UNIVERSITY of CALGARY a Fossilized Turtle Egg Clutch With

Home , Adocidae, Adocus, Basilemys, Macrobaenidae, Nanhsiungchelyidae, Neurankylus

UNIVERSITY OF CALGARY

A fossilized turtle egg clutch with embryos from the Upper Cretaceous Oldman

Formation, southeastern Alberta: Description, taxonomic identity, and embryonic staging

Amanda Rea McGee

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF GEOSCIENCE

CALGARY, ALBERTA

JANUARY, 2012

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FACULTY OF GRADUATE STUDIES

The undersigned certify that they have read, and recommend to the Faculty of Graduate

Studies for acceptance, a thesis entitled "A fossilized turtle egg clutch with embryos from the Upper Cretaceous Oldman Formation, southeastern Alberta: Description, taxonomic identity, and embryonic staging" submitted by Amanda Rea McGee in partial fulfilment of the requirements of the degree of Master of Science.

Supervisor, Darla Zelenitsky, Department of Geoscience

François Therrien, Department of Geoscience

Jason Anderson, Faculty of Veterinary Medicine

External Examiner, Donald Brinkman, Royal Tyrrell Museum

External Examiner, Chris Sheil, John Carroll University

Date

ii Abstract

Fossilized turtle clutches containing embryos are heretofore undescribed. Here the description of such a specimen from the Upper Cretaceous of Alberta sheds light on the taxonomic identity and paleobiology of the turtle that laid the clutch. Examination of the clutch reveals 33 rigid-shelled, spherical eggs arranged in a layered cluster. Clutch size and the paleoenvironment of the nest site suggest that the fossil clutch belongs to a large, freshwater turtle. Numerous embryonic bones described from virtual reconstructions of computed tomographic data reveal that the embryonic bones are well-ossified.

Comparisons with modern turtles indicate the fossil embryos were at a late stage of development, near hatching, at the time of death. Morphology of the maxillae, dentary, and plastron suggests affinities with Adocidae, an extinct freshwater turtle clade.

Additional lines of evidence, including egg and eggshell morphology, the predicted size of the female, and geographic and stratigraphic location, also suggest that the fossil clutch belongs to adocids.

iii Acknowledgements

I have developed a substantial amount of gratitude to many individuals during my time at the University of Calgary, and would like to thank everyone who has helped me.

Firstly, I am grateful to my supervisor, Darla Zelenitsky, for providing me with such a valuable learning experience. Her guidance, knowledgeable comments, and feedback throughout the duration of my project has increased my passion for palaeontology, as well as improved my writing style. I greatly appreciate the time and effort she put into editing and improving each chapter of my thesis. I would also like to thank the members of my defence committee: Christopher A. Sheil, François Therrien, Donald Brinkman, and Jason Anderson for their invaluable advice in their respective areas of expertise, which greatly improved the quality of this thesis. I would like to thank Chris Sheil for providing me the opportunity to visit his lab at John Carroll University, and his continued advice throughout this thesis. Chris is an authority on turtle embryology and his expertise has greatly improved my understanding of the subject. I am very appreciative to Don

Brinkman for the valuable discussions we have had, and his knowledgeable advice in regards to fossil turtle taxa from Alberta, as well as access to said specimens. For input into the geological interpretation of the clutch site I thank François Therrien, and for access to unpublished revised radiometric dates I thank Dave Eberth. I am grateful to

Walter Joyce for his assistance in helping me navigate through the complicated subject that is turtle phylogeny. I thank Wendy Sloboda for collecting and preparing the fossil clutch that was the focus of this study, and I thank Matthew Colbert from the University of Texas, Austin, for micro-CT scanning the specimen. For their advice and guidance on the software program Amira, I am grateful to Jessica Theodore and Julian Divay. I would

iv like to extend my appreciation to the Royal Tyrrell Museum and the Biodiversity

Research Institute at the University of Kansas for access to fossil and extant turtle specimens, respectively. Additionally, I would like to thank the Royal Tyrrell Museum for access to their scanning electron microscope. For the preparation of eggshell thin sections, I would like to thank the staff at Calgary Rocks and Material Services Inc. For inspirational discussions, motivation, and unwavering support I would like to thank my parents, Chris and Laura McGee, Kaylee Anderson, Caleb Brown, Ashleigh Cadogan,

Shirley Cheng, Greg Dean, Chris Farrow, Dani Fraser, Melissa Freeman, Meg Gavilanez,

Kristina Koutras-Virlas, Magdalene Leung, Josh Ludtke, Chris McGarrity, Kelly

McGarrity, Heather McGee, Sheila McGee, Katie Quinney, Patty Ralrick, Jessica

Theodore, Cam Tsujita, Victoria Walker, and Melissa Zieglar. I would like to especially thank Kate Zubins-Stathopoulos, Annie Quinney, Kohei Tanaka, and Kirstin Brink; you guys rock (pun intended).

This research was supported by an Alberta Graduate Student Scholarship, a

University of Calgary Thesis Research Grant, the Grant Spratt Graduate Scholarship in

Geology, two Queen Elizabeth II Graduate Scholarships, as well as a teaching assistant position.

TABLE OF CONTENTS

Approval Page ...... ii Abstract ...... iii Acknowledgements ...... iv Table of Contents ...... vi List of Tables ...... viii List of Figures and Illustrations ...... ix List of Plates ...... x

CHAPTER ONE: INTRODUCTION ...... 1

CHAPTER TWO: CLUTCH, EGGS, AND EGGSHELLS ...... 4 2.1 Introduction ...... 4 2.2 Materials and Methods ...... 6 2.2.1 Collection and Preparation of the Clutch ...... 6 2.2.2 Analysis of the Fossil Eggs and Eggshells ...... 6 2.2.3 Comparative Extant Turtle Eggshell ...... 9 2.2.4 Creation of a Stratigraphic Column ...... 10 2.3 Geologic Setting and Paleoecology ...... 11 2.4 Description of Clutch, Eggs, and Eggshell ...... 17 2.4.1 Description of TMP 2008.27.1 ...... 17 2.4.2 Comparison of Fossil Eggs and Eggshell ...... 19 2.4.3 Description of Eggs and Eggshell of Extant Turtles ...... 22 2.4.4 Comparisons of Extant Turtle Eggs and Eggshell with TMP 2008.27.1 ...... 24 2.5 Discussion ...... 27

CHAPTER 3: EMBRYONIC REMAINS OF TMP 2008.27.1 ...... 41 3.1 Introduction ...... 41 3.1.1 Literature Review of Studies on Extant Turtle Embryos ...... 43 3.1.2 Sequence of Ossification ...... 45 3.2 Materials and Methods ...... 49 3.2.1 Computed Tomography ...... 49 3.2.2 Three-Dimensional Reconstructions of Embryonic Remains ...... 49 3.2.3 Description of Embryonic Remains ...... 50 3.2.4 Comparative Extant Turtle Embryos ...... 50 3.2.5 Comparative Fossil Turtles ...... 51 3.3 Description of Embryonic Elements ...... 53 3.3.1 Premaxilla ...... 53 3.3.2 Maxilla ...... 54 3.3.3 Prefrontal ...... 56 vi

3.3.4 Dentary ...... 57 3.3.5 Plastron ...... 58 3.3.6 Dorsal Ribs ...... 59 3.3.7 Humerus ...... 60 3.3.8 Femur ...... 61 3.3.9 Zeugopodial Elements ...... 61 3.3.10 Ilia ...... 62 3.3.11 Ischia ...... 62 3.3.12 Possible Vertebrae ...... 63 3.3.13 Phalanges ...... 63 3.3.14 Unidentifiable Elements ...... 63 3.4 Discussion ...... 666 3.4.1 Ossification and Staging of Embryos ...... 66 3.4.2 Mortality of the Embryos ...... 70 3.4.3 Taxonomic Affinity of TMP 2008.27.1 ...... 71

CHAPTER 4: CONCLUSIONS ...... 89

REFERENCES ...... 91

APPENDIX A: EGGSHELL CHARACTERS OF FOSSIL AND EXTANT TAXA ...... 104

APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES . 106

APPENDIX C: BODY SIZE OF THE FOSSIL TURTLE ...... 111 Introduction ...... 111 Materials and Methods ...... 112 Results ...... 116

APPENDIX D: REVIEW OF TURTLE EMBRYOLOGY STUDIES ...... 118

APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES ...... 121

vii

List of Tables

Table 1.1: Fossilized turtle eggshell fragments, isolated eggs, in situ clutches, and gravid turtles from the fossil record ...... 2

Table 2.1: Fossil and modern eggshell fragment specimens examined in this study ...... 7

Table 2.2: Eggshell thicknesses and preservation of each egg from the fossil clutch ...... 18

Table 2.3: Eggshell comparisons of TMP 2008.27.1 with two other gravid Adocus sp. specimens and a fossil clutch from China ...... 21

