters (Hoyle et al., 1984); Scotland river waters (Hoyle et al., 1984); anoxic waters (Elderfield and Sholkovitz, 1987); and current research, and C. carcharias, I. hastalis, C. angustidens, C. auriculatus, C. megalodon, and O. obliquus, and C. mantelli. Oceanic, Coastal, and Continental boxes are based on values given by Reynard et al. (1999). Blue circles indicate samples with seawater-like REE N patterns, green circles indicate samples with shale-like REE N patterns, and red circles indicate samples with mixed seawaterand shale-like REE N patterns.

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41 large Ce anom. (greater than -0.3), and an (La/Yb) N ratio that falls within coastal waters. O. obliquus is the only sample that has a (La/Sm) N ratio greater than oceanic waters. The second group, which includes C. angustidens and C. mantelli, have shale-normalized REE patterns similar to the modern shark pattern of C. carcharias (UF211351) and close to seawater but with some flattening (Fig. 3-6B), show a minimum at Nd (disregarding Ce), (La/Yb) N and (La/Sm) N ratios that fall within or just outside of coastal waters (Fig. 3-7), and small negative Ce anom. (less than -0.3). The third group, which includes C. auriculatus, shows a minimum in HREE, shale-normalized REE pattern between seawater and the modern C. carcharias (UF211351) pattern (Fig. 3-6B), (La/Yb) N ratio above marine and continental waters, (La/Sm) N ratio within coastal and oceanic waters (Fig. 3-7), and a small negative Ce anom. (less than -0.3). The fourth group, which includes C. megalodon and I. hastalis, show a minimum at Yb, a flat shale-normalized pattern or a maximum in the heavy-middle REE (Fig36C), a high (La/Yb) N ratios (above seawater and continental waters), and (La/Sm) N ratio within coastal and oceanic waters (Fig. 3-7). Discussion Mineralogical Characterization of Centra Fossil specimens have lost most, if not all, of their organic content through diagenetic processes and contain less absorbed (3430 cm -1 band) and structural H 2 O (3330 cm -1 band; Holcomb and Young, 1980; Michel et al., 1995) than modern specimens. The weak intensity of the absorption band near 1660 cm -1 corresponding to CONH of the amide group (amide I), and the absence of the two other amide bands (amide II and III) signify a significant loss of organics (Reiche et al., 2003) in the fossils. In contrast, the FT-IR spectra of the modern specimens show intense amide I, II, and III bands and thus indicate the presence of organic matter (Reiche at al., 2003).

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42 When comparing the two modern specimens, the CIs are similar, but the carbonate content differs, with the South African C. carcharias shark (BTO433) having a higher carbonate content than the one from Florida. This may be due to due to natural variability within a shark species or overlapping absorption peaks that have both carbonate substitution in the A-site for OH and B-site for PO 4 3(i.e., peaks 1460 cm -1 and 870 cm -1 ) are not included in the estimation of carbonate content. Since, the carbonate content estimation is only normalized by a phosphate peak, the use of A-site substituted carbonate would not be an accurate representation of carbonate content. Usually in modern specimens the OH band at 3567 cm -1 is masked by the water bands and is completely absent in fossil specimens (Fig. 3-2), making it difficult for normalization. Lower C/P and higher CI in fossil specimens (Fig. 3-4) indicate diagenetic loss of carbonate during recrystallization and possibly dissolution of the mineral phase. Implications for Diagenetic and Biological Signal Reconstruction The shale normalized REE patterns of the two modern shark specimens (Fig. 3-6) have similar patterns to seawater, however, C. carcharias (BTO433) from South Africa has a more negative Ce anom and greater HREE enrichment than UF211351 from Florida. The larger negative Ce anom in C. carcharias (BTO433) may indicate that sharks from the coast of Africa live and/or spent most of their time at greater depths than sharks from the East coast of North America. Alternatively, larger great white sharks may feed and/or spend a majority of their time at shallower depths than smaller great whites (UF211351 was 12 in length when captured and BTO433 was 6 in length when captured). Further study of modern great white shark behavior is needed to fully understand the bulk chemistry variations within this species.

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43 The first fossil group, which includes the two O. obliquus specimens, have seawater-like patterns indicating that REE enrichment involved quantitative uptake of REE without fractionation. In other words, there was no absorption taking place, no indications of a terrestrially derived diagenetic signature (flattening of the shale-normalized REE patterns; Grandjean et al., 1987), or no fractionation of REE (i.e. no MREE enrichment or bell shape pattern; Reynard et al., 1999). The La/Yb ratio of about 7 and a larger negative Ce anom. points to deposition in an environment with a deepwater influence (Grandjean, 1987; Grandjean et al., 1988; German and Elderfield, 1990; Pipegras and Jacobsen, 1992). Results from DSDP Leg 41 off the Moroccan coast have identified the onset of abundant chert deposition during the early Tertiary reflecting the input of cold bottom water (Lancelot and Seibold, 1978). Cappetta (1981) showed that Ypresian fish associations from the Ouled Abdoun basin (Oued Zem, location of O. obliquus, is located in the eastern part of the Ouled Abdoun basin) were indicative of greater depth than previous periods. All this evidence indicates either upwelling of deep water onto the continental shelf or progressive deepening of the troughs and channels in which the phosphorite was deposited (Grandjean, 1987) and suggests an influence of deepwater during the diagenesis of O. obliquus. The original REE signal of the O. obliquus centra have been replaced during diagenesis at/or near the sediment/seawater interface with the seawater signal present at time of deposition of the centra. Therefore, the two O. obliquus centra preserved a seawater signal at or near the seawater/sediment boundary and will be useful in reconstructing paleoceanographic environments. The second diagenetic grouping, which includes C. angustidens and C. mantelli, have shale-normalized REE patterns similar to the modern C. carcharias (UF211351)

