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Electronic Theses, Treatises and Dissertations The Graduate School

2011

Skeletochronology of the AmericanAlligator (Alligator Mississippiensis):Examination of the Utility of Elements forHistological StudyBonnie Joan Garcia

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

SKELETOCHRONOLOGY OF THE AMERICAN ALLIGATOR (ALLIGATOR

MISSISSIPPIENSIS): EXAMINATION OF THE UTILITY OF ELEMENTS FOR

HISTOLOGICAL STUDY

By

BONNIE JOAN GARCIA

A Thesis submitted to the

Department of Biological Science

in partial fulfillment of the

requirements for the degree of

Master of Science

Degree Awarded:

Summer Semester, 2011

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The members of the committee approve the thesis of Bonnie Joan Garcia defended on July 1,

2011.

_______________________________________

Gregory Erickson

Professor Directing Thesis

_______________________________________

William Parker

Committee Member

_______________________________________

Brian Inouye

Committee Member

_______________________________________

Emily DuVal

Committee Member

Approved:

_____________________________________

P. Bryant Chase, Chair, Department of Biological Science

The Graduate School has verified and approved the above-named committee

members.

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For my family.

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ACKNOWLEDGEMENTS

The list of those involved with this project is vast but I would be remiss without starting

that list with Gregory Erickson. He took a chance on a kid fresh out of high school that wanted to

do “dinosaur research” and then let her chop up raptors. Thank you for your infinite patience,

always encouraging me throughout my time at Florida State, and being my greatest champion.

Second in that list is William Parker. As an undergraduate, Dr. Parker never failed to give me the

advice I needed to hear and the encouragement to keep on keepin’ on. The amount of gratitude I

feel for your support couldn’t be contained within a TARDIS. I would also like to thank my

other committee members, Brian Inouye and Emily DuVal, for their comments, suggestions, and

support throughout this research and for bringing an outside eye to what I’ve been doing.

I also need to thank Paul Gignac, Matt Kolmann, and Aki Watanabe for providing me

with thoughtful comments and suggestions throughout and being the best lab family a gal could

ask for. You guys are simply tops. Special mention needs to be made for Christina Kwapich, Lee

Eng Tan, Patrick Griffith, Anna Strimaitis, and Neil Aschliman who helped keep me sane when

it seemed all hope was lost. Thank you to Ken Womble for your artwork. And of course, a BIG

thanks to the E&E graduate community. What an amazing, talented, and kind group of people to

constantly be surrounded by – you all have changed my life in ways I’ll never be able to fully

express.

The Askew Student Life Center and its staff, in particular, Bob Howard, have also largely

influenced my time at Florida State. I feel so incredibly fortunate to have met you, Bob, during

my time here. Thank you for always having an open door and an ear to listen.

All of those friends and family who have heard me whine and groan over the past few

years: Thank You. I know that I can be a right witch. I’m working on that.

To my father; thank you for teaching me to never go to bed upset or angry. Words to live

by – also, they helped to get the majority of this written. Not a day goes by, dad, not a single day.

To my mother; thank you for always being my personal cheerleader and helping me to make

sense of life’s priorities. I’m getting emotional just typing this. Haha! To my siblings; anything is

possible as you all continue to teach me each and every day. No one has it better than me

because I have all of you. To my nieces and nephews; thank you all for being cute and letting me

dote on you for the rest of your lives. That’s right, the rest of your lives.

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And lastly I want to thank Kenneth Gloeckner who continues to believe in me even when

I do not. “I hope that I don’t sound insane when I say / There is darkness all around us / I don’t

feel weak but I do need sometimes for him to protect me / And reconnect me to the beauty that

I’m missin’” I can’t wait to have more adventures with you. I am eternally grateful for the

random circumstances that brought us together.

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TABLE OF CONTENTS

List of Tables ................................................................................................................................ vii

List of Figures .............................................................................................................................. viii

Abstract ............................................................................................................................................x

1. INTRODUCTION...................................................................................................................1

2. HISTOLOGY OF ALLIGATOR MISSISSIPPIENSIS .............................................................3

APPENDIX....................................................................................................................................38

REFERENCES ..............................................................................................................................39

BIOGRAPHICAL SKETCH .........................................................................................................42

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LIST OF TABLES

2.1 Specimen ID and measurements taken by the Florida Fish and Wildlife Commission during

the female’s lifetime. ....................................................................................................................33

2.2 Measurements and label ranking for thin-sectioned skull. ....................................................34

2.3 Measurements and label ranking for thin-sectioned vertebral elements. ..............................35

2.4 Measurements and label ranking for thin-sectioned girdle elements. ...................................35

2.5 Measurements and label ranking for thin-sectioned limb elements ......................................36

2.6 Measurements and label ranking for thin-sectioned pes elements ........................................36

2.7 Measurements and label ranking for thin-sectioned manus elements. ..................................37

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LIST OF FIGURES

2.1 Histological map of Alligator mississippiensis. Bone areas colored in green indicate highest

preservation of skeletochronological information. .........................................................................7

2.2 Section through nasal bone......................................................................................................9

2.3 Section through angular bone. ..............................................................................................10

2.4 Section of chevron removed from caudal vertebra #4. .........................................................11

2.5 Section of vertebral rib. . .......................................................................................................12

2.6 Section of scapula..................................................................................................................16

2.7 Section of coracoid.………….……………………………………………………………..17

2.8 Section of ishium.. . ...............................................................................................................18