Table 2.4: Parataxonomic names of fossil turtle eggshell ...... 22

Table 3.1: Specimens of staged extant turtle embryos used for comparative purposes in this study ...... 51

Table 3.2: Bones identified in from eggs 4-6, 11, 12, 14, 22, 24-26, 29 and 30 and the predicted stage of development of each egg ...... 66

Table 3.3: Stage of development at which various bones ossify in six diverse species of extant turtle ...... 69

Table 3.4: Fossil turtle of the Judith River Group, Alberta, Canada ...... 72

Table A.1: References used to create phylogenetic tree in Mesquite ...... 115

viii List of Figures and Illustrations

Figure 2.1: The three different types of turtle eggshell (pliable, flexible, and rigid) in radial view ...... 5

Figure 2.2: Schematic drawing of the orientation of radial (red) and tangential (blue) sections through the eggshell...... 9

Figure 2.3: Map of Alberta showing approximate turtle clutch locality...... 12

Figure 2.4: Stratigraphy column of the formations of the Judith River Group ...... 13

Figure 2.5: Photograph of the outcrop in the Milk River Natural Area where the clutch was discovered ...... 14

Figure 2.6: Stratigraphic section of the fossil clutch site ...... 15

Figure 3.1: Fossilized turtle embryo in isolated egg from the Late Cretaceous of Mongolia ...... 41

Figure 3.2: Fossilized turtle embryo in isolated egg from the Late Cretaceous of Brazil ...... 42

Figure 3.3: Ventral surface baenid species Boremys pulchra and a fossil embryo from the clutch ...... 73

Figure A.1: Phylogenetic tree of 171 species of extant turtle taxa ...... 113

ix List of Plates

Plate 1: Photograph of the top view of fossil clutch ...... 29

Plate 2: Schematic drawing of top view of the fossil clutch ...... 31

Plate 3: Photograph of bottom view of fossil clutch ...... 33

Plate 4: Schematic drawing of bottom view of the fossil clutch ...... 35

Plate 5: Egg and eggshells of the fossil clutch ...... 37

Plate 6: Eggshell of extant turtle taxa ...... 39

Plate 7: Embryos from Eggs 1, 2, and 3 of the clutch ...... 77

Plate 8: Embryonic cranial elements ...... 79

Plate 9: Embryonic mandibular and plastral elements ...... 81

Plate 10: Embryonic plastral elements and dorsal ribs ...... 83

Plate 11: Embryonic limb bones ...... 85

Plate 12: Embryonic pelvic girdle elements and vertebrae ...... 87

1 CHAPTER ONE: INTRODUCTION

Turtle eggs and eggshell fragments are relatively common in the fossil record, although in situ clutches and embryos are rare; the latter of which have yet to be described (Table 1.1). Turtle eggshell has a unique acicular microstructure and aragonitic composition that allows it to be readily identified as turtle, even in the absence of skeletal material (Erben, 1970; Hirsch, 1983, 1996). However, associated skeletal remains, of either an embryo or a gravid female, allows for more precise taxonomic identification than the eggshell alone.

In this thesis, I describe an in situ clutch containing embryos, which was recovered from the Upper Cretaceous Oldman Formation of southeastern Alberta (Zelenitsky et al., 2008). The clutch (TMP 2008.27.1) was briefly described and assigned to the turtle Adocus sp. (Adocidae), although this taxonomic assignment was tentative as it was based on eggshell structure (Zelenitsky et al., 2008). The clutch also contains the remains of several fossilized embryos, which were not reported in the preliminary description by Zelenitsky et al. (2008). This study is the first to describe fossilized turtle embryos, determine their taxonomic affinity, and approximate their stage of embryonic development using extant turtle embryos as a proxy.

The objectives of this thesis are to: 1) describe the geology of the site where the fossil clutch was preserved in order to reconstruct the paleoenvironment in which the clutch was lain; 2) describe the clutch, eggs, eggshells, and embryonic elements of the fossil clutch; 3) assign the clutch and embryos to a turtle taxon based on characters of the eggs and skeletal remains; and 4) compare the fossil eggs and embryos to those of modern turtle species to determine the stage of embryonic development of the embryos and comment on the nesting habits of the fossil turtle that laid the clutch.

2 Table 1.1 - Reports of fossilized turtle eggshell fragments, isolated eggs, in situ clutches, and gravid turtles from the Mesozoic and Cenozoic. Asterisk indicates embryonic remains were reported or observed. Specimen Number of Eggs Age Location Egg1, 15, 17 2 Jurassic, Middle England Egg2, 15 1 Jurassic, Middle England Eggshell9, 19 n/a Jurassic, Late Portugal, Guimarota Gravid female22 4+ Jurassic, Late Germany, Solnhofen Eggshell11, 17 n/a Jurassic, Late USA, Colorado Clutch24, 29 ~27 Cretaceous, Early China (Albian) Egg4,11 1 Cretaceous, Early England (Albian) Eggshell25 n/a Cretaceous, Early Japan Egg10 1 Cretaceous, Late Hokkaido, Japan (Coniacian) *Clutch27 ~33 Cretaceous, Late Canada, Alberta (Campanian) *Clutch23, 28 13 Cretaceous, Late USA, Montana (Campanian) Gravid female31 ~25–30 Cretaceous, Late USA, Utah (Campanian) Gravid female27 ~19 Cretaceous, Late Canada, Alberta (Campanian) Eggshell14 n/a Cretaceous, Late Canada, Alberta (Campanian) Eggshell30 n/a Cretaceous, Late USA, New Mexico (Campanian) Egg18, 19, 29 3 Cretaceous, Late India (Maastrichtian)

3 Specimen Number of Eggs Age Location *Egg11, 13, 16, 29 2 Cretaceous, Late Mongolia, Oolongy- Ulan-Tsav *Egg20 1 Cretaceous, Late Brazil, São Paulo Egg29 1 Cretaceous, Late China Eggshell21 n/a Cretaceous, Late France Eggshell7, 11 n/a Paleocene Belgium Egg and n/a Oligocene Belgium, Gaimersheim eggshell7, 8, 29 Egg3, 6, 11, 15 1 Oligocene USA, South Dakota Egg4 1 Oligocene USA, Nebraska Egg12 6 Miocene France Egg6, 11, 15 0.5 Miocene USA, Colorado Eggshell19 n/a Miocene Czech Republic Eggshell7, 11 n/a Miocene Belgium Eggshell26, 29 n/a Miocene, Late Venezuela, Estado Falcon Egg4 7 Pliocene Ethiopia Egg and 2 Pliocene Canary Islands, Gran eggshell5, 11 Canaria Eggshell4 n/a Pliocene Ethiopia Eggshell15 n/a Pleistocene USA, Florida Sources: 1Buckman, 1859; 2Carruthers, 1871; 3Hay, 1908; 4Hirsch, 1983; 5Hirsch and Lopez-Jurado, 1987; 6Hirsch and Bray, 1988; 7Schleich and Kastle, 1988; 8Schleich et al., 1988; 9Kohring, 1990; 10Fukuda and Obata, 1991; 11Mikhailov, 1991; 12Kohring, 1993; 13Mikhailov et al., 1994; 14Zelenitsky, 1995; 15Hirsch, 1996; 16Mikhailov, 1997; 17Bray and Hirsch, 1998; 18Mohabey, 1998; 19Kohring, 1999; 20Azevedo et al., 2000; 21 Garcia, 2000; 22 Joyce and Zelenitsky, 2002; 23 Jackson et al., 2002; 24Fang et al., 2003; 25 Isaji et al., 2006; 26 Winkler and Sanchez-Villagra, 2006; 27Zelenitsky et al., 2008; 28Jackson and Schmitt, 2008; 29Jackson et al., 2008; 30 Tanaka et al., 2011; 31Knell et al., 2011.

4 CHAPTER TWO: CLUTCH, EGGS, AND EGGSHELLS

2.1 Introduction

Fossilized turtle eggs were initially identified 140 years ago (Carruthers, 1871), based on macromorphological features such as size, shape, and surface texture (Buckman, 1859; Carruthers, 1871; Hay, 1908). As spherical eggs are common among extant turtles, many small fossilized eggs of this shape were suggested to belong to turtles in the older literature (for review see Hirsch and Bray, 1988). Buckman (1859) described the first fossil turtle eggs, although he mistakenly ascribed them to a “teleosaurian” reptile because the eggs were ellipsoidal, rather than spherical in shape (Hirsch, 1996). Carruthers (1871) was the first to ascribe spherical fossil eggs to turtles and observed that the surface sculpturing was similar to that of modern turtle eggs. Later, Hay (1908) assigned spherical fossil eggs to a specific fossil tortoise species, Stylemys nebrascensis, based on similarities in size and shape to eggs of the modern tortoise Gopherus polyphemus. More recent studies on the fossil eggs originally described by Buckman (1859), Carruthers (1871), and Hay (1908) have examined the eggshell microstructure, and were able to confirm that these specimens belonged to turtles (Hirsch, 1996; Hirsch and Bray, 1988).