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44 and close to seawater but with some flattening (Fig. 3-6B). C. angustidens was deposited in glauconitic shelf sand (Kokoamu Greensand) at a water depth of 50-100 m. The presence of glauconite indicates slow sedimentation and low inputs of detritus from land. New Zealand, as a whole, was low-lying and almost fully submerged at this time and therefore the terrestrial input was minor (Ayrees, 1993). Minor flattening of the C. angustidens shale-normalized REE pattern relative to seawater supports that the diagenetic signature, which is produced by large pore water concentration gradients that allow fluxes of REE from sediments to seawater, has had a small influence from terrestrial derived sediments. C. mantelli was deposited in the Smoky Hill Chalk Member of the Niobrara Formation in a water depth between 30-180 m (Hattin, 1981). The sediments of the Smoky Hill Chalk Member are from the mid to outer shelf of the Cretaceous epicontinental seaway. Once again, slight flattening of C. mantelli shale-normalized REE pattern relative to seawater supports a small influence from a terrestrially derived diagenetic signature. Neither C. angustidens nor C. mantelli have any indications of REE fractionation (bell-shaped shale-normalized REE pattern). C. angustidens and C. mantelli centra have the potential to be used for a general paleoceanographic and paleoenvironmental reconstruction, but with caution, because they do have a mixture of seawater signal and diagenetic signature, which will decrease the Ce anom. as well as flatten the shale normalized REE pattern. The third group, which includes C. auriculatus, has a shale-normalized REE pattern between seawater and the modern C. carcharias (UF211351) (Fig. 3-6B). The C. auriculatus centrum was deposited in the Brussels Sand in a shelf/nearshore environment (Hooyberghs, 1990; Herman et al., 2000). The shale-normalized REE pattern, unlike the

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45 second fossil group and C. carcharias (UF211351), has a high at Gd and is more depleted in Dy and Yb. The flattening of the shale-normalized REE pattern, the (La/Yb) N ratio greater than seawater, and the depletion of Dy and Yb in the C. auriculatus centrum indicates a diagenetic signature that is more strongly derived from terrestrial sediments than that of the second fossil group. The C. auriculatus centrum has the potential to be used for a general paleoenvironmental reconstruction, but with caution, because of the terrestrially influenced diagenetic signature, which will decrease the Ce anom. and flatten the shale-normalized REE pattern. The fourth group, which includes C. megalodon and I. hastalis have a flat shale-normalized pattern or a maximum in the heavy-middle REE (Fig36C). The C. megalodon centrum was deposited in sandy siltstone near the Kanto Mountains in Japan (Hayashi et al., 2003). The shale-normalized pattern of C. megalodon is almost completely flat indicating a diagenetic signature that is extensiveljy derived from terrestrial sediments. The I. hastilas centrum was deposited in a shallow bay sandstone and has a flat shale-normalized REE pattern, which indicates a diagenetic signature derived primarily from terrestrial sediments and/or major influence from river water. This is supported by fresh water diatoms in the sediments of the Pisco Formation suggesting the influx of river water entering the basin (Brand et al., 2004). The high (La/Yb) N ratios found in C. megalodon and I. hastalis can be explained by the extensive terrigenous influence (Grandjean et al., 1987). C. megalodon and I. hastalis do not show any indications of late diagenesis (bell-shaped shale-normalized REE pattern). These two centra indicate the quantitative intake of REEs during early diagenesis with a strong influence of continental sedimentary supply in a near-shore environment; therefore they

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46 are not useful in paleoceanographic reconstruction (global controls) but the record of the diagenetic signature indicates the sedimentary environment (local controls) can be reconstructed. For the fossil shark centra used in this study the major control of the shale-normalized REE pattern would be the diagenetic signature and how the terrestrially derived sediments generate concentration gradients between pore waters and seawater. The more prominent the terrestrial sediments the flatter and more shale-like the shale-normalized REE signal become. Each depositional environment must be assessed in order to determine which fossils can be used in paleoceanographic studies versus depositional environment reconstructions. Conclusions While certain variables independently provide information about diagenesis, the simultaneous use of FT-IR and elemental concentrations gives a much better picture of depositional environment and the extent of diagenesis. There is no doubt that diagenetic alteration has affected the REE composition of these seven fossil shark centra. Through the processes of diagenesis, the centra have been imprinted with an REE seawater and diagenetic REE pattern at the sediment/seawater interface. The REE seawater signature was incorporated into the biogenic apatite via a transfer from a short-lived phase made of oxy-hydroxides and organic detritus (Grandjean et al., 1987). The diagenetic signature is caused by the development of pore water concentration gradients, which allow fluxes of REE from sediments to seawater. The amount of terrigenous input and therefore the REE composition of diagenetic signature controls whether the shale-normalized REE patterns and Ce anomalies are representative of the original seawater signal for these seven fossil shark centra. This is clearly seen for I. hastalis, and C. megalodon, where these two

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47 specimens have a strong terrestrially influence diagenetic signature, which disturbs the oceanic signal and Ce anom. In contrast, the two O. obliquus centra preserve a REE seawater signal at the time of deposition and have no indication of a diagenetic signature derived from terrestrial sediments. The remaining three centra (C. angustidens, C. megalodon, and C. auriculatus) have diagenetic signatures that have some influence from terrestrial sediments, which is evident by the slightly flattened shale-normalized REE patterns. These later kinds of samples would require extreme care when interpreting Ce anomalies because it is difficult to determine how much the Ce anom. has been reduce by the diagenetic signature. Hence, even if there is no negative Ce anom. that does not necessarily indicate an anoxic environment but may represent a strong continental influence in the depositional environment. In summary, geochemical data from biogenic apatite of fossil marine vertebrates, like lamnid sharks, have the potential to be used to understand diagenesis, depositional environments (local controls), and/or paleoceanography (global controls).