2.9 Section of ilium. ....................................................................................................................19

2.10 Section of pubic shaft. ...........................................................................................................20

2.11 Section of femur. ...................................................................................................................21

2.12 Section of tibia.......................................................................................................................22

2.13 Section of fibula. ...................................................................................................................23

2.14 Section of humerus. ..............................................................................................................24

2.15 Section of radius. ...................................................................................................................25

2.16 Section of ulna.......................................................................................................................26

2.17 Section of radialis. .................................................................................................................27

2.18 Section of pisiform bone. .....................................................................................................28

2.19 Section of metatarsal, Digit #1. ............................................................................................29

2.20 Section of bone in claw, Digit #4. ........................................................................................30

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ABSTRACT

Neontological studies of reptilian growth are important as they provide a proxy allowing

investigation of the life history of extinct relatives. As such, finding modern correlates for bone

growth and histological types that can then be used in a skeletochronological capacity are

important for unraveling prehistoric mysteries that involve the growth of the extinct relatives of

modern taxa. Most skeletochronology studies have focused on the lines of arrested growth, or

LAGs, generated in the femur, as femoral size is large enough to study in even small reptiles and

round in cross-sectional shape such that growth occurs evenly in all directions in the transverse

plane of reference. No single study has yet to section every bone type in the body of an animal.

This would allow for exploration of the extent to which other elements preserve the growth

record and provide alternative elements to study growth, which may prove useful when the

femur is not available. Modern archosaurs such as crocodilians, and in particular the American

alligator (Alligator mississippiensis), not only allow for not only an interesting modern system

but also provide a proxy for the past given their close evolutionary ties to fossil archosaurs such

as dinosaurs. With the goal of generating a histological map of elements useful for aging in

archosaurs, I conducted a histological analysis of an alligator previously in the care of the Florida

Fish and Wildlife Commission was conducted. A representative of every type of bone was

sectioned at multiple points and chemical label counts and tissue type characterizations were

made. Aside from major long bone elements commonly used in histological studies, other

skeletal structures such as ribs and phalanges exhibit areas of excellent LAG deposition that

make them potentially useful in skeletochronologic analysis. From this data a “map” of the

alligator skeleton was constructed regarding where along skeletal elements researchers are likely

to find unobstructed deposition of LAGs. The results of this study elucidate which bones are best

suited for analysis, as well as where along those bones information is preserved. Because

histological analysis is a destructive technique, this information will allow researchers to make

more informed decisions with regards to which skeletal elements to sample, thus reducing the

potential for damaging more elements than is necessary. This will also open the possibility to age

partial skeletons in which the femur is missing or unavailable for sampling.

1

CHAPTER ONE

INTRODUCTION

Studies of reptilian growth are important for two reasons; not only do they allow us to

better understand the biology of modern day taxa, they also allow for a proxy for their prehistoric

ancestors. If the growth rates and life histories of extant archosaurs was better understood, our

understanding of the life spans and maturity rates of extinct archosaurs would be greatly

enhanced. Vertebrate fossil remains are often limited to bones and teeth. As such, finding

modern organisms with similar bone growth and histological types is important for unraveling

questions involving these ancestors of modern taxa. However, detailed studies regarding the

growth of these modern organisms are required to answer such questions and often entail

arduous mark and recapture methods (McIlhenny, 1934; Chabreck & Joanen, 1979; Brandt,

1991).

Modern archosaurs such as crocodylians, and in particular the American alligator

(Alligator mississippiensis), allow for not only an interesting modern experimental system but a

proxy for the past given their close evolutionary ties to fossil archosaurs, such as extinct

crocodylians and dinosaurs. The crocodylian lineage itself dates back to the Late Cretaceous

(roughly 85 million years ago) and has, on the whole, conserved its body form over time

(Brochu, 2003). Alligators are also an easy resource for study given their availability. The yearly

deposition of growth lines in their bones is common among reptiles (Castanet, 1994). Among

fossil archosaurians (including dinosaurs), lines of arrested growth, or LAGs, are apparent and

have been well documented (Horner, et al., 2000; Klein & Sander, 2008).

Though several studies have been done regarding growth and growth lines, much of the

focus of these studies has been on limited elements, such as major long bones like the femur

(Erickson & Tumanova, 2000; Horner, et al., 2000). Therefore, a study regarding the growth of

an organism where multiple elements were used to document the appearance of growth lines and

rates stands to provide a rich source of new and relevant data.

2

The focus of this study is to answer two primary questions. First, do bones other than

femora show utility for aging these reptiles? Second, is the skeletochronologic information in

long bones mirrored in other parts of the body? My goal is to determine not only which bones in

the A. mississippiensis are most appropriate for aging and reconstructing the animal’s growth,

but also where along those bones the growth rate of these animals can best be replicated and has

been preserved. Such knowledge would be extremely beneficial given that skeletochronology is

a rather destructive process and so limiting destructive steps as much as possible is often not only

wanted but also needed. It would also be beneficial given that fossil specimens are sometimes

missing elements (such as the femur). This information can then be applied to reconstructing the

growth of other extinct and extant taxa and could open the gateway for many more histological

studies that have previously not been attempted due to a lack of “appropriate” material.