Examination of eggshell structure using microscopy is the current technique to identify turtle eggshell in the fossil record. One of the first detailed descriptions of turtle eggshell microstructure was conducted by Erben (1970), who found that the eggshell was unique and consisted of acicular aragonite crystals that radiate from the organic core to form the individual shell units that comprise the eggshell. Subsequently, Hirsch (1983) conducted a detailed comparative study of the microstructure of modern and fossil turtle eggshells and recognized three different types of eggshell (Fig. 2.1): 1) pliable, or parchment-like eggshell, composed of loosely arranged shell units that are wider than they are high, resulting in a collapsible eggshell (e.g., Cheloniidae and Dermachelyidae); 2) flexible, or moderately pliable eggshell, composed of shell units that are as high as they are wide and are arranged in groups, but are not interlocking (e.g., Chelydridae and most Emydidae); and 3) rigid eggshell composed of interlocking shell units that are higher than they are wide, thus forming a hard, non-collapsible egg (e.g., Carettochelyidae, Chelidae, Dermatemydidae, Kinosternidae, Testudinidae, and Trionychidae). Microstructural studies

5 of fossil egg fragments have allowed for the definitive assignment of many specimens to turtles (Table 1.1).

Figure 2.1. Three different types of turtle eggshell drawn to approximately the same scale. A) pliable or parchment-like eggshell of Lepidochelys kempi; B) flexible or moderately pliable eggshell of Chelydra serpentina; C) rigid eggshell of Geochelone elephantopus (Hirsch, 1983: Figure 2).

Egg clutches of extinct turtles have only recently been identified in the fossil record (Jackson et al., 2008; Jackson and Schmitt, 2008; Zelenitsky et al., 2008). Little is known about reproduction in extinct turtle taxa because few in situ clutches are known, and previous specimens could not be definitively assigned to a taxon or clade. One turtle clutch (TMP 2008.27.1), from the Oldman Formation of southeastern Alberta, has tentatively been assigned to the taxon (Adocus sp.), based on a comparison of its eggshell to the eggshell found within a gravid specimen of Adocus sp. (TMP 1999.63.2), also from the Late Cretaceous of southeastern Alberta (Zelenitsky et al., 2008). In this chapter, I describe the clutch, eggs, and eggshells, as well as the geological setting of this specimen. Comparisons are made to living turtle species in order to shed light on reproductive traits and nesting habits of an extinct turtle.

6 2.2 Materials and Methods

2.2.1 Collection and preparation of the clutch

The fossil turtle clutch (TMP 2008.27.1) was collected by Darla Zelenitsky and Wendy Sloboda in 2005. Subsequently, the clutch was prepared by Wendy Sloboda from both the top and the bottom. Fine hand tools, including dentistry tools, fine tipped needles, scalpels, and brushes were used to manually prepare the specimen under a binocular microscope. During preparation of TMP 2008.27.1, the specimen was divided into twelve blocks that have been labelled B1–B12 for the purposes of this study. Three eggs were revealed to contain embryonic elements during initial preparation (E1, E2, and E3). These eggs were removed from the clutch for further examination using micro-computed tomography (CT). The clutch was further prepared manually after the three eggs were examined using micro-CT.

2.2.2 Analysis of the Fossil Eggs and Eggshells

The number of eggs present in TMP 2008.27.1 was estimated based on the clustering of eggshell fragments in the matrix, as well of their curvature and orientation. The extent of each egg was documented by photographing both the top and bottom of the clutch. Each egg was then outlined and numbered using the brush tool in Adobe Photoshop CS4. As the majority of eggs in the clutch are crushed, egg diameter was measured from two relatively intact eggs using digital callipers. Egg volume was calculated using the maximum egg radius, and egg mass was calculated by multiplying egg volume by 1.13g/cm3, which is the calculated density of rigid-shelled eggs among extant taxa (Iverson and Ewert, 1991). Clutch mass was calculated as the product of clutch size and egg mass. Eggshell thicknesses for each egg in the clutch were measured using digital callipers, and eggshell thicknesses of fragments were measured in thin section using the measurement tool in Adobe Photoshop CS4.

Fragments of eggshell were removed from TMP 2008.27.1 for microscopic analysis. Radial and tangential thin sections of eggshell fragments from TMP 2008.27.1 were prepared by Calgary Rocks and Materials Services Inc., Calgary, AB (Table 2.1). Radial and tangential thin sections bisect the eggshell perpendicular and parallel to the surface,

7 respectively (Fig. 2.2). The eggshell fragments were embedded in clear epoxy in order to strengthen and reinforce the fragment before thin sectioning. One side of the fragment was polished and affixed to a glass slide before the other side was ground down and polished to the desired thickness. The thin sections were examined using a Leica DM2500 P petrographic light microscope, and photographs were taken using a Leica DFC290 HD digital camera. The thin sectioned eggshell fragments were examined under plane polarized light (PPL) and cross polarized light (XPL). The height and width of shell units were measured from digital images in Adobe Photoshop CS4.

Table 2.1 – List of eggshell fragments examined from fossil and extant taxa in this study, and the manner in which they were examined (e.g., SEM or thin sectional view). Family/Genus/Species Catalogue # Cross Sectional View/SEM Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-001-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-002-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-003-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-004-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-005-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-006-001 Tangential thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-007-001 Tangential thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-009-001 Radial thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-015-001 Tangential thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-016-001 Tangential thin section Adocus sp. (gravid) (TMP 1999.63.2) ZEC 059-017-001 Radial thin section Nest with embryos (TMP 2008.27.1) ZEC 352-001-001 Radial thin section Nest with embryos (TMP 2008.27.1) ZEC 352-002-001 Radial thin section Nest with embryos (TMP 2008.27.1) ZEC 352-003-001 Radial thin section Nest with embryos (TMP 2008.27.1) ZEC 352-004-001 Tangential thin section Nest with embryos (TMP 2008.27.1) ZEC 352-005-020 SEM Nest with embryos (TMP 2008.27.1) ZEC 352-006-001 Radial thin section

8 Family/Genus/Species Catalogue # Cross Sectional View/SEM Nest with embryos (TMP 2008.27.1) ZEC 352-007-001 Radial thin section Nest with embryos (TMP 2008.27.1) ZEC 352-008-001 Tangential thin section Nest with embryos (TMP 2008.27.1) ZEC 352-009-001 Tangential thin section Nest with embryos (TMP 2008.27.1) ZEC 352-010-001 Tangential thin section Carettochelyidae - Carettochelys sp. ZEC 293-001-001 Radial thin section Carettochelyidae - Carettochelys sp. ZEC 293-002-001 Tangential thin section Carettochelyidae - Carettochelys sp. ZEC 293-003-020 SEM Dermatemydidae - Dermatemys sp. ZEC 294-001-001 Radial thin section Dermatemydidae - Dermatemys sp. ZEC 294-003-020 SEM Kinosternidae - Claudius sp. ZEC 292-002-001 Radial thin section Kinosternidae - Claudius sp. ZEC 292-003-020 SEM Kinosternidae - Kinosternon integrum ZEC 308-001-001 Radial thin section Kinosternidae - Kinosternon integrum ZEC 308-002-020 SEM Kinosternidae - Kinosternon odoratum ZEC 289-001-001 Radial thin section Kinosternidae - Staurotypus triporcatus ZEC 354-001-001 Radial thin section Kinosternidae - Staurotypus triporcatus ZEC 354-002-020 SEM Kinosternidae - Sternotherus minor ZEC 255-001-001 Radial thin section Kinosternidae - Sternotherus odoratus ZEC 254-001-001 Radial thin section Trioncyhidae - Amyda cartilaginea ZEC 307-001-001 Radial thin section Trioncyhidae - Amyda cartilaginea ZEC 307-001-020 SEM Trionychidae - Apalone mutica ZEC 448-001-001 Radial thin section Trionychidae - Apalone mutica ZEC 448-002-020 SEM Trionychidae - Apalone spinifera ZEC 258-001-001 Radial thin section Trionychidae - Apalone spinifera ZEC 258-002-001 Radial thin section Trionychidae - Apalone spinifera ZEC 258-003-001 Tangential thin section Trionychidae - Apalone spinifera ZEC 258-004-020 SEM Trionychidae - Lissemys punctata KT 101-1 Radial thin section Trionychidae - Lissemys punctata KT 101-2 Tangential thin section

Figure 2.2. Schematic drawing of the orientation of radial (red) and tangential (blue) sections through the eggshell. After Mikhailov (1991).

Eggshell fragments were also examined using a Hitachi TM-1000 scanning electron microscope (SEM) at the Royal Tyrrell Museum (Table 2.1). Prior to scanning, the eggshell fragments were cleaned using a GemOro ultrasonic bath and were mounted on stubs so that the inner, outer, and radial surfaces could be examined and photographed.