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CHAPTER 4 OXYGEN ISOTOPIC AND RARE EARTH ELEMENTAL ANALYSIS OF MODERN LAMNID SHARKS: DETERMINATION OF LIFE HISTORY? Introduction Lamnid sharks, Family Lamnidae (great white sharks and their relatives), are of great interest not only to the scientific community but the public as well. Scientists have spent a great deal of time trying to study and understand the life history of great whites (Carcharodon carcharias) and their relatives. Because great whites do not survive well in captivity, tagging and recapture studies and captured sharks from fishermen have been the main source of study. Sharks deposit light and dark bands on their vertebral centra throughout their lives (Fig. 4-1). It is known that in most sharks the darker, denser portions are deposited during slower growth times (e.g., winter) and lighter portions are deposited during more rapid growth (e.g., summer). The problem is that the growth rate is affected by the physical environment (including temperature and water depth), food availability, and stress (Branstetter et al., 1987). Therefore, it cannot be assumed that a band pair (one light and one dark band) reflects a single year (called annulus). Wintner and Cliff (1999) estimated ages of great white sharks from the East coast of South Africa by counting growth rings in the centra. The vertebrae of 61 females and 53 males were x-rayed and counts were made from the x-rays. X-rays enhance the visibility of growth rings in shark centra and have been used successfully to accurately determine ontogenic age of several species (Cailliet et al., 1983; Yudin and Cailliet, 48

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49 1990; Ferreira and Vooren, 1991). Of particular interest from Wintner and Cliffs (1999) study was the one shark that was injected with oxytetracycline (OTC) on October 10, 1994, and was recaptured on May 27, 1997 (specimen BTO433). BTO433 was tagged at 140 cm and grew 69 cm within that two year, seven month and 27 day period. The OTC indicated annual growth ring deposition in most of the centra from BTO433; however, this could not be confirmed from growth ring counts of the entire sample (Wintner and Cliff, 1999). Figure 4-1. Scanned contact print of BTO433 centra. (Top) Dark growth bands, OTC mark and birth mark have all been indicated. Dark growth rings on the contact print show up as white bands, which can be seen in the lower image of the centra. Bomb Carbon and Oxygen Isotopes Atmospheric testing of atomic bombs in the 1950s and 1960s resulted in a rapid increase in radiocarbon ( 14 C) in the worlds oceans (Druffel and Linick, 1978). The period of radiocarbon increase was almost synchronous in marine carbonates such as

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50 corals, bivalves, and fish otoliths around the world (Kalish, 1993; Campana, 1997; Baker and Wilson, 2001), providing a date marker in calcified structures exhibiting incremental growth. More recently, analysis of bomb radiocarbon has been used to validate age estimates derived from vertebral centra of sharks (because sharks do not contain otoliths that grow incrementally; Campana et al., 2002). Campana et al. (2002) used bomb carbon dating as an age validation method for long-lived sharks. They compared radiocarbon assays in young, known-age porbeagles (Lamna) collected in the 1960s with corresponding growth bands in old porbeagles collected later. With this method Campana et al. (2002) confirmed the validity of porbeagle vertebral growth band counts as accurate annual indicators. Because shark vertebrae grow incrementally, the oxygen isotopic signal preserved should reflect the seawater conditions at the time of formation. Oxygen isotopes vary with temperature and salinity (Hoefs, 1988), consequently variations preserved in the vertebral centra could indicate migration into various water bodies and/or water depths. Also, it might be possible to determine the frequency of the migration and, if annual, the oxygen isotopic signals could be used to estimate ontogenic age of the individual. Rare Earth Elements The rare earth elements (REE) consist of fifteen elements which form a series from the lightest REE, lanthanum (La), to the heaviest, lutecium (Lu). With the exception of multiple oxidation states for Ce and Eu, the other REEs have trivalent oxidation state in most natural waters. Cerium may undergo oxidation in seawater from the solvated Ce 3+ state to insoluble Ce 4+ consequently Ce fractionates relative to other REE (German and Elderfield, 1990). Europium may undergo reduction from the Eu 3+ to Eu 2+ which substitutes readily for Ca 2+ inCabearing minerals such as apatites (Elderfield, 1988).

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51 The residence time of REEs in seawater is 10 2 -10 3 years and is therefore shorter than the mixing time of the oceans (1600 years) making these elements useful tracers of oceanographic events and processes (Elderfield and Greaves, 1982; Bertram and Elderfield, 1993; Nozaki et al., 1999; Lacan and Jeandel, 2001). Because most REE concentrations increase with water depth (Elderfield and Greaves, 1982; deBaar et al., 1985a; deBaar et al, 1985b; Dubinin, 2004) they may act as a proxy to indicate the relative water depth at which these individual sharks are living This study tests whether the chemistry of vertebral centra can be used to improve our understanding of the life history of sharks. The possibility exists to elucidate shark migration, the relative depth of habitat, and determination of individual age using rare earth elemental compositions, bomb carbon, and oxygen isotopes. Background Five species of lamnid sharks live today, Carcharodon carcharias (great white), Isurus paucus (longfin mako), Isurus oxyrinchus (shortfin mako), Lamna ditropis (salmon shark), and Lamna nasus (porbeagle). This study focuses only on the largest three members of Lamnidae, the great white, longfin mako, and shortfin mako (Table 4-1), due to ease in sampling larger vertebral centra. Great White (Carcharodon carcharias) The great white is the largest extant lamnid shark and has one of the broadest distributions of all modern sharks. The great white is cosmopolitan in cold temperate to tropical seas, living primarily in coastal and offshore habitats of continental and insular shelves. However large individuals have been recorded off oceanic islands. The known depth range of great whites is from the surface to at least 6,150 ft (1.875 km). Great whites maintain a body temperature up to 27F (15C) warmer than surrounding waters