3

CHAPTER TWO

HISTOLOGY OF ALLIGATOR MISSISSIPPIENSIS

Introduction

Skeletochronology (Castanet, et al., 1977) is a tool that has been more used in recent

years to study growth in fossil organisms as a result in the shift in paleontological studies from

collecting as many specimens as possible to actually learning about the life histories of these

organisms. It is a technique using the cross section of skeletal elements and counts the concentric

rings generated in the bone tissue layers of reptiles and amphibians to age the specimens. It also

allows researchers to make assessments of bone growth and histological types as well as

osteological links to environmental shifts (Erickson, et al., 2007). It has been used to estimate

growth and age in a variety of both extinct and extant taxa (Chinsamy, et al., 1994; Erickson,

2003). Bone is excellent for aging studies, as periods of arrested growth are easily

distinguishable from times where growth is actively occurring, generating stretches of

uninterrupted bone deposition separated by lines. These lines of arrested growth, or LAGs, are

deposited with a predictable frequency and can therefore be used for aging specimens (Castanet,

et al., 1977; Erickson, 2005).

Most skeletochronology studies on tetrapods have focused on the LAGs generated in long

bones, primarily the femur due to its large size. Currently no one study has sectioned a

representative of every type of bone in the body of an animal and so the scope of what elements

could be useful in histological studies is limited to major long bones such as the femur. The use

the American alligator (Alligator mississippiensis) in this study would be beneficial since these

animals are commonly used in comparative biology to represent typical reptilian, as well as basal

archosaurian growth (Erickson & Tumanova, 2000). The findings would allow researchers to

establish what bones show efficacy for aging that might be applied to other taxa as well as

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facilitate exploration of how scaling occurs on an intra-elemental basis, that is how elements

change their shape during an organism’s lifetime.

Materials and Methods

One female alligator was used in this study. It was raised by the Florida Fish and Wildlife

Commission (FF&WC: 4005 South Main Street, Gainesville, Florida 32601) at a research center

located in Gainesville, FL, USA, in outdoor pens. Pelleted food was regularly provided but the

animal was still exposed to environmental fluxes in climate. This is an important consideration

since wild alligators are more prone to fluctuating diets, parasite infestations, and disease, all of

which can affect bone growth (Coulson, et al., 1973; Joanen & McNeasse, 1976; Buffrenil,

1982). If an alligator not exposed to these fluxes had been used, an understanding of how these

animals are developing in their first decade of life would not be properly recorded with this

study. It should also be noted that although this specimen is female, this should bear little

consequence on the study overall as sexual dimorphism does not appear to occur until later in life

(Chabreck & Joanen, 1979).

While in the FF&WC’s care, the alligator was periodically administered intramuscular

injections of the fluorochromic “chemical labels”, Alizarin Red (Sigma-Aldrich Co. LLC., St.

Louis, MO). This dye mark the mineralization front in bones and teeth within hours of being

administered (Frost, 1958) and stain the bone that is being deposited during the time of injection

(up to 24 hrs), making regions of growth during that time period easily identifiable in cross

section and best viewed with the aid of fluorescent microscopy (Vilmann, 1969). They have also

been used in other histological studies regarding growth in alligators (Erickson 1996; Erickson,

et al. 2004) and penguin chicks (de Margerie, et al. 2003). Four injections were administered

over the lifetime of the animal with the first occurring roughly one year and five months after

hatching, the second at two years and seven months after hatching, the third at seven years and

three months after hatching, and the last being approximately six months before it was sacrificed,

seven years and ten months after hatching (Table 2.1). Alizarin Red is visible in the bone and

teeth of specimens even without the use of fluorescent microscopy. As such it can be assumed

that if all four labels of Alizarin Red are visible in thin sections of the specimen, the entire

growth record, that is growth zones and LAGs, should also be preserved, and a correct aging of

5

the specimen can be made. In addition to the labeling, regular yearly measurements of total

length, snout to vent length, and weight were also collected for this animals (Table 2.1). With

these measurements, growth rates, in terms of weight and total length, can be calculated for

every year of the animal’s life. The alligator was sacrificed or died during their eighth or ninth

year and were then kept frozen.

The specimen was provided to the Erickson lab at Florida State University for analysis.

The specimen was stripped of muscle and all bones were separated for future analysis. All bone

types of the specimen were thin-sectioned using a slow-speed bone saw (Buehler IsoMet 1000

Precision Saw) and analyzed under plain light (Olympus SZX12 Stereo Microscope) and

fluorescent microscopy (Leica MZ10F Stereo Microscope) to determine the presence of

fluorescent markers. Fluorescent microscopy was done at the Biological Science Image Resource

at FSU. Elements of the axial skeleton sectioned in this study include: the skull and right

mandible, cervical vertebra #4, thoracic vertebra #5, lumbar vertebra #3, caudal vertebra #4, the

chevron removed from caudal vertebra #4, and a vertebral rib. Elements of the appendicular

skeleton sectioned in this study include: the left scapula, the right coracoid, the right ilium,

ischium, and pubis, the right femur, tibia, and fibula, the left humerus, right radius and ulna, the

right calcaneum and astragalus, right radialis, ulnare, and pisiform bone, all digits of the right

pes, and all digits of the right manus. The skull was sliced in 10 mm sections, along the frontal

plane, in order to preserve tooth rows for future analysis. Long bones were sectioned in roughly

2 mm sections in the transverse plane. The transverse processes, neural spines, and centra of

vertebrate were sectioned in the transverse plane in 2 mm increments. Due to the size of the

bones in the manus and pes, only the metacarpals and metatarsals were sectioned in 2.0 mm

increments. All other bones (phalanges and claws) were simply cut in half. Measurements of

length and measurements of the skull were taken for every bone type following Dodson’s criteria