The eggshell from TMP 2008.27.1 was compared to fossil eggshells from within an articulated gravid Adocus sp. specimen (TMP 1999.63.2), because TMP 2008.27.1 was tentatively assigned to Adocus sp. (Zelenitsky et al., 2008) based on preliminary comparisons of the two specimens.

2.2.3 Comparative Extant Turtle Eggshell

Because it was not possible to examine the eggshell microstructure from all extant turtle families for this project, those living taxa most closely related to adocids were examined for comparison (Table 2.1). Species of trionychids (Apalone spinifera, Apalone mutica, Amyda cartilaginea, and Lissemys punctata) and carettochelids (Carettochelys

10 insculpta) were the focus of the comparison because they are the closest living relatives to adocids (Danilov and Parham, 2006, 2008; Joyce, 2007). Kinosternids and dermatemydids were examined because morphological phylogenetic analyses generally place Kinosternoidea as the sister taxon to Trionychia, forming the superfamily Trionychoidea (Gaffney, 1975; Meylan and Gaffney, 1989); however, this sister group relationship is not supported by molecular analyses (Bickham and Carr, 1983; Shaffer et al., 1997; Cervelli et al., 2003; Fujita et al., 2004; Near et al., 2005; Parham et al., 2006). Eggshell from the clutch was not compared to that of Pleurodira, Testudinidae, Emydidae, Geoemydidae, or Platysternidae because there are no fossil representatives of these groups in the Late Cretaceous of Alberta (Eberth et al., 2001; Brinkman, 2003, 2005).

2.2.4 Creation of a Stratigraphic column

The upper six meters of section where the clutch was found was trenched in order to reveal a fresh, unweathered surface, and the thickness of each bed was measured using a tape measure and Brunton compass. Lithologic features (e.g., grain size, sorting, sedimentary structures, and erosional surfaces) and fossil occurrences (e.g., ichnofossils, bone fragments, and mollusc fragments) were documented, and a stratigraphic section was drafted.

11 2.3 Geologic Setting and Paleoecology

The fossil clutch (TMP 2008.27.1) was discovered in the Milk River Natural Area (MRNA) of southeastern Alberta, in the uppermost Oldman Formation of the Judith River Group (JRG) (Fig. 2.3).The Judith River Group is comprised of the Foremost, Oldman and Dinosaur Park formations (DPF) (Fig. 2.4), and represents an eastward-thinning clastic wedge that was deposited during the Late Campanian in the Western Canada Basin (Eberth and Hamblin, 1993; Hamblin, 1997). The Oldman Formation is fully terrestrial, lacking marine deposits, and records the maximum regressional phase of the Judith River Group (Eberth and Hamblin, 1993; Eberth et al., 2001). The formation is interpreted to have been deposited by a low sinuosity, ephemeral fluvial system (Eberth and Hamblin, 1993; Eberth, 2005). The sandstone deposits of the formation are interpreted to have been formed by shallow rivers (< 5 m deep) that were vertically aggrading and influenced by seasonal rainfall (Eberth and Hamblin, 1993), whereas the finer-grained deposits represent overbank or floodplain deposits (Eberth and Hamblin, 1993; Eberth et al., 2001). Small fossils, including egg and embryonic remains, are usually found in these finer-grained facies (Zelenitsky, pers. comm. 2011).

Figure 2.3. Map of Alberta showing approximate location of turtle clutch (TMP 2008.27.1) site.

Figure 2.4. Stratigraphy of the Judith River Group in the southern plains of Alberta. After Eberth and Hamblin (1993).

The fossil clutch was recovered from the ‘upper muddy unit’ of the Oldman Formation, the uppermost of three informally designated units of the formation (Hamblin, 1997; Brinkman et al., 2004). Approximately 70 m of the ‘upper muddy unit’ is exposed in the MRNA, and the fossil clutch was found in the upper few meters, close to prairie level (Fig. 2.5). The contact between the ‘upper muddy unit’ of the Oldman Formation and overlying Dinosaur Park Formation is not exposed in the MRNA, but is exposed near Onefour, Alberta, approximately 20 km northeast of the MRNA. In the Onefour area, a bentonite layer approximately 6 m above the Oldman-DPF contact has been radiometrically dated at around 74.9 ± 0.1 Ma, and a layer approximately 74 m below the contact was dated at around 76.2 ± 0.2 Ma (Eberth and Hamblin, 1993). These dates are currently under revision with the Oldman-DPF contact being 40Ar/39Ar radiometrically dated at around 75.86 ± 0.54 Ma, and a tuff at the base of the ‘upper muddy unit’ being radiometrically dated at approximately 76.40 ± 0.54 Ma (Eberth, pers. comm. 2011). As the nest was found

14 in the uppermost Oldman Formation, likely just a few meters below the Oldman-DPF contact, the age of the clutch is estimated to be close to 75.86 Ma.

Figure 2.5. Exposure in the uppermost Oldman Formation of the MRNA where the fossil clutch (black arrow) was discovered.

The section from which the clutch was collected represents a succession of overbank and channel deposits (Fig. 2.6). The coarse-grained deposits in the section consist of well sorted, fine- to very fine-grained lithic arenites, many of which contain fine laminations and small trough cross-beds. Additionally, many of the arenitic layers are fining upwards which, combined with the sedimentary structures, suggest they are fluvial deposits. The section is composed of alternating layers of siltstone and mudstone, and fine-grained and very fine-grained sandstone. The mudstone, siltstone, and sandstone layers contain rootlet traces, small calcareous nodules, burrows, and local concentrations of bivalve and gastropod fragments.

Figure 2.6. Measured stratigraphic section through the site of the fossil turtle clutch in the uppermost Oldman Formation.

16 The mudstone and siltstone deposits found in the measured section (Fig. 2.6), likely represent levee deposits. Most siltstone and mudstone layers contain features suggestive of subaerial exposure, including bioturbation, evidence of terrestrial plant growth in the form of rhizocretions, and the presence of poorly-developed calcareous paleosols. The aforementioned features are common in both floodplain and levee deposits; however, the presence of alternating fine-grained and coarse-grained layers in section is suggestive of levee deposits (Nichols, 2003; Boggs, 2006), as floodplains typically consist of thin sheets of fine-grained sediment that settle out of suspension (Nichols, 2003; Boggs, 2006). Though floodplains may contain fine sand if they are adjacent to rapidly flowing rivers, sand is usually less abundant than silt or mud. In contrast, levee deposits generally consist of horizontally-stratified fine sands overlain by laminated muds (Boggs, 2006).

The clutch was discovered in the middle of a 1.5 m-thick unit that consists of grey, structureless sandy siltstone. The unit features small burrows at its base, and contains bivalve, gastropod, and bone fragments in the lower half of the unit. The upper part of the unit contains two hadrosaur eggs, the distal portion of a hadrosaur femur, and small carbonate nodules. The unit is capped by a sideritic layer, and it should be noted that siderite is common in, but not restricted to, levee deposits (Dodson, 1971).

17 2.4 Description of Clutch, Eggs, and Eggshell

2.4.1 Description of TMP 2008.27.1

The clutch (TMP 2008.27.1) was preserved in a grey sandy siltstone that lacks evidence of sedimentary structures. Only one egg from the periphery of the nest was exposed in outcrop at the time of discovery, indicating that most of the clutch is preserved. During preparation, the specimen was separated into 12 different blocks (B1–B12) (Plates 1–4). The eggs and eggshell fragments of the clutch are cream-coloured and stained brownish-orange, which contrasts with the grey matrix and dark brown embryonic bone.

The clutch is oval-shaped in outline, approximately 43 cm wide X 32 cm long X 1.5– 4 cm thick, and contains approximately 33 in situ eggs (E1-E33) (Plates 1–4), which is a relatively large clutch size compared to modern turtle species (mean = 13.5 eggs; n = 161 species). Embryonic remains are present in E1–E6, E11, E12, E14, E22, E24–E26, E29, and E30. The eggs in the clutch are heavily crushed, although most eggs can be delimited by the position and orientation of the eggshell fragments. Portions of three eggs (E20, E27, and E31) are intact and retain the original spherical shape (Plate 5, Fig. A). Eggs 31 and 27 have approximate diameters of 4.10 cm and 4.25 cm, respectively. Egg volume and egg mass for a spherical egg with a diameter of 4.25 cm are estimated to be 40.19 cm3 and 45.42 g, respectively.

It is evident that the eggs in the clutch are arranged in two layers because E7, E9, E13, E15, E16, E18, E21, E23 and E24 are only visible from the top of the clutch (Plate 1 and 2), whereas E31, E32, and E33 are only visible from the bottom of the clutch (Plates 3 and 4). The clutch size estimate of 33 eggs is a minimum number because additional eggs may be present in the matrix between the top and bottom layers of the clutch. If additional eggs are present between the top and bottom layers, there are likely only a few because the amount of matrix remaining in the clutch is minimal. In cross section, B4–B9 and B11 exhibit multiple layers of superimposed eggshell, which either represent eggshells from a single, collapsed egg, or eggshell from collapsed superimposed eggs (Plate 5, Fig. B).