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52 by an adaptation to its circulatory system that does not allow the heat generated within the body to escape through the gills (Compango, 2002). Great whites prefer waters with a sea surface temperature between 59-72F (15-22C). Most individuals are 12-16 ft (3.7-4.9 m) long with a maximum length of 20 ft (6.1 m). At birth a great white is between 3 to 4 in length. Juvenile sharks feed on bottom-dwelling teleost fish, small sharks, and rays, while adult sharks feed on sharks, rays, teleost fish, seals, sea lions, dolphins, whale blubber (scavenged), squid, seabirds, marine turtles, crabs, and snails (Campagno, 2002). Longfin Mako (Isurus paucus) The longfin mako was first described in 1966 and is one of the least-known lamnids. It is also the second largest member of Lamnidae, after the great white. Longfin makos have an appearance similar to shortfin makos but have a slimmer body, larger eyes, and larger pectoral fins. Most specimens are about 7 ft (2.2 m) long and the maximum known length is 14 ft (4.3 m), which is based on a male specimen taken from 15 mi (24 km) off Pompano Beach, FL, in February 1984 (Compango, 2002). At birth a longfin mako is between 3 to 3 (92 to 120 cm) in length. Longfin makos are widely distributed in tropical to warm temperate seas. They are fairly common in the western Atlantic (Gulf Stream waters, northern Cuba to southeast Florida) and possibly in the central Pacific (near Phoenix Island and north of Hawaii), although rare, longfin makos have been recorded off northwestern Africa and the Iberian Peninsula, from the northern Gulf of Mexico to the Grand Banks, Bahamas and off New South Wales Australia (Compango, 2002). Most specimens of longfin makos are caught on long-lines in deep tropical waters, from depths of 360-720 ft (110-220 m). The long broad pectoral fins suggest that longfin makos are slower and less active than shortfin makos. This inference

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53 is also supported by the fact that longfin makos have the same heat-retaining modifications to the circulatory system as other lamnids; however longfin makos are unique among the members of its family in that this species is not warm bodied. The diet of a longfin mako consists of schooling fish and pelagic squid (Compagno, 2002). Table 4-1. Lamnid specimens used in this study. Taxon Specimen Location of Capture Number of Centra Body Length (ft) Carcharodon carcharias BTO433 Capetown, S. Africa 1 6 Carcharodon carcharias UF211351 Islamorada, Florida 1 129 Carcharodon carcharias UF211352 Marathon, Florida 1 12 Carcharodon carcharias UF31648 Florida 1 N/A Isurus paucus UF211355 Miami, Florida 3 8 Isurus paucus UF211354 Pompano Beach, Florida 3 84 Isurus paucus UF211353 Pompano Beach, Florida 1 14 Isurus oxyrhincus UF47943 Florida 1 N/A Shortfin Mako (Isurus oxyrinchus) The shortfin mako is the fastest swimming shark and has a global distribution in tropical and temperate waters. Shortfin makos are common in coastal and oceanic regions of tropical and temperate seas but seldom occur in waters less than 61F (16C). They range from California to Chile in the Pacific Ocean and from the Grand Banks of the Bahamas to Brazil, including the Gulf of Mexico and the Caribbean Sea in the Atlantic Ocean. In the eastern Atlantic, shortfin makos range from Norway to South Africa, including the Mediterranean, and is found throughout the Indian Ocean from South Africa to Australia. In the western Pacific it can be found from Japan to New

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54 Zealand and in the central Pacific it occurs from the Aleutian Islands to the Society Islands (Compango, 2002). The known depth range is from the surface down to at least 1,300 ft (400 m). Shortfin makos tend to follow movements of warm water in extreme northern and southern parts of its range. Tagging studies off the northeastern US show a seasonal pattern of abundance along the western margin of the Gulf Stream, moving inshore and into higher latitude waters as the stream shifts northward from April-October, possibly wintering in the Sargasso sea from November to March (Compango, 2002). At birth a shortfin mako is 2 to 2 long with most individuals 6-8 ft (1.8-2.5 m) long and a maximum recorded length of 12.8 ft (3.9 m). Shortfin makos maintain body temperatures 12.5-18F (7-10C) warmer than the ambient water and are capable of rapid acceleration and bursts of speed when hooked or in pursuit of prey. Adults have been clocked at 31 mph (50 km/hr) (Compango, 2002). Materials and Methods X-radiograph Analyses X-radiographs were used to enhance the visibility of the growth rings. X-radiographs of whole centra were taken at the C.A. Pound Human Identification Laboratory at the University of Florida. The x-rays are set at 78 kV for 2 minutes. The x-rays are used to make contact prints, which are a reversed pattern of the x-ray (i.e., dark lines on the x-ray are the light lines on the contact print). The dark and light alternating growth rings are easily seen on the contact prints (Fig. 4-1). These contact prints were digitally scanned and Adobe Photoshop TM was used to enhance the images. Growth ring counts were made from the scanned contact prints and interpretation of the vertebral growth bands was made using published criteria for porbeagles and Pacific shortfin makos. Campana et al. (2002) has proven with the use of bomb carbon, annual growth

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55 band pairs form in porbeagles, and Cailliet at al. (1983) demonstrated that a single band pair formed each year in Pacific shortfin mako based on growth ring counts. Oxygen Isotopic Preparation and Analyses Oxygen isotopes can be used for the interpretation of temperature. For each of the centra, about 1 mg was drilled with a foredoom slow speed drill approximately every 1 mm across the growth axis starting from the birthmark and ending at the external margin. An interval of 1 mm insured that each primary growth band was sampled. Sample powders were treated with standard isotope preparation techniques (e.g., MacFadden et al., 1999). At least 0.75 mg of each treated sample powder was then measured into individual metal boats and placed in the carousel of the isocarb device for introduction into the VG Prism mass spectrometer in the Stable Isotope Laboratory in the UF Department of Geological Sciences. The sample runs were calibrated to internal laboratory and NBS 19 standards. The oxygen isotopic results are reported in the standard convention: (parts per mil, ) = (R sample /R standard )-1) x 1,000), where R = 13 C/ 13 C or 18 O/ 16 O, and the standard is VPDB. Ontogenic ages based on the oxygen isotopic data were estimated by counting the number of peaks or valleys present long the growth axis of the centra. Bomb Carbon Dating Preparation and Analysis Atmospheric testing of atomic bombs in the 1950s and 1960s produced a time-specific radiocarbon marker, which allows for material formed between the 1950s and the present to be dated (Kalish, 1993; Weidman and Jones, 1993). To determine if bomb carbon dating would work on great white centra, only one shark centra, BTO433, was chosen for dating. BTO433 was chosen because the capture date is known and this shark