(1975) and can be found in Tables 2.2 through 2.7. Histological characterizations were made for

each of these bones following the terminology of Francillon-Vieillot, et al.(1990) so that later

comparisons can be made across other alligators and broader taxa. Once sectioned, bones were

ranked according to the number of chemical labels visible to determine a subset of bones that

preserve entire growth line records. Because the last two chemical labels only vary in their

injection dates by 7 months, there tended to be a rather solid line of Alizarin Red instead of two

distinct lines on the outside edge of the bones (see results). As such, anytime the results mention

6

having seen all four labels, they are actually stating that the first two and last set can be seen

(three lines). Though rare, some bones did exhibit two distinct lines at their periphery and these

occurrences were noted in the results. As such, the bones were ranked on a scale of Zero to

Three, where a Three indicates a bone in which the first two labels and the last set are visible, a

Two indicates a bone where the second label and last set are visible, a One indicates a bone

where only the last set of injections is visible (seen along the edge of the bone and again, is much

more solid in color than the other two labels), and finally a Zero is a bone with no chemical

labels remaining recorded. This information can be found in Tables 2.2 through 2.7. Bones

scoring at a Two or higher were then analyzed to see what regions of those bones show

preservation for the entire growth line record and where and to what degree information begins

to be lost. This information is expressed in the Results Section as a percentage of the bone’s total

length (relative to it’s proximal or distal ends, sometimes with reference made to the diaphysis,

middle portion, of that bone) to allow standardization to other specimens regardless of size. The

overall result is a map that future histological studies can use for navigating where it is

appropriate to section A. missisippiensis and presumably other archosaurs and reptiles to achieve

accurate recovery of skeletochronological information (Fig. 2.1).

7

Figure 2.1. Histological map of Alligator

mississippiensis. Bone areas colored in green

indicate highest preservation of

skeletochronological information (Scores of

Three). Yellow areas are slightly less

informative but still useful (Scores of Two).

Red areas are uninformative (Scores of One

or Zero). White areas are ones where

potential for aging are yet to be known.

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Results for Axial Skeleton

Analysis of the skull shows six areas that were scored as Threes (i.e. all four Alizarin Red

markers are visible). These include midway through the nasal bones (Fig. 2.2), through the

frontal between the orbits, along the jugal (posterior to the postorbital bar), and in the squamosal

(near the point of contact with the postorbital). As for the right mandible, the presence of four

fluorescent labels appears to be isolated to the surangular and angular (Fig. 2.3), above and

below the middle of the external mandibular fenestra. With the exception of the base of the spine

of lumbar vertebra #3 (scored Two), all elements were uninformative, ranked as Ones, and

appeared heavily remodeled. Remodeling is the result of calcium being broken down in the

diaphysis of the bone and being redeposited as the bone grows and changes shape. It is evident

that remodeling has taken place when growth zones and LAGs are only partially visible at the

center of a cross-section. It should be noted that the chevron removed from caudal vertebra #4

was scored as a Two. It showed the last two chemical labels approximately a quarter of the

length from the spine. The last three labels were present in the proximal 40% of the chevron

nearest its attachment to the centrum of caudal vertebra #4, including the hemal arch (Fig. 2.4).

This was also one of two bones in the specimen that actually showed distinct, separate lines for

labels three and four.

The vertebral rib has the potential for quite a bit of preserved information, compared to

the rest of the axial skeleton elements. While the first set of Alizarin Red labels was nonexistent,

the bone remained solid with little remodeling to near the top of the capitulum (Fig. 2.5).

Measurements and chemical label ranking for axial elements can be found in Tables 2.2

and 2.3.

9

Figure 2.2. Section through nasal bone in regular light. Scored as a Three, fluorescent markers

are labeled with arrows. The first marker is labeled in yellow, the second in green, and the third

and fourth in red.

10

Figure 2.3. Section through angular bone in regular light. Scored as a Three, fluorescent markers

are labeled with arrows (only three arrows as the last set of injections are so close). The first

marker is labeled in yellow, the second in green, and the third and fourth in red.

11

Figure 2.4. Section of chevron removed from caudal vertebra #4 viewed in regular light. Growth

zones are not clearly defined though the last three labels are present (and noted with arrows).

This was one of the few times in which the last set of markers were each visible meaning that

growth occurred between the last two injection dates. Some longitudinal vascularization is

present. Bone scored as a Two. The second marker is labeled in green, and the third and fourth in

red.

12

Figure 2.5. Section of vertebral rib viewed in regular light. Some longitudinal vascularization

present, though generally low. Remodeling at interior of bone has destroyed the first two

injections of labels. At least four zones of growth still present (with LAGs identified with white

bars). Bone scored as a One. Third and fourth fluorescent markers have been labeled a red arrow.

13

Results for Appendicular Skeleton

Though remodeling took place at the proximal and anterior ends of the left scapula, the

middle portion (approximately a fourth of the bone’s total length) scored as a Three due to the

presence of the second and last set of chemical labels. At the skinniest portion of the scapula,

under fluorescent microscopy, all four labels could be seen (Fig. 2.6). The left coracoid shares a

similar presence of Alizarin Red. The blade of the coracoid is also remodeled, as in the scapula,

and the last set of marker lines begin their visibility roughly a quarter of the way from the blade.