The eggshell from the clutch is rigid, and varies in thickness between and within individual eggs, ranging from 0.76–0.92 mm (n = 43) (Table 2.2). The outer surface of the

18 eggshell is smooth, lacks ornamentation, and individual shell units are not visible (Plate 5, Fig. C). The eggshell has domed shell units and a shell unit width that ranges between 0.13–0.31 mm, with an average width of 0.21 mm (n = 24). The shell unit height to width ratio ranges from 3.1–4.8, with an average of 3.5 (n = 24). In radial view, the shell unit margins are well defined and parallel, with lateral feathering along the outer half to third of the eggshell (Plate 5, Fig. D). The pore canals are straight and simple (Plate 5, Fig. E), with an approximate diameter of 26.4 µm (n = 11). Tangential thin sections show that the pores are rare and irregular in shape and distribution (Plate 5, Fig. F). Eggshell porosity calculated from tangential thin sections averages 0.48% (n = 4).

Table 2.2 – Eggshell thickness and state of preservation of eggs in the fossil clutch, TMP 2008.27.1. Asterisks indicates the presence of embryonic bone in the egg. Egg Block Eggshell Thickness Completeness (mm) 1* 1 0.80–0.81 scattered fragments 2* 2 0.80–0.85 clearly outlined 3* 3 0.76–0.86 scattered fragments 4* 4 0.84–0.87 clearly outlined, relatively uncrushed 5* 5 0.78–0.84 clearly outlined 6* 6 0.82 clearly outlined, relatively uncrushed 7 7 0.80–0.81 partially outlined 8 7 0.9 clearly outlined 9 8 0.8 clearly outlined 10 8 0.77–0.89 clearly outlined 11* 8 0.85 clearly outlined 12* 8 0.9 clearly outlined 13 8 0.81–0.9 clearly outlined 14* 9 n/a clearly outlined 15 9 n/a scattered fragments 16 9 0.92 scattered fragments

19 Egg Block Eggshell Thickness Completeness (mm) 17 9 n/a scattered fragments 18 9 n/a scattered fragments 19 9 0.79–0.85 clearly outlined 20 9 n/a two thirds of egg, uncrushed 21 9 0.84–0.89 partially outlined 22* 9 n/a scattered fragments 23 9 0.85 partially outlined 24* 9 0.82–0.84 partially outlined 25* 9 0.77–0.84 scattered fragments 26* 9 0.81–0.88 scattered fragments 27 10 0.78–0.79 nearly complete, uncrushed 28 11 0.81–0.85 partially outlined 29* 11 0.82–0.89 partially outlined 30* 12 0.78–0.81 clearly outlined, only half of egg 31 9 n/a nearly complete, uncrushed 32 9 0.78–0.85 partially outlined 33 9 n/a partially outlined

2.4.2 Comparison of Fossil Eggs and Eggshell

Eggs from the clutch are similar in size and shape to those preserved inside a gravid specimen of Adocus sp. (TMP 1999.63.2), recovered from the upper Dinosaur Park Formation (Campanian, Late Cretaceous) of southeastern Alberta (Zelenitsky et al., 2008) (Appendix A and Table 2.3). The shell units of the eggshell are higher than they are wide, and are feathered along the upper half of the shell unit margins (Zelenitsky et al., 2008: Figure 1d). The shell unit width of the eggshell from the clutch (range, 0.13–0.31mm; mean, 0.21) includes the range of the eggshell from the gravid specimen (range, 0.14–0.22; mean, 0.17). The eggshell from the gravid female is thinner (0.5–0.63 mm) than that of the

20 clutch (0.76–0.92 mm), and has a lower shell unit height to width ratio (2.3) than the eggshell in the clutch (3.5), which is likely due, in part, to the fact that the eggshell from the gravid specimen is not fully formed (Zelenitsky et al., 2008). Similarities between the eggs of the clutch and of the gravid specimen suggest that the clutch belongs to Adocus, as previously suggested by Zelenitsky et al. (2008).

A second gravid specimen of Adocus sp. containing eggshell was recently discovered from the Kaiparowits Formation (Campanian, Late Cretaceous) of Utah (Knell et al., 2011) and can also be compared to the clutch (Appendix A; Table 2.3). The eggs of the Utah specimen are similar in shape, although slightly smaller, with a diameter of 3.5 cm. The eggshell from the Utah specimen is also much thinner (0.25–0.28 mm) than the eggshell from the clutch and has a smaller shell unit height to width ratio (2.5). The eggshell from this gravid specimen also lacks domed shell units and the feathered structures along the upper shell unit margins, both of which are observed in the eggshell from the clutch (Knell et al., 2011: Figure 2d). The difference in these features suggests that the eggshell in the Utah specimen was in the very early stage of development and that the outer portion of the eggshell had not formed, making it difficult to compare with the eggshell of TMP 2008.27.1.

The eggs from clutch TMP 2008.27.1 were also compared to another clutch recently discovered from the Liangtoutang Formation (Albian, Early Cretaceous) of the Zhejiang Province of China (Appendix A and Table 2.3), which was assigned to the ootaxon Testudoolithus jiangi (Fang et al., 2003; Jackson et al., 2008). The two clutches have similar dimensions and exhibit layering of the eggs, with three layers of eggs observed in the Chinese clutch (Jackson et al., 2008). The eggs within the clutches are similar in size and shape, and the eggshell thickness (0.7–1.0 mm) of T. jiangi is comparable to that of the clutch from Alberta. Both of their eggshells consist of shell units that are higher than they are wide, although the shell unit height to width ratio is greater in TMP 2008.27.1 (3.5) than in the Chinese clutch (2.5–3.0). Neither lateral feathering along the upper margins of the shell units nor domed shell units are observed in the eggshell of the Chinese clutch (Jackson et al., 2008: Figure 2c), both of which are present in the Alberta specimen.

21 Table 2.3 - Comparison of eggs and eggshell of TMP 2008.27.1 with two other gravid Adocus sp. specimens and a fossil clutch from China.

Fossil Clutch Gravid Adocus Gravid Adocus Fossil Clutch (TMP sp. (TMP sp. (LBA-06-7)2 (ZMNH 2008.27.1) 1999.63.2)1 M8713)3 Egg shape Spherical Spherical Spherical Spherical Egg diameter (cm) 4.1–4.25 3.5–4.0 3.5 4.2–4.5 Eggshell thickness 0.76–0.92 0.5–0.63 0.25–0.28 0.7–1.0 (mm) Shell unit height: 3.1–4.8 2.3 2.5 2.5–3.0 width Shell unit width 0.13–0.31 0.14–0.22 0.10–0.11 0.20–0.35 (mm) Feathered shell units Yes Yes No n/a Domed shell units Yes Yes No n/a Sources: 1Zelenitsky et al. (2008); 2Knell et al. (2011); 3Jackson et al. (2008).

In comparison to fossil turtle eggs that have been assigned to a parataxon (Table 2.4), the eggs of the fossil clutch can be assigned to the oogenus Testudoolithus (oofamily Testudoolithidae), based on the interlocking adjacent shell units that form rigid eggshell. The eggshell is substantially thicker than that of T. rigidus (0.22–0.25 mm; Hirsch, 1996) and T. hirschi (0.15 mm; Kohring, 1999), but is comparable in thickness to T. jiangi (0.7– 1.0 mm; Fang et al., 2003; Jackson et al., 2008). Potential differences in the eggshell microstructure discussed previously between TMP 2008.27.1 and T. jiangi, however, preclude assignment of this specimen to T. jiangi, so the clutch is referred to as Testudoolithus sp.