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56 was injected with oxytetracycline (OTC) on October 30 th 1994. The OTC mark gives a second reference for the accuracy of the bomb carbon dates generated. About 60 mg of sample was extracted from BTO433 first formed growth band (corresponding to the first year of growth) and the last growth band. The external surface of the centrum was removed in order to minimize surface contamination. The sample was weighed to the nearest 0.01 mg in preparation for assay with Accelerator Mass Spectrometry (AMS). The sample was assayed at the Keck Carbon Cycle AMS Laboratory, UC Irvine for 13 C (to determine the carbon source) and 14 C (measure of radiocarbon), with 14 C calculated per Stuiver and Polach (1977). To assign dates of formation to an unknown sample, it is necessary that the 14 C of the unknown sample be compared with a 14 C of a known-aged material. BTO433 14 C data were compared to the 14 C values of Pagrus auratus otolith collected off the East coast of North Island, New Zealand (Kalish, 1993). An age for BTO433 based off the Campana et al. (2002) reference curve was assigned by correcting the 1997 14 C value of BTO433 to the1997 porbeagle reference curve value generated by Campana et al. (2002), then the same correction was applied to the first growth band 14 C value and that value was compared to the porbeagle curve to determine a year for deposition of the first growth band (Fig. 4-2). This was done to determine if the second method would produce similar results to the first, because reference curves are not always available for the area in which the specimen was recovered and this would allow for the use of previously produced reference curves. It is important to have a local reference curve since the carbon was not uniformly distributed at the time of atomic bomb testing, especially

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57 between the Northern and Southern Hemisphere as most of the bomb testing was done in the Northern Hemisphere. Figure 4-2. BTO433 bomb carbon data plotted vs. two reference curves (a) fish otolith from New Zealand (Kalish, 1983) and (b) porbeagle (Campana, 2002). BTO433 is plotted on the fish otolith curve and BTO433B is plotted on the porbeagle curve. Inductively Coupled Plasma Mass Spectroscopy (ICPMS) Inductively coupled plasma mass spectroscopy (ICPMS) allows for the quantification of elemental abundances within the shark centra. Approximately 5-10 mg of bulk sample was drilled from each of the twelve centra. 5 mg of each sample were weighed into 3 mL Savillex vials and dissolved in 1 mL of 3M HNO 3 and heated overnight. Samples were allowed to cool and then dried. Then 2 mL of 1% HNO 3 was added, heated overnight, and allowed to cool. Samples were analyzed on an Element 2 High Resolution Inductively Coupled Plasma Mass Spectrometer (HR-ICP-MS) at the UF Department of Geological Sciences. All samples were corrected by subtracting the blank, corrected for instrumental drift based on internal machine standards that were analyzed during the run, and correcting ion counts to a constant response to the known amount.

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58 All REE values were shale-normalized to PAAS (Post-Archean Shale Standard) in order to eliminate the odd-even effect of the natural abundances of the REE. The Ce anomaly (Ce anom ) was calculated with the Elderfield and Greaves (1982) formula, Ce anom =Log[3Ce N /(2La N +Nd N )]. Results and Discussion Ontogenic Age Determinations The ontogenic ages determined from oxygen isotopic cyclicity, growth ring counts and the 14 C of the eight sharks are presented in Table 4-2. The ontogenic ages estimated based on the oxygen isotopic cyclicity (Fig. 4-3) give ages that are too young with the exception of UF31648 and BTO433 (Table 4-2) when compared to growth ring counts. For UF211351, UF211352, and UF47943 the oxygen isotopic signal gave an ontogenic ages of 5+ years, 7+ years, and 4+ years, respectively, which are all several years too young compared to growth ring counts. For specimens UF211354 and UF211353 the oxygen isotopic data gave ages of 6+ and 8+, respectively, which are about half of the ages estimated from the growth ring counts. The three UF211355 have age estimates of 3+, which is 8+ years too young according to the growth ring counts. One problem with the oxygen isotopic data is that sampling every growth ring is challenging. As sharks get older, their growth rate decreases and therefore the size of the growth rings become narrower making it difficult to sample only one growth ring at a time. Also, the location of the centra in the sharks vertebral column (i.e., towards the head or towards the tail) will affect the age estimate for both oxygen isotopic analysis and growth ring counts. Sampling the exact same location for multiple centra within an individual is difficult to accomplish because not all the vertebrae grow at the exact same time. This accounts for the offset of the isotopic data in sharks UF211354 and UF211355

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59 Table 4-2. Ontogenic age estimates based growth ring counts (GR), oxygen isotopic ( 18 O) cyclicity, and bomb carbon ( 13 C). Ages estimated from growth ring counts should be considered minimum ages since the last growth band may not have been formed completely at time of capture. Taxon Specimen GR counts 18 O age 14 C age Carcharodon carcharias BTO433 3+ 3+ 3 Carcharodon carcharias UF211352 9+ 7+ Carcharodon carcharias UF211351 7+ 5+ Carcharodon carcharias UF47943 4+ 3+ Isurus paucus UF211355A 11+ 3+ Isurus paucus UF211355B 11+ 3+ Isurus paucus UF211355C 11+ 4+ Isurus paucus UF211354C 11+ 4+ Isurus paucus UF211354B 13+ 5+ Isurus paucus UF211354A 12+ 5+ Isurus paucus UF211353 16+ 8+ Isurus oxyrhincus UF31648 3+ 3+ of the three centra analyzed (Fig. 4-3). Choosing the largest centra and/or centra that are closer to the head than the tail would probably alleviate some of these effects. Another possibility is that the sharks studied did not migrate into bodies of water with enough temperature variation to alter the oxygen isotopic signal. Also, great whites and shortfin makos are warm-bodied and maintain a body temperature above the surrounding water temperature, which will affect the oxygen isotopic signal, (i.e., the isotopic variation might not reflect the actual water temperature change, but the changes in body temperature as the sharks encounter colder or warmer waters). It has been shown that muscle temperature of lamnid sharks changes in response to changes in ambient water temperature (Carey et al., 1982; Tricas and McCosker, 1984; and Carey et al. 1985). Oxygen isotopic signals may reflect changes in body temperature; these changes may accentuate or dampen the oxygen isotopic signal preserved in the centra. The shark centra analyzed in this study do not seem to have a dampened oxygen isotopic signal (i.e., oxygen isotopic values along the growth axis have similar absolute values) indicating that