The inner 50% of the coracoid contains the most information; with the last three markers present

in roughly 40% of the bones total length from the scapular articulation surface (Fig. 2.7). With

the aid of fluorescent microscopy, all four labels can be seen at the inner 20% of the bone.

The right ischium, though heavily remodeled, did appear to have some bone with LAG

preservation at the middle 20% of the bone though the first set of chemical markers were not

visible (Fig. 2.8). The right ilium was the most uninformative of the pelvic girdle bones, as it was

heavily remodeled throughout, and only had one possible portion showing LAGs, that being

along the medial projection (Fig. 2.9). However, that region, though small, did contain all four

labels when viewed under fluorescent microscopy and so at least that part of the bone could be

potential useful and was scored as a Three. The pubic shaft of the right pubis is the single most

informative element in the girdle. Though the ends are heavily remodeled, the middle 40% of the

bone contains markers and visible LAGs. All four markers are distinct and separate and are

present at the thinnest portion of the bone, approximately 60% from the end of the pubic shaft

(Fig. 2.10).

Not surprisingly, the femur, tibia, fibula, humerus, radius, and ulna all proved to be

highly informative and preserved a great amount of the growth sequence. The femur scored as a

Three (all labels were present) and all labels were visible in the middle 25% of the bone’s

diaphysis (Fig. 2.11). In the tibia, the first appearance of markers occurs in the middle 45% of

the bone, with all markers best represented at the middle 25% (Fig. 2.12). One of the most

exquisitely informative bones was the fibula, which contained not only clearly visible markers,

but also marker lines that retained their spacing around the entire bone. The fibula’s middle 35%

is where markers first appeared, and the middle 10% is where they best retained their shape (Fig.

2.13). The first two and last set of markers were present in the middle third of the humerus,

14

though some remodeling of the bone has begun and so rings do not travel around the whole bone

(Fig. 2.14). In the radius and ulna, we again see that the inner third of each bone has all marker

lines present (Fig. 2.15 and 2.16).

In contrast to the bastion of information preserved in the long bones of the limbs, the

calcaneum, astragalus, and ulnare were found to be useless for skeletochronological purposes.

This is because all of them are solely composed of spongy bone. Though the first set of Alizarin

Red lines were not visible in the radialis, the center of the bone had undergone minimal

remodeling and may still be useful for aging (Fig. 2.17). The pisiform bone, though heavily

remodeled on the ends, did have all marker lines present at the middle of the bone when viewed

under fluorescent microscopy and was scored a Three (Fig. 2.18).

Each digit of the pes (metatarsal, phalange(s), and claw) was sectioned. Overall, claws

were heavily remodeled, uninformative, and only the very outside edge contained any trace of

Alizarin Red. The rest of the elements in the pes (metatarsals and phalanges) did contain all of

the markers at the middle of the elements. The first digit’s metatarsal had four markers highly

visible starting 25% away from the distal end and ending about 40% from proximal end (Fig.

2.19). The first digit’s phalanx yielded the all markers. The second digit’s metatarsal had all

markers first visible at the middle 50% of the bone, it’s phalanges each only had all markers

visible in the dead middle of each bone. The third digit had all markers present in its metatarsal

starting 30% away from the distal end and ending at roughly 35% away from the proximal end.

The first (proximal) phalanx of the third digit had all markers present starting 27% from the

distal end and ending 45% from the proximal end. The last two (distal) phalanges of the third

digit both had all markers, again, at the middle of the bone. The fourth digit’s metatarsal was

informative in its middle 40% of the bone, containing all markers, while it’s most proximal

phalanx is informative with all markers only at its center, and it’s most distal phalanx has all

markers visible at it’s center, but are weakly visible.

The first digit’s metacarpal had all markers visible at its center. The same can be said for

the first digit’s phalanx. The claw of the first digit, however, was the same as that seen in the

claws of the pes, where only the very outside edge of the bone actually contained Alizarin Red.

This is also the case for the claws of the second, third, and fifth digits. The second digit’s

metacarpal was informative at its middle 40%, while its two phalanges had all four markers only

at their direct centers. The third digit’s metacarpal had all marker information best preserved at

15

it’s middle third. The third digit’s two most proximal phalanges also had all four markers present

at their diaphyses while it’s most distal phalanx only had the last set of Alizarin injections

visible, even at dead center. The fourth digit was unique in that its claw actually had both sets of

markers visible (Fig. 2.20). In fact, all of the bones in the fourth digit had all four markers

present. Within its metacarpal, markers appear at approximately 27% away from the distal end

and disappear roughly 50% away from its proximal end. Lastly, the fifth digit’s metacarpal and

phalanx both had all four markers visible throughout their diaphysis.

Measurements and chemical label rankings for appendicular elements can be found in

Tables 2.4, 2.5, and 2.6.

16

Figure 2.6. Section of scapula viewed in regular light (A) and under fluorescent microscopy (B).

Longitudinal vascularization, as well as some radial vascularization, present. Growth zones

visible (LAGs denoted with white bars), as well as chemical labels (arrows). Bone scored as a

Three. The first marker is labeled in yellow, the second in green, and the third and fourth in red.

Blue box in A denotes area enlarged in B.

17

Figure 2.7. Section of coracoid viewed in regular light (A) and under fluorescent microscopy

(B). Longitudinal vascularization common with some remodeling at center. Growth zones visible

(LAGs denoted by white bars), as well as chemical labels (arrows). Bone scored as a Three. The

first marker is labeled in yellow, the second in green, and the third and fourth in red. Blue box in

A denotes area enlarged in B.