22 Table 2.4 – Parataxonomic names assigned to fossil turtle eggs. Structural Oofamily Oogenus Oospecies Junior Morphotye Synonym Spherurigidis Testudoolithidae Testudoolithus T. rigidus (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) Spherurigidis Testudoolithidae Testudoolithus T. hirschi (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) (Kohring, 1999) Spherurigidis Testudoolithidae Testudoolithus T. jiangi Tiantaioolithus (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) (Jackson et jiangi (Fang et al., 2008) al., 2003) Spherurigidis Testudoolithidae Haininchelys H. curiosa (Hirsch, 1996) (Hirsch, 1996) (Schleich et al., (Schleich et 1988) al., 1988) Spherurigidis Testudoolithidae Testudinarum T. ovum (Hirsch, 1996) (Hirsch, 1996) (Schleich et al., (Schleich et 1988) al., 1988) Spherurigidis Testudoolithidae Testudinovum T. sp. (Hirsch, 1996) (Hirsch, 1996) (Mikhailov et al., (Mikhailov et 1997) al., 1997) Spheruflexibilis Testudoflexoolithidae Testudoflexoolithus T. Oolithes (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) bathonicae bathonicae (Hirsch, (Buckman, 1996) 1859) Spheruflexibilis Testudoflexoolithidae Testudoflexoolithus T. agassizi (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996) (Hirsch, 1996)

2.4.3 Description of Eggs and Eggshell of Extant Turtles

Turtles of the family Trionychidae lay rigid, spherical eggs that range from 20–35 mm in diameter, and generally have a clutch size ranging from 6–30 eggs per clutch,

23 depending on the species (Appendix A and B). The eggshell varies in thickness within the family, ranging from 0.10–0.37 mm. Trionychid eggs lack surface ornamentation and have a smooth outer surface (Ewert, 1979; Packard and Packard, 1979; Kohring, 1999). The outline of the shell units is visible on the outer surface of the eggshell (Packard and Packard, 1979; Ewert, 1985). Species examined here (A. spinifera, A. mutica, A. cartilaginea, and L. punctata) reveal that the shell units are relatively high, domal, and interlocking (Plate 6, Fig. A), except for A. mutica, which has interlocking, blocky shell units. Although not previously reported in trionychid eggshell, the shell unit margins of these species show lateral feathering of crystallites in the upper half of the eggshell (Plate 6, Fig. A). The shell unit height to width ratio varies among trionychid species examined within this study, ranging from 1.1 (A. mutica) to 2.3 (A. cartilaginea and L. punctata). Despite having similar shell unit height to width ratios, A. cartilaginea has thicker eggshell than L. punctata, with thicknesses of 0.327 mm and 0.261 mm, respectively. Pores are irregular in size and shape, and occur where four or more shell units intersect (Packard and Packard, 1979; Ewert, 1985). Near the inner surface, the shell units taper to a conical tip, which is embedded into the shell membrane (Packard and Packard, 1979; Ewert, 1985).

Turtles of the family Carettochelyidae lay rigid, spherical eggs that range in diameter from 36–46 mm, and have clutch sizes that range from 4–39 eggs (mean = 13 eggs; Appendix A and B). Carettochelyid eggs lack surface ornamentation and have smooth, although slightly dimpled, outer surfaces (Erben, 1970; Ewert, 1979; Webb et al., 1986). The margins of the shell units are visible on the outer surface of the eggshell, and pore openings are rare. The eggshell is slightly thicker than that of their sister taxon, Trionychidae, with thicknesses ranging from 0.43–0.45 mm. The shell units have parallel and distinct margins with lateral feathering in the upper half of the eggshell (Plate 6, Fig. B), similar to that of trionychids (Plate 6, Fig. A). Additionally, the shell units are columnal in shape and domed along the upper surface. The shell unit height to width ratio is similar to that of A. cartilaginea (2.1), and is much higher than the ratio of both Apalone species examined.

Turtles of the family Kinosternidae lay rigid, elongated eggs that range in size from 24–45 mm X 13–26 mm, depending on the species (Appendix A and B). Clutch sizes are

24 often between 2–5 eggs, but can range from 1–16 eggs, depending on the species. Eggshell thickness varies between 0.13–0.42 mm within the family, and the eggshell of kinosternids has a sculptured outer surface that is unique to the clade (Packard et al., 1984a; Packard et al., 1984b; Iverson and Ewert, 1991; Kohring, 1999). The morphology of the outer surface is variable with small, needle-like crystallites visible in some areas, and blocky crystalline material visible in other areas of the same egg (Packard et al., 1984a). Additionally, the shell unit margins are not visible from the outer surface, except where pores are present (Packard et al., 1984a; Packard et al., 1984b). The contact between shell units is parallel and distinct, and lacks the lateral feathering that was observed in the trionychids and carettochelyids (Plate 6, Fig C). Species examined in this study have blocky shell units (Claudius sp., Kinosternon intergrum, K. hirtipes, K. flavescens, K. odoratum, Staurotypus triporcatus, Sternotherus minor, Sternotherus odoratus), rather than the columnal, doming shell units of the trionychids (Plate 6, Fig C). These species also have a smaller range of variation in the shell unit height to width ratio than trionychids, ranging from 0.8 (K. intergrum) to 1.7 (S. tripocatus). On the lower surface, the shell units taper to a conical tip, which is embedded into the shell membrane (Packard et al., 1984a; Packard et al., 1984b; Iverson and Ewert, 1991).

Dermatemys (Dermatemydidae) lays rigid, elongate eggs that are 54.1–72 mm X 32.4–49.8 mm in size (Appendix A and B). Additionally, Dermatemys lays between 2–20 eggs per clutch, with an average of 15 eggs. The eggshell varies in thickness from 0.42– 0.45 mm. Unlike kinosternid eggshell, the outer surface is smooth and lacks surface ornamentation; however, like kinosternid eggshell, the shell unit margins are not visible on the outer surface of the eggshell, except where pores are present, which are rare (Ewert, 1979). Dermatemys eggshell has distinct, blocky shell units with parallel margins that do not feather laterally (Plate 6, Fig D). These shell units have a height to width ratio of 1.3, well within the range of kinosternids (Table 4.2).

2.4.4 Comparisons of Extant Turtle Eggs and Eggshell with TMP 2008.27.1

Eggs from the fossil clutch were compared to those of extant trionychians (Trionychidae + Carettochelyidae) because they are the closest living relatives to the extinct family Adocidae (Meylan and Gaffney, 1989; Danilov and Parham, 2006, 2008;

25 Syromyatnikova and Danilov, 2009), the clade to which the fossil clutch was tentatively assigned (Zelenitsky et al., 2008). Similar to the eggs of TMP 2008.27.1, trionychians lay rigid-shelled, spherical eggs that have a smooth outer surface that lacks ornamentation. In terms of clutch size, the fossil specimen is relatively large (33 eggs) compared to those of trionychians, many of which rarely lay clutches above 30 eggs (Appendix A and B). However, the basalmost extant trionychian (C. insculpta) can lay large clutches of up to 39 eggs. In terms of egg size, most trionychian eggs are smaller (2–3 cm) than those of the clutch (4.1–4.3 cm), although C. insculpta lays eggs similar in size to those of the clutch (3.6–4.6 cm) (Appendix A and B). Additionally, modern trionychian eggshell is much thinner (0.10–0.45 mm) than the eggshell from the fossil clutch (0.76–0.92) (Appendix A). Whereas C. insculpta eggshell is thin relative to the fossil clutch (0.43–0.45mm), it is among the thickest eggshell observed in extant taxa in this study. The shell unit height to width ratio of extant trionychians (range = 1.1–2.3) is also much less than that of the clutch (3.5), with A. cartilaginea and L. punctata exhibiting the largest ratio among the modern taxa, with a ratio of 2.3 in each species. Additionally, trionychian eggshell exhibits lateral feathering along the upper margin of the shell units, as well as doming of shell units along the outer surface of the eggshell, similar to eggshell from the clutch (Plate 5, Fig. D and E; Plate 6, Fig. A and B).

The eggs from TMP 2008.27.1 were also compared to eggs of Kinosternoidea (Kinosternidae + Dermatemyidae) because this taxon has traditionally been considered the sister taxon to Trionychia in most morphological phylogenetic analyses (Gaffney, 1975; Meylan and Gaffney, 1989; Joyce, 2007; Zelenitsky et al., 2008). The only similarity between the kinosternoid eggs and those of the fossil clutch is the rigid eggshell consisting of interlocking shell units (Plates 5, Fig. D and E; Plate 6, Fig. C and D). Kinosternoid eggs are elongate, unlike the spherical eggs of the fossil clutch and extant trionychians (Appendix A and B). Additionally, most kinosternoid eggshell exhibits whorled or ridged sculpturing on the outer surface of the eggshell, unlike the eggshell from the fossil clutch. Kinosternoids also generally lay small clutches, with an average of 2–5 eggs for kinosternids, and an average of 15 eggs for Dermatemys. Eggshell thickness varies in kinosternoids, with thicknesses that range from 0.13–0.42 mm in kinosternids and 0.42– 0.45 mm in Dermatemys, both of which are much thinner than that of the fossil clutch

26 (0.76–0.92 mm). Additionally, the shell unit height to width ratio of kinosternids (0.8–1.7) and dermatemydids (1.3) eggshell is also much less than that of the fossil eggshell (3.5). Furthermore, kinosternoid eggshell lacks the feathering along the margins of the shell units present in eggshell from the fossil clutch (Plates 5, Fig. D and E; Plate 6, Fig. C and D).

In summary, the eggs of the fossil clutch are most similar to the eggs and eggshells of trionychians, rather than kinosternoids. More specifically, the fossil eggs most resemble those of the basalmost extant trionychian, C. insculpta, which has relatively large, thick, spherical eggs that lack surface ornamentation. C. insculpta lays relatively large clutches, the range of which includes the clutch size of TMP 2008.27.1. The eggshell of C. insculpta also has parallel, distinct shell unit margins that feather laterally in the upper half of the eggshell, similar to the eggshell of the fossil clutch.