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60 great whites and shortfin makos do not maintain a constant body temperature (Fig. 4-3). Unlike the shortfin mako and great white, the longfin mako is not warm-bodied and therefore does not maintain a body temperature above that of its surroundings and consequently the oxygen isotopic signal should reflect the temperature of the surrounding water. BTO433 age is estimated by the 14 C values, which are plotted against the two reference curves in Fig. 4-2. BTO433 14 C values were plotted against the first reference curve, otoliths from fish off the coast of New Zealand (Kalish, 1993), and no correction was necessary because the 1997 age plotted on the curve. The second method entailed subtracting the 1997 14 C reference curve value generated from the porbeagle centra (Campana, 2002) from the BTO433 1997 14 C value and then subtracting that same amount from the BTO433 first growth ring 14 C value. Both methods produce the same ontogenic age of 3 years. More work needs to be done in order to determine if subtracting the difference of the 14 C values of the known age of the specimen to the reference curve works for sharks that are older. However, the fact that both methods(growth ring counting and bomb carbon dating) produced an ontogenic age of 3 years for BTO433 indicates that at least at young ages great whites deposit growth bands on an annual basis. Also, if two growth band pairs form per year, this shark would only be 1 years old and that is impossible since 2.6 years passed before recapture of BTO433 after the OTC injection (Wintner and Cliff, 1999). However one individual cannot be used to provide a definitive growth rate for great whites, further radiocarbon assays of lamnid shark vertebrae is needed to accomplish this goal.

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61 Figure 4-3. Oxygen isotopic data (VPDB) for the shark centra analyzed. UF211354 and UF31155 both have three centra per specimen. Rare Earth Elements The elemental concentrations are given in Table 4-3 and the shale-normalized REE patterns are shown in Fig. 4-4. The shale-normalized REE concentrations are enriched relative to seawater by about 104 -105. For all the sharks captured off the coast of Florida, Eu is enriched relative to seawater, Eu was not analyzed for the specimen caught

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62 off the coast of South Africa. Eu is known to replace Ca in apatite more readily than other REE since Eu2+ has the same oxidation state and a similar ionic radius to Ca2+ (Elderfield, 1988), therefore expectations are for marine biogenic apatite to be enriched in Eu relative to seawater. Specimens UF211353, UF31648, UF47943, and UF211352 all have depletion in Gd relative to seawater. The degree of enrichment of Eu and depletion of Gd varies between specimens. BTO433 and UF211352 have shale normalized REE patterns similar to average seawater, ignoring the Eu enrichment (Fig. 4-4). UF211351, UF211354, and UF211355 have shale-normalized REE patterns between seawater and coastal waters (Fig. 4-4), ignoring the Eu enrichment. UF211353, UF31648, and UF47943 have shale normalized REE patterns similar to coastal waters (Fig. 4-4), ignoring the Eu enrichment. The REE patterns differ with location along the Florida coast, indicating that REE shale-normalized patterns may serve as a provence tool (i.e., location determination). In this study the two centra, UF31648 and UF47943, belong to sharks without capture location data but exhibit REE distributions similar to that of UF211353. To use bulk samples as a provenace tool numerous sharks from an area should be analyzed, which is evident from the differences between UF211354 and UF211353. Both of these sharks were caught off the coast of Pompano Beach, Florida but they show different shale-normalized REE patterns, because they probably spent time in different waters/depths throughout their lives. The REE pattern throughout life will be averaged when bulk samples are used; therefore changes in the REE shale-normalized patterns along the growth axis of the centra (i.e., migration patterns) should be shown when microsampling is utilized.

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63 The REE content variation in seawater correlates to the circumcontinental zonality (Dubinin and Rozanov, 2001; Tachikawa et al., 1999) due to an exchange between the dissolved REE and absorbed complex of terrigenous suspended matter. The decrease in terrigenous particulate content toward the pelagic area leads to the increase of the dissolved REE concentration (Dubinin, 2004). Dissolved REE content increases with water depth by several times in the Atlantic Ocean (de Barr et al., 1985; Sholkovitz, 1994; Dubinin, 2004), therefore REE concentrations could be a proxy for relative water depth for these shark centra. The plot of La+Sm+Yb (ppm) versus (Sm/Yb) N (Fig. 4-5) indicates that (a) UF211351, UF211353,UF211355, UF47943, and UF31648 lived at similar depth closest to the surface; and (b) BTO433, UF211352, and UF211354 also lived at similar depth, but deeper than the previously mentioned group. UF211353, UF211354, and UF211355 should plot at similar depths (i.e., have similar REE concentrations) since it is known that most longfin makos live and feed between 360-720 ft and expectations are that the REE values would not vary much within this depth range since it is only a difference of 360 ft. Shortfin makos have a larger depth range in which they feed and inhabit than longfin makos, however according to these data UF47943 lived or ate at a fairly shallow depth relative to the other sharks analyzed. Conclusions This is the first study to analyze the chemical and light stable isotopic composition of lamnid shark centra. Because lamnid sharks cannot be kept in captivity, REE concentrations, oxygen isotopes, and bomb carbon are new approaches to gaining knowledge about these animals. Bomb carbon dating is an effective way to accurately date shark centra, and this has been tested with an individual with a known capture date.