18

Figure 2.8. Section of ischium viewed in regular light. Heavy remodeling throughout with radial

vascularization taking place along periphery. Some growth zones visible (LAGs denoted with

white bars), as well as last set of chemical labels (red arrow). Bone scored as a One.

19

Figure 2.9. Section of ilium viewed in regular light (A) and under fluorescent microscopy (B).

Again, heavy remodeling with radial vascularization along periphery. Some growth zones visible

(LAGs denoted with white bars), as well as chemical labels (arrow). Bone scored as a Three. The

first marker is labeled in yellow, the second in green, and the third and fourth in red. Blue box in

A denotes area enlarged in B.

20

Figure 2.10. Section of pubic shaft viewed in regular light. Moderate remodeling and radial

vascularization present though not in region where growth zones are highly visible (LAGs

denoted with white bars). Note presence of all chemical labels (arrows). The first marker is

labeled in yellow, the second in green, and the third and fourth in red. Bone scored as a Three.

21

Figure 2.11. Section of femur viewed in regular light. Remodeling has taken place in interior of

the bone. Longitudinal vascularization is prevalent. Growth zones clearly visible (LAGs denoted

by white bars). Note presence of all chemical labels (arrows). The first marker is labeled in

yellow, the second in green, and the third and fourth in red. Bone scored as a Three.

22

Figure 2.12. Section of tibia viewed in regular light. Remodeling at center with growth zones

highly visible (LAGs denoted by white bars). Longitudinal vascularization present. Note

presence of all chemical labels (arrows). The first marker is labeled in yellow, the second in

green, and the third and fourth in red. Bone scored as a Three.

23

Figure 2.13. Section of fibula viewed in regular light. Growth zones (LAGs denoted with white

bars) and labels highly visible. Note presence of all chemical labels (arrows). The first marker is

labeled in yellow, the second in green, and the third and fourth in red. Bone scored as a Three.

24

Figure 2.14. Section of humerus viewed in regular light. Growth zones highly visible (LAGs

denoted with white bars). Longitudinal vascularization prevalent. Note presence of all chemical

labels (arrows). The first marker is labeled in yellow, the second in green, and the third and

fourth in red. Bone scored as a Three.

25

Figure 2.15. Section of radius viewed in regular light. Growth zones visible (LAGs denoted with

white bars). Longitudinal vascularization present mostly along periphery. Note presence of all

chemical labels (arrows). The first marker is labeled in yellow, the second in green, and the third

and fourth in red. Bone scored as a Three.

26

Figure 2.16. Section of ulna viewed in regular light. Remodeling at center with primary osteons

visible along outer growth zones (LAGs denoted with white bars). Note presence of all chemical

labels (arrows). The first marker is labeled in yellow, the second in green, and the third and

fourth in red. Bone scored as a Three.

27

Figure 2.17. Section of radialis viewed in regular light. Outer growth zones present (LAGs

denoted by white bar). Large interior cavity has destroyed inner growth zones. Last set of

chemical labels denoted by red arrow. Bone scored as a One.

28

Figure 2.18. Section of pisiform bone viewed in regular light (A) and under fluorescent

microscopy (B). Second label weakly visible in A though growth zones are highly visible (LAGs

denoted with white bars). Bone appears to have a woven-fiber matrix. Chemical markers denoted

by arrows. The first marker is labeled in yellow, the second in green, and the third and fourth in

red. Bone scored as a Three.

29

Figure 2.19. Section of metatarsal viewed in regular light, The first digit of the pes. All markers

visible (arrows), as well as a fair number of growth zones (LAGs denoted with white bars). The

first marker is labeled in yellow, the second in green, and the third and fourth in red. Bone scored

as a Three.

30

Figure 2.20. Section of bone in claw of the fourth digit of the manus viewed in regular light (A)

and under fluorescent microscopy (B). Markers very weakly visible (arrows). Woven fibered

matrix throughout. Individual growth zones not visible.

31

Discussion

This work reaffirms the utility of skeletal elements previously used in histological

studies, such as the femur, which preserve an excellent growth line record in Alligator

mississippiensis. However, this work also shows evidence of preserved growth records in other

major long bones such as the humerus, ulna, radius, and fibula, as well as the scapula, coracoid,

pubis, pisiform, and phalanges. This evidence therefore broadens the scope of what skeletal

elements can be used in histological studies. Though the latter are not major long bones, they still

exhibit the high aspect ratio of length to width, which characterizes long bones. Long bone

growth is characterized by ossification taking place within cartilage, where the cartilage starts

degenerating and calcification begins (Enlow, 1963). The middle of the bone is often the best

place to find preserved growth records because new layers of bone are being added to the

periphery, resulting in a compacted bone with, in the case of archosaurs, rings. These bones also

all contained six to seven growth zones each meaning that nearly, if not all, of the growth record

can be extracted and the animal can be properly aged. Although the rib did not show the first set

of chemical labels, it did preserve nearly five full growth zones near its capitulum meaning that

most of the growth record of this animal can be found within a very small segment of the

element, and so could potentially be used for aging. However, the ribs are also under constant

pressure from the organism and have to change rapidly during development and it’s likely that

remodeling would end up destroying much of the growth record if this animal had lived any

longer. The cylindrical shape of long bones is likely the reason for why different bones varied in

their percentages of showing a useful signal. If a bone was more cylindrical and elongated, it

tended to have a higher the percentage of a useful signal within that bone. If a bone had an area

that suddenly flattens, such as the pubis, we see a decrease in the amount of bone that contains a

useful signal. Although these elements have not been used previously, likely due to them not

being particularly large, perhaps not always being available, or simply thought to have been

completely uninformative, they certainly have always contained the potential for

skeletochronological study with regard to their shape and development.