27 2.5 Discussion

Based on the geology at the site where TMP.2008.27.1 was discovered, the clutch was laid in a sandy siltstone matrix on a levee deposit, near a river. Based on the arrangement of the eggs, the fossil clutch was likely laid in a pit or depression. Despite being crushed, the eggs of the clutch are closely associated and stacked in at least two layers, an arrangement also reported in a fossil turtle clutch from China (Jackson et al., 2008). If the eggs were deposited on flat ground rather than in a pit or depression, they likely would not have retained the stacked arrangement. It was difficult to determine if the eggs were buried by the female because the nest structure is not preserved and the clutch is in a homogeneous sandy siltstone unit that extends approximately 50 cm below and 100 cm above the clutch. However, the close association of the eggs, as well as the large clutch size (n = 33 eggs), suggest the fossil clutch was buried because extant turtles that lay large clutches generally bury their eggs in closely associated, stacked layers (Ackerman, 1980; Seymour and Ackerman, 1980; Orenstein, 2001).

The fossil clutch likely was laid by a freshwater turtle because it has a clutch size more comparable to freshwater turtles (n = 33); terrestrial turtles tend to lay small clutches of large eggs (rarely over 20 eggs per clutch) and marine turtles tend to lay large clutches of small eggs (often over 100 eggs per clutch) (Wilbur and Morin, 1988; Elgar and Heaphy, 1989; Iverson, 1992; Orenstein, 2001). The hypothesis that the clutch was laid by a freshwater turtle, as opposed to a marine turtle, is also supported by the fact that the clutch was discovered within a levee deposit. Furthermore, the eggs of the fossil clutch are similar in size, shape, and eggshell microstructure to those found within a gravid Adocus sp. (TMP 1999.63.2), suggesting that the fossil clutch also belongs to the Adocidae, which were freshwater turtles (Danilov and Parham, 2006, 2008; Syromyatnikova and Danilov, 2009). In comparison to extant taxa examined, the eggs and eggshell of the fossil clutch are most similar to those of extant trionychians (feathering along shell unit margins, doming of shell units, clutch size), which are also freshwater turtles. This result is thus consistent with the assignment of the fossil clutch to a trionychian adocid. Among extant trionychians, the clutch size, egg size, egg shape, and eggshell microstructure of the fossil clutch and eggs are most similar to those of C. insculpta.

28 Based on clutch mass of extant taxa, the predicted carapace length of the fossil turtle that laid the clutch is approximately 71.4 cm (Appendix C). This body size is within the known size range of freshwater turtles (Iverson et al., 1993; Orenstein, 2001). It is also consistent with the carapace length reported for Adocus (Syromyatnikova and Danilov, 2009), although it is on the larger end of the range. Adocus, like many trionychians, are relatively large turtles and have been reported to reach sizes or 100 cm in Kazakhstan, although generally are between 40–65 cm (Syromyatnikova and Danilov, 2009). Therefore, estimated carapace length, as well as features of the clutch, eggs, and eggshell, indicates that the clutch most likely belongs to Adocus.

29 PLATE 1

Photograph of the top view of the fossil clutch (TMP 2008.27.1). Eggs 1–30 are outlined. Grey eggs are the eggs removed after initial preparation for micro-CT scanning. Scale is 5 cm.

Plate 1 30

31 PLATE 2

Schematic of the top view of fossil clutch (TMP 2008.27.1). Arrangement of the 12 blocks (numbered in black) and the position of eggs (1–30) are illustrated. Eggs labelled in red are only visible from the top of the clutch. Scale is 5 cm.

4 5 14 5 4 15 28 11 3 11 3 16 10 10 17 27 29 12 8 6 18 12 30 6 9 9 19 13 20 21 7 8 1 23 22 7 1

2 24 25 26

Plate 2 32

33 PLATE 3

Photograph of the bottom view of the fossil clutch (TMP 2008.27.1). Eggs are outlined. Scale is 5 cm.

34 Plate 3

35 PLATE 4

Schematic of the bottom view of fossil clutch (TMP 2008.27.1). Arrangement of the 12 blocks (numbered in black), and position of eggs 1–6, 8, 10–12, 14, 17, 19, 20, 22, 25–33 are illustrated. Eggs labelled in red are only visible from the bottom of the clutch. Scale is 5 cm.

4 28 11 4

31 14 29 10 5 27 3 3 5 32 11 12 30 19 20 10 12

9 8 6 33 6 1

1 8 22 26 7 25 2 2

Plate 4 36

37 PLATE 5

Figure A. Top view of a well preserved, spherical egg (E27, B10) from the clutch (TMP 2008.27.1). Scale is 1 cm.

Figure B. Cross sectional view of Block 8 (TMP 2008.27.1). Multiple layers of eggshell fragments either from multiple stacked eggs or a single egg are shown.

Figure C. Outer surface of the eggshell from the fossil clutch (TMP 2008.27.1). Pores are rare. Scale is 1 mm.

Figure D. Radial view (PPL) of the eggshell from the fossil clutch (TMP 2008.27.1). Outer surface is up. Note the lateral feathering of shell units (arrow) in the upper portion of the eggshell. Scale is 500 µm.

Figure E. Radial view (SEM) of the eggshell from the fossil clutch (TMP 2008.27.1). Outer surface is up. Note the two pores (arrows) bisecting the eggshell.

Figure F. Tangential view (PPL) the eggshell from the fossil clutch (TMP 2008.27.1). Pores are rare (arrows) and irregular in shape and distribution.

38 A B

C D

E F

Plate 5

39 PLATE 6

Figure A. Radial view (SEM) of Amyda cartilaginea eggshell (ZEC 307). Outer surface is up. Note the lateral feathering of shell units (white arrow), doming of the shell units, and simple pores (black arrow).

Figure B. Radial view (SEM) of Carettochelys insculpta eggshell (ZEC 293). Outer surface is up. Note the lateral feathering of the shell units (arrow), as well as doming of the shell units.

Figure C. Radial view (SEM) of Claudius sp. eggshell (ZEC 292). Outer surface is up. Eggshell has the blocky shell units, with parallel, distinct margins.

Figure D. Radial view (PPL) of Dermatemys sp. eggshell (ZEC 294). Outer surface is up. Eggshell has blocky shell units, with parallel, distinct margins. Scale is 200 µm.

A B

C D 40 Plate 6

41 CHAPTER 3: EMBRYONIC REMAINS OF TMP 2008.27.1

3.1 Introduction

Fossilized turtle embryos are extremely rare and all previous reports lack descriptions and taxonomic assignments (Mikhailov et al., 1994; Azevedo et al., 2000; Clouse, 2001; Jackson et al., 2002; Jackson and Schmitt, 2008). Previous reports of fossil turtle embryos include: an embryo in an isolated egg from the Ologoy-Ulan-Tsav locality (Late Cretaceous) of Mongolia (Fig. 3.1; Mikhailov et al., 1994); an embryo in an isolated egg from the Bauru Group (Late Cretaceous) of Brazil (Fig. 3.2; Azevedo et al., 2000); and embryonic remains in a clutch from the Judith River Formation (Upper Cretaceous) of Montana (Clouse, 2001; Jackson and Schmitt, 2008). These studies focused on the eggs and eggshell structure, and only mentioned the presence of embryonic remains, and thus require further study in order to identify and describe the skeletal elements.

Figure 3.1. Upper and lower view of a reported fossilized turtle embryo in an isolated egg from the Late Cretaceous of Mongolia that was described as well-developed (Mikhailov et al., 1994: Figure 7.19). Details of anatomy not published.

Figure 3.2. Reported fossilized turtle embryo in isolated egg from the Late Cretaceous of Brazil (Azevedo et al., 2000: Figure 5). A) Computerized tomography image of specimen; B) Schematic drawing of CT image. e: embryo, h: head, v: vertebrae.

The fossil clutch (TMP 2008.27.1) from the Oldman Formation of southeastern Alberta is only the fourth report of embryos inside fossilized turtle eggs. The clutch was tentatively assigned to the genus Adocus, based on similarities with the eggs and eggshell of a gravid Adocus specimen (Zelenitsky et al., 2008); however, the embryonic material was not examined in that study. Here, these embryos are studied and I provide the first detailed description of fossilized turtle embryos and assign them to a fossil turtle taxon. In order to aid in the description, identification, and staging of the embryonic remains of the

43 fossil specimens, comparisons are made with ossified embryonic elements of numerous species of modern turtles.