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64 Table 4-3. Elemental data (in ppm), and oxygen isotopic and bomb carbon dating ages. N/A represents elements that were not analyzed and () indicates that the concentrations were below detection limits of the ICPMS. Taxon Specimen Na Mg Al Mn Fe Ni Cu Zn Sr Y Ba Pb U P C. carcharias BTO433 4053.43 1752.92 266.01 4.80 177.22 N/A N/A 904.81 352.49 0.56 1.47 9.11 0.28 59982.29 C. carcharias UF211352 8391.58 3196.68 82.32 6.95 35.19 1.54 2.13 255.64 1428.63 0.05 3.20 1.04 2.42 144448.89 C. carcharias UF211351 6559.68 2678.65 30.78 6.43 408.77 7.91 6.65 29.51 1120.88 0.03 1.76 0.54 0.14 116058.87 C. carcharias UF47943 5012.73 2822.44 75.03 4.82 19.52 5.72 8.62 36.04 1181.54 0.05 3.47 2.47 0.11 119935.33 I. paucus UF211355A 6012.29 3053.64 88.06 9.80 42.78 0.66 1.15 52.40 1541.16 0.06 6.62 0.19 0.10 131921.99 I. paucus UF211355B 5001.04 1868.17 52.46 10.60 16.28 0.75 0.93 31.45 1545.61 0.04 8.27 0.15 0.11 135493.72 I. paucus UF211355C 5992.55 3050.59 83.15 9.25 14.71 0.86 1.81 46.09 1478.26 0.04 6.67 0.27 0.09 130841.17 I. paucus UF211354C 4915.74 2651.50 46.06 8.94 79.74 0.84 1.09 35.57 1417.09 0.08 6.78 0.64 0.21 131198.65 I. paucus UF211354B 4548.74 2327.36 21.16 7.01 41.85 0.38 0.60 28.17 1226.32 0.04 6.05 0.32 0.18 117790.59 I. paucus UF211354A 4782.93 2627.17 49.99 7.77 54.53 0.76 0.95 32.09 1337.93 0.08 7.43 0.67 0.21 125275.09 I. paucus UF211353 7522.55 2775.05 11.37 10.52 16.66 0.45 1.05 54.17 1596.44 0.01 4.52 24.26 0.17 137821.84 I. oxyrhincus UF31648 5869.75 3483.01 20.78 7.62 14.73 1.61 5.87 54.54 1563.24 0.09 5.87 0.57 0.21 142857.41 Taxon Specimen K Ca La Ce Pr Nd Sm Eu Gd Dy Yb Lu Ce anom. C. carcharias BTO433 1133.63 73202.7 0.23 0.09 N/A 0.14 0.02 N/A 0.04 0.04 0.03 N/A -0.68 C. carcharias UF211352 322.38 302628.1 0.168 0.028 0.033 0.135 0.025 0.014 0.007 0.024 0.016 0.003 -1.08 C. carcharias UF211351 261.62 246712.3 0.044 0.068 0.01 0.035 0.009 0.006 0.005 0.008 0.005 0.002 -0.11 C. carcharias UF47943 185.32 250650.4 0.055 0.138 0.011 0.044 0.011 0.013 0.008 0.005 0.001 0.1 I. paucus UF211355A 187.57 280707.6 0.081 0.097 0.018 0.078 0.014 0.022 0.029 0.014 0.008 0.003 -0.25 I. paucus UF211355B 187.16 279298.98 0.086 0.132 0.021 0.087 0.018 0.023 0.012 0.016 0.009 0.002 -0.15 I. paucus UF211355C 217.55 277130.3 0.185 0.17 0.041 0.16 0.029 0.026 0.018 0.027 0.009 0.002 -0.35 I. paucus UF211354C 131.12 280654.2 0.476 1.45 0.13 0.62 0.145 0.047 0.156 0.183 0.103 0.019 0.1 I. paucus UF211354B 105.88 252319.5 0.2223 0.444 0.057 0.245 0.051 0.024 0.036 0.055 0.034 0.007 -0.05 I. paucus UF211354A 109.88 270683.3 0.271 0.773 0.073 0.336 0.07 0.033 0.1 0.089 0.058 0.009 0.08 I. paucus UF211353 639.50 291824.4 0.05 0.071 0.012 0.047 0.01 0.017 0.01 0.006 0.001 -0.18 I. oxyrhincus UF31648 192.68 30000.6 0.055 0.131 0.013 0.051 0.013 0.018 0.007 0.006 0.001 0.05

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65 Figure 4-4. Post Archean Australian Shale normalized rare earth element plots for the eight sharks analyzed compared with average seawater (Elderfield and Sholkovitz, 1987) and Connecticut coastal waters (Elderfield et al., 1990).

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66 Figure 4-5. Depth estimates for the eight lamnid sharks. Arrow indicates direction of increasing water depth. Both methods of bomb carbon age estimation (correcting to a known curve and plotting against a known curve) seem to work for at least the one great white analyzed here. BTO433 has annual growth ring deposition giving it an age of 3+ years, which coincides with the growth ring counts from the contact prints of the centra. Oxygen isotopes give an indication of changes in body temperature for shortfin makos and great white sharks, and water temperature for longfin makos throughout their lives. Ontogenic age estimated based on the oxygen isotopic data did not give similar ages for most sharks when compared to the growth ring counts. Either the eight sharks studied were not encountering waters that shifted their body temperature enough to change the oxygen isotopic signal seasonally, or during sampling some growth bands were missed and/or more than one band was sampled averaging the oxygen isotopic data. Annual growth deposition in lamnid sharks is supported by bomb carbon dating from Camapana (2002) and from the great white BTO433 from this study. Also, UF211353 a

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67 14 longfin mako, which is twice the usual size, would only be 8 years old if two growth band pairs were deposited annually, indicating that the longfin mako, which is the slow moving non-warm bodied lamnid, would grow at a faster rate than the warm-bodied fast moving shortfin mako. REE shale normalized patterns have the potential to be utilized for assessing the general location of sharks that have unknown capture locations. Elemental analysis was done on bulk samples, which would average the REE values along the growth axis. Sharks with similar REE patterns most likely lived in similar waters. UF31648 and UF47943 were captured off the coast of Florida and no other location data were given. When compared to the other centra analyzed from the coast of Florida UF31648 and UF47943 REE shale-normalized patterns are almost identical to UF211353, indicating that these sharks may have spent a significant amount of time in similar waters. Also with microsampling the changes of the shale-normalized REE patterns along the growth axis of the centra will allow for the determination of migration timing. The use of La+Sm+Yb vs. (La/Sm) N demonstrates the possibility of estimating relative water depth for sharks. According to this interpretation (Fig. 4-5), the great whites in this study have a large depth distribution and longfin makos in this study have a fairly restricted depth distribution, which is supported by that fact that great whites have been seen from the surface down to 6,150 ft and longfin makos are usually captured depths between 360-720 ft. More shark centra must be analyzed in order to determine whether REE in shark centra accurately represent water depth. This study demonstrates the potential of using elemental and isotopic analysis to learn more about the life histories of sharks from the chemical make-up of their vertebral centra.