Though the skull did have points were growth was preserved, much of the skull remained

largely uninformative. The appearance of markers in the surangular and angular is perhaps a

32

result of them being some of the most robust bones in the dentary, and even they were victims of

heavy remodeling. It should also be noted that many of the elements that scored as Threes in the

skull and mandible were elements that were near fenestrae, and so had less structural pressure,

likely enabling them to grow in a more rounded shape, like that of long bones. This may be a

reason why those elements were able to capture the moments of dye injection though a more

thorough analysis of the skull is needed to confirm this statement. It’s also unlikely that if given

the option to section a skull or to section a phalanx, that the institution would choose to section

the skull. Most are prized possessions and are more likely to be put on display than elements of

the appendicular skeleton. However, a partial skull could still be useful along these points,

especially if the ends of these bones were already exposed.

All of these bones types are bones that are found in all archosaurs and so overall, this

study has the potential to vastly increase the knowledge base to pull from regarding what can and

cannot be used for skeletochronological study of archosaurs. Of course, like all science, there is

room for more. This study could be greatly enhanced with the addition of more alligators of

varying sexes and ages. As mentioned earlier, captive alligators are more prone to fluctuations in

their diets and are more susceptible to disease, which can change bone growth and deposition,

and although this alligator was kept in an outdoor pen and experienced climate fluxes, it was still

a captive animal. It would be interesting to see if purely wild alligators show the same signal in

their bones. Though not likely to change the types of bones that were found to be useful for

aging (as preliminary analysis of other alligators show the same elements to be useful),

quantifying inter-individual variation would help to verify which areas within these bones that

are consistently informative and provide further detail for the alligator map presented here.

Beyond that, the methods used here could just as easily be applied to a wider range of

crocodilian taxa to see if these results hold true. Further, it could prove interesting to apply these

methods to a diversity of herpetofauna. Not only could researchers compare and contrast the

utility of various elements for aging reptiles, but also study how elements change during the

lifetime of an animal.

33

Table 2.1. Specimen ID and measurements taken by the Florida Fish and Wildlife Commission

during the female’s lifetime. Measurements include total length, snout to vent length, and

weight, as well as the date all the measurements were taken and with what chemical label the

alligator was injected.

ID Total Length

(cm)

S.V. Length

(cm)

Weight (g) Date Label Comments

35905 25.0 Aug 31, 1994 Hatched; TL estimated

35905 64.5 30.0 750 Jan 31, 1996 Alizarin Red

35905 76.0 36.1 1100 Mar 11, 1997 Alizarin Red

35905 98.5 48.0 2675 Aug 12, 1998

35905 124.2 60.2 5800 Nov 2, 2001 Alizarin Red

35905 125.7 61.5 6000 Jun 12, 2002 Alizarin Red

35905 130.6 63.0 6500 Dec 11, 2002 Sacrificed

35905 Jun 21, 2003 Transferred to Greg Erickson

34

Table 2.2. Measurements and label ranking for thin-sectioned skull.

Skull

Measurements

(mm)

Label Ranking

Skull width at posterior border of external nares 51.30

Skull width at 4th

maxillary tooth 64.75

Skull width at anterior border of orbit 73.52

Skull width at posterior border of quadratojugals

(estimated based on half of skull still intact)

88.88

Skull width across occipitals (estimated based on half

of skull still intact)

50.06

Skull length from posterior border of orbit to external

condyle of quadrate

41.00

Maximum depth of jaw 29.20

External mandibular fenestra length 37.20

External mandibular fenestra width 12.90

Retroarticular process length, from crest of ridge

posterior to articular cotylus to tip of process

22.8

1: Though all the labels appear in

six sections of the skull, they do

not appear with enough frequency

to make them beneficial.

Throughout the skull, the most

prevalent set of markers were those

of the last two injections, on the

periphery. The exceptions to this

were the nasals, frontal jugal,

squamosal, surangular, and angular

which all scored as Threes (as

discussed in the results)

35

Table 2.3. Measurements and label ranking for thin-sectioned vertebral elements.

Table 2.4. Measurements and label ranking for thin-sectioned girdle elements.

Bone Type Length (mm) Label Ranking

Scapula (left) 42.2 3

Coracoid (left) 44.5 3

Length

(mm)

Width

(mm)

Ilium (right) 44.6 26.6 3 (only along medial projection)

Ischium (right) 44.4 23.3 1

Pubis (right) 38.7 19.1 3

Bone Type Vertebra

Length (mm)

Vertebra

Width (mm)

Vertebra

Height (mm)

Length

across

Transverse

Process

(mm)

Label Ranking

Cervical #4 16.00 11.41 34.76 31.35 1

Thoracic #5 17.67 12.17 26.73 52.33 1

Lumbar #3 20.59 13.81 30.20 44.02 2

Caudal #4 18.51 11.68 29.56 41.29 1

Chevron Height (mm)

Chevron

removed from

Caudal #4

29.40 2

Total Length (mm)

Vertebral Rib 54.0 1

36

Table 2.5. Measurements and label ranking for thin-sectioned limb elements.

Table 2.6. Measurements and label ranking for thin-sectioned pes elements. All are from right

pes. Numbers denote digit placement in phalangeal formula and letters denote proximal versus

distal phalanx, with “A” being most proximal.

Bone Type Total Length (mm) Label Ranking

Metatarsal #1 44.2 3

Phalanx #1 16.2 3

Claw #1 20.4 1

Metatarsal #2 48.2 3

Phalanx #2A 17.1 3

Phalanx #2B 12.0 3

Claw #2 16.6 1

Metatarsal #3 47.3 3

Phalanx #3A 18.0 3

Phalanx #3B 12.7 3

Phalanx #3C 9.8 3

Claw #3 14.7 1

Metatarsal #4 41.8 3

Phalanx #4A 14.9 3

Phalanx #4B 9.9 3

Claw #4 13.8 1

Calcaneum 21.9 0

Astragalus 19.5 0

Bone Type Total Length (mm) Label Ranking

Femur (right) 101.8 3

Tibia (right) 75.4 3

Fibula (right) 74.7 3

Humerus (left) 78.1 3

Radius (right) 58.7 3

Ulna (right) 63.9 3

37

Table 2.7. Measurements and label ranking for thin-sectioned manus elements. All are from right

manus. Numbers denote digit placement in phalangeal formula and letters denote proximal

versus distal phalanx, with “A” being most proximal.

Bone Type Total Length (mm) Label Ranking

Metacarpal #1 17.9 3

Phalanx #1 10.5 3

Claw #1 16.6 1

Metacarpal #2 21.5 3

Phalanx #2A 11.5 3

Phalanx #2B 8.3 3

Claw #2 14.1 1

Metacarpal #3 23.0 3

Phalanx #3A 10.5 3

Phalanx #3B 7.9 3

Phalanx #3C 6.2 1

Claw #3 11.5 1

Metacarpal #4 19.1 3

Phalanx #4A 8.7 3

Phalanx #4B 6.3 3

Claw #4 11.0 3

Metacarpal #5 11.5 3

Phalanx #5 7.3 3

Claw #5 11.1 0

Ulnare 9.8 0

Radialis 16.6 1

Pisiform 10.4 3

38

APPENDIX

FF&WCC LETTER

39

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42

BIOGRAPHICAL SKETCH

PERSONAL

Born 18 March, 1986; Newton, New Jersey

EDUCATION

B. S., Geology (2008) - Florida State University, Tallahassee, Florida.

Honors Thesis Title: Longevity and Growth Patterns in the Dromaeosaurid Velociraptor

mongoliensis Osborn Inferred from Long Bone Histology.

TEACHING EXPERIENCE

Education Department Volunteer, Director of Education: Jennifer Golden. Tallahassee Museum,

Tallahassee, FL. (Summer 2010)

Graduate Teaching Assistantship. Animal Diversity (BIO2011L), Instructor: Walter Tschinkel.

Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2008,

Fall 2010)

Graduate Teaching Assistantship, Animal Diversity (BIO2011L), Instructor: Trisha Spears.

Department of Biological Sciences, Florida State University, Tallahassee, FL. (Summer

2010, Summer 2011)

Laboratory Coordinator, Comparative Vertebrate Anatomy (ZOO4343C), Instructor: Gregory M.

Erickson. Department of Biological Sciences, Florida State University, Tallahassee, FL.

(Spring 2010, Spring 2011)

Graduate Teaching Assistantship, Animal Diversity (BIO2011L), Instructor: Gavin J.P. Naylor.

Department of Biological Sciences, Florida State University, Tallahassee, FL. (Fall 2009)

Graduate Teaching Assistantship. Comparative Vertebrate Anatomy (ZOO3713C), Instructor:

Gregory M. Erickson. Department of Biological Sciences, Florida State University,

Tallahassee, FL. (Spring 2009)

Biology Teaching/Learning Workshop, Florida State University, Department of Biological

Sciences. 22 August, 2008. Workshop Director: Dr. Ann S. Lumsden.

43

RESEARCH EXPERIENCE

Graduate Student, Department of Biological Science, Florida State University; Tallahassee, FL,

August 2008 - Present.

Undergraduate Research Assistant, Department of Biological Sciences, Florida State University;

Tallahassee, FL, January 2006 - December 2007.

Fossil Preparation Assistant, Academy of Natural Sciences; Philadelphia, Pennsylvania, March

2005.

ACADEMIC AWARDS

Dean’s List at Florida State University, Tallahassee, FL (2006, 2007)

Phi Eta Sigma Honors Fraternity; Florida State University, Tallahassee, FL (2007)

GRANTS RECEIVED

Travel Grant, Council of Graduate Students, Florida State University, October 2009.

PROFESSIONAL SOCIETIES

Society of Vertebrate Paleontology

SCIENCE-RELATED ORGANIZATIONS

Florida State University Geological Society; 2004 – 2008. Elected to President; 2006, 2007.

Ecology and Evolution Research Discussion Group (EERDG). Florida State University,

Tallahassee, Florida; 2008 – Present. Elected to Secretary; 2009.

CONFERENCE PRESENTATIONS

Garcia, B.J., G.M. Erickson, K. Curry Rogers, and M.A. Norell. October 2007. Longevity and

Growth Patterns in the Dromaeosaurid Velociraptor mongoliensis Osborn Inferred from

Long Bone Histology. Society of Vertebrate Paleontology Annual Meeting, Austin,

Texas (Poster Presentation)