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CHAPTER 5 SUMMARY AND CONCLUSIONS Diagenesis is pervasive in fossil bones and ancient sedimentary environments even though in paleontology this process is all too often ignored. Understanding how diagenesis affects the biological signal preserved in fossils is imperative to correctly interpret analytical findings. The study presented here gives several procedures (including FT-IR, ICMPS, and stable isotope analysis) to help unravel the diagenetic story. A modern analog must always be understood before the fossil data can be interpreted. Therefore, if the end memebr composition of unaltered specimens is not known there is no way to interpret the signal preserved in the fossil (all fossil undergo diagenesis just to varying degrees). Otodus obliquus centra from the Eocene of Morocco demonstrate that a biological oxygen isotopic signal remains preserved along the growth axis even though diagenesis has taken place. The oxygen isotopic signal does not seem to be annual in this case, because if annual growth ring deposition is assumed as in other lamnids, then the oxygen isotopic age is eight years too young. Given that the oxygen isotopes vary with temperature, this distribution relative to the growth rings may indicate that these sharks were not migrating annually into waters with temperature shifts detectable by the oxygen isotopes. Another possibility is that Otodus obliquus, like modern lamnids, may be warm-bodied. If this were the case, then the oxygen isotopic data represent body temperature and not water temperature and O. obliquus may be migrating on an annual basis but it may not be encountering waters that raise or lower its body temperature. The 68

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69 oxygen isotopic analysis of Otodus obliquus centra have prospective broad ramifications for understanding the evolution of growth rates and developmental strategies in fossil sharks; however, analytical techniques that assess diagenesis should be used in combination with isotopic studies in order to produce the most insightful analysis of fossil shark paleobiology. Diagenesis can be quantified by the use of multiple variable analyses. The use of ICPMS and FT-IR data provide a clearer picture of the depositional environment and the extent of diagenesis. Seven fossil lamnid shark centra from all over the world were analyzed for elemental and mineralogical composition. All seven fossil centra are diagenetically altered, which is evident from the FT-IR spectra indicating the presence of fluorine and the decrease in carbonate content, and ICPMS data which show an enrichment in REE, Y, and U. However, through diagenesis the centra have been imprinted with the seawater signal at/near the sediment/water interface. The type of diagenetic fluids expelled from sediments into the water column determines how representative the shale-normalized REE patterns and Ce anomalies are to the original seawater signal. The more terrigenous sediments present in the depositional environment the flatter and more shale-like the normalized REE patterns of the centra. Therefore samples that contain a terrestrial influence would require extreme care when interpreting Ce anomalies because it is difficult to determine how much the Ce anomaly has been reduced by continental input. In these cases, the chemistry of depositional environment is determined rather than the chemistry of the seawater. These seven shark centra illustrate that geochemical data from biogenic apatites of fossil marine vertebrates have

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70 the potential to be used to understand diagenesis, depositional environments, and/or paleoceanography. Modern lamnid sharks, which include great whites, longfin makos, and shortfin makos, have vertebral centra composed of carbonate hydroxyapatite similar to other vertebrates. The oxygen isotopic signals preserved along the growth axis of great whites and shortfin makos represent changes in body temperature as they encounter varying water temperatures, while in the longfin mako the actual water temperature is represented. This is because great whites and shortfin makos are warm-bodied and longfin makos are cold-bodied (Campana, 2002). Unfortunately, the oxygen isotopic signal recorded in the eight modern sharks studied did not demonstrate annual cyclicity. Growth ring counts made on all specimens did indicate annual growth, but the oxygen isotopic data usually gave an age of about half of the actual age. Annual growth ring deposition in lamnid sharks was supported by bomb carbon dating of lamnid sharks (BTO433) and a comparison of growth rates for three different lamnid species. Rare earth elemental data illustrate that shale-normalized rare earth elements can be used as to determine habitat for sharks with an unknown capture site off the coast of Florida and can be used for relative depth estimation of the eight sharks in this study. These studies will potentially serve as a general model for other researchers interested in assessing the extent of diagenesis of their fossils in a particular study area. These studies therefore have broad applicability to paleobiologists, paleoclimatologists, paleoceanographers, and archaeologists. Plans to continue this study include geochemical modeling of the diagenetic system using thermodynamics and kinetics to better understand the stability of biogenic apatites

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71 and chemical pathways for diagenesis. Also a comparison of the accuracy of laser ablation techniques with conventional dissolution and dilution methods currently used to quantify the elemental concentrations on the ICPMS. The precision of laser ablation will allow for fine scale resolution along the growth axis of the sharks centra and will minimize destruction to the specimen. Future work will include applying the techniques presented in this dissertation to the terrestrial system in order to determine how diagenesis affects the orthodentine of Edentates (sloths and armadillos). Edentates, unlike other mammals, do not have enamel on their teeth and are therefore more prone to diagenesis. Finally, future work will be to expand the techniques used here to other groups of fishes (including modern and fossil) and possibly mososaurs and plesiosaurs (which preserve incremental growth in their vertebrae).

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BIOGRAPHICAL SKETCH Joann Labs Hochstein was born in Rockville Center, NY, on January 13, 1977. She graduated from Commack High School on Long Island in June 1995. In August 1995 she attended South Dakota School of Mines and Technology in Rapid City, SD, where her research was focused on vertebrate paleontology. In 1999 she graduated with honors with her Bachelor of Science degree in geology. After, Joann attended the University of Florida, focusing her research on the high resolution study of the variations in paleointensity of the Earths magnetic field. In May 2001 she received her Master of Science degree in geological sciences. In August 2001 Joann entered the Ph.D. program at the University of Florida. Her research focused on understanding how diagenesis affects the chemistry of fossils. Joann will complete her Ph.D. at the University of Florida in 2005. 82