3.1.1 Literature Review of Studies on Extant Turtle Embryos

Turtle embryology has been studied for well over 150 years. Pioneers in the field include Rathke (1848) and Agassiz (1857), whose comprehensive studies describe the sequential appearance and development of morphological characters, including the formation and development of the organs, the nervous system, and the circulatory system. These authors focused on the early stages of development, primarily gastrulation and early organogenesis, with little emphasis on the later stages of development. Therefore, descriptions of the development of the skeletal system are cursory in early studies, with only a few regions of the skeleton examined, namely the limb bones, ribs, and vertebrae (Rathke, 1848; Agassiz, 1857). Many other early studies examined early aspects of embryonic development (e.g., migration of the germ cells, formation of the gonads; for review see Risley, 1933), and grouped embryos based on size and approximate age in order to document the sequential changes in morphology throughout development. In the latter half of the 20th century, studies began to increase emphasis on the later stages of embryological development, including differentiation of digits, formation of the carapace, and pigmentation of the eye, carapace, and scutes (Penyapol, 1958; Yntema, 1968; Domantay, 1968; Mahmoud et al., 1973; Crastz, 1982; Ewert, 1985; Miller, 1985). Additionally, these later studies recognised that the length of incubation is variable and therefore an unreliable index unless temperature is considered (Yntema, 1968; Mahmoud et al., 1973; Crastz, 1982; Ewert, 1985; Miller, 1985). These studies used various criteria to establish a relative sequence of arbitrary stages of development, which are essentially “the progressive appearance of discrete morphological characters” (Beggs et al., 2000).

The first researcher to establish an embryonic staging scheme for turtles was Yntema (1968), who developed a table of normal embryonic stages for the species Chelydra serpentina using timed periods of development at given constant temperatures. Body length and carapace length were measured at various time intervals throughout development, and 27 stages were established based on these measurements and on the duration of incubation. For each stage, Yntema (1968) identified novel features and changes in morphological

44 characters, such as the extent to which the lower eyelid covers the eye, pigmentation of the carapace, and forelimb morphology. A similar staging method was proposed by Mahmoud et al. (1973); however, morphological changes and novel features were used as the primary criteria to establish relative developmental stages, rather than duration of incubation and the size of the embryos. The methods used by Yntema (1968) and Mahmoud et al. (1973) are similar in that morphological features, incubation time, and embryo size are all documented for each stage; however, the primary criterion used to define the relative stages differs between the two methods. The morphological characters outlined by Yntema (1968) have since been used to stage embryos of turtle species from several families, including: Chelidae; Pelomedusidae; Cheloniidae; Dermochelyidae; Chelydridae; Trionychidae; Carettochelyidae; Kinosternidae; and Emydidae (Appendix D and references therein).

Although Yntema’s (1968) staging scheme for turtle embryos has been widely used as a model for staging other turtle species (Burke and Alberch, 1985; Webb et al., 1986; Burke, 1989; Rieppel, 1993; Guyot et al., 1994; Cherepanov, 1995; Beggs et al., 2000; Gilbert et al., 2001; Tokita and Kuratani, 2001; Greenbaum, 2002; Greenbaum and Carr, 2002; Sheil, 2005; Sheil and Greenbaum, 2005; Sanchez-Villagra et al., 2007a; Sanchez- Villagra et al., 2007b), applying a staging scheme specific to C. serpentina to other turtle species has proven problematic. Yntema (1968) established 27 stages for C. serpentina, although subsequent studies have revealed that other turtle species have a different number of embryonic stages when staged using the same methodology. For example, Chrysemys picta has 23 stages of development (Mahmoud et al., 1973), Lepidochelys olivacea has 31 stages (Crastz, 1982; Miller, 1985), and Pelodiscus sinensis has 23 stages (Tokita and Kuratani, 2001) (Appendix D). Another potential issue with staging embryos of various species, based on observations from a single species, is that morphological features may appear within a single developmental stage in one taxon, but occur over multiple stages in another taxon, thus confounding interspecies comparisons (Greenbaum and Carr, 2002). Another factor to consider when using a single species model for stage determination in other species is the variation in external morphology observed among taxa. For example, Greenbaum and Carr (2002) outlined external morphological criteria for the trionychid Apalone spinifera, following the morphological criteria of Yntema’s (1968) staging scheme. Trionychids, however, have a highly divergent morphology among turtles,

45 including a lack of carapacial scutes, reduced keratinized integumentary structures, and claws on only three of their digits (Cherepanov, 1995; Tokita and Kuratani, 2001), and thus lack some of the primary criteria used in Yntema’s staging series. Although morphological diversity among turtle species exists, forelimb skeletal morphology is highly conservative among turtle taxa, and forelimb characters span all the later developmental stages (Ewert, 1985; Greenbaum, 2002, Greenbaum and Carr, 2002; Sheil, 2003; Sheil and Portik, 2008). As such, forelimb morphology may be the most useful character for staging well-developed embryos and could be the basis of an all-inclusive staging scheme for turtles (Greenbaum, 2002; Greenbaum and Carr, 2002).

Recently, a new staging system for vertebrate embryos was introduced which attempts to redefine developmental events in a comparative approach, while avoiding “staging tables” based on “model organisms”, in order to avoid potential issues with sequence heterochrony (Werneburg, 2009; Werneburg et al., 2009). Essentially, the new system consists of a checklist of 104 developmental characters of external morphology that are common to all vertebrates. The developmental characters observed in a specimen, or several specimens within a single developmental event, are keyed into a “type-in-formula” that documents the character set at that “stage”. Thus, embryonic series are arranged in defined Standard Event System (SES) stages that are described and illustrated in a SES- formula. This system for “staging” embryos is still in its infancy so it has yet to be applied and tested using a number of turtle or other vertebrate species.

3.1.2 Sequence of Ossification

The study of the sequence of ossification of the skeletal system of turtle embryos is mostly recent (Shaner, 1926; Rieppel, 1993; Cherepanov, 1995; Gilbert et al., 2001; Sheil, 2002, 2003, 2005; Sheil and Greenbaum, 2005; Sanchez-Villagra et al., 2007a + b, 2009; Tulenko and Sheil, 2007; Sheil and Portik, 2008; Bona and Alcalde, 2009). Many studies that examine the sequence of ossification focus on a specific structural unit of the skeleton, such as the appendicular skeleton, or carapace and plastron, rather than the entire skeleton (Burke, 1989; Burke, 1991; Lee, 1993; Cherepanov, 1995; Gilbert et al., 2001; Nagashima et al., 2007; Sanchez-Villagra et al., 2007a +b; Moustakas, 2008; Sheil and Portik, 2008). Ossification sequences of the entire skeleton are known for C. serpentina (Rieppel, 1993;

46 Sheil, 2002; Sheil and Greenbaum, 2005), A. spinifera (Sheil, 2002, 2003), Eretmochelys imbricata (Sheil, 2002), Macrochelys temminckii (Sheil, 2002, 2005), P. sinensis (Sanchez- Villagra et al., 2009), and Phrynops hilarii (Bona and Alcalde, 2009) (Appendix E). For these species, the ossification of each skeletal element was correlated to a specific stage of embryonic development in order to determine its sequence of ossification.

Variation in the Sequence of Ossification of Turtles: Recent studies on the ossification of turtle embryos show that variations exist in the sequence of ossification among and within species, primarily in the skull bones (Appendix E; Sheil, 2002). The first bones to ossify in the skull are dermal; however, which dermal bone ossifies first differs between species. In E. imbricata, the prefrontal is the first bone to ossify, whereas in C. serpentina and M. temminckii the postorbital is the first to ossify (Sheil, 2002, 2005; Sheil and Greenbaum, 2005). Within the trionychids, there is considerable variation in the sequence of ossification of cranial elements, more than in the postcranial skeleton. In P. sinensis, the parietal ossifies several stages before the frontal (Sanchez-Villagra et al., 2009), whereas the frontal ossifies slightly before the parietal in A. spinifera (Sheil, 2002, 2003). Variation in the sequence of ossification of skull bones was also observed within a single species, C. serpentina, in two different studies. Reippel (1993) reported the ossification of the postorbital at Stage 17, and the ossification of the parietal, pterygoid, and squamosal at Stage 18. Later studies on C. serpentina report ossification of the aforementioned skull bones at Stage 19 (Sheil, 2002; Sheil and Greenbaum, 2005).

There is also variation in the direction of ossification of the dorsal ribs and cervical vertebrae between species. In A. spinifera, C. serpentina, M. temminckii, and E. imbricata, the dorsal ribs ossify at the midbody first, and then ossification progresses anteriorly and posteriorly so that dorsal ribs I and X are the last to ossify (Sheil, 2002, 2003, 2005; Sheil and Greenbaum, 2005). However, in P. sinensis ossification begins anteriorly and progresses to the posterior ribs (Sanchez-Villagra et al., 2009). The direction of ossification of the cervical vertebrae also varies between species, with A. spinifera ossifying in a rostral-caudal direction, and C. serpentina, M. temminckii, and E. imbricata ossifying in a caudal-rostral direction (Sheil, 2002, 2003, 2005; Sheil and Greenbaum, 2005). In most species, the centra are the first postcranial elements to ossify, followed by the neural arches

47 and ribs; the exception is P. sinensis, which ossifies plastral bones first (Sheil, 2002; Sanchez-Villagra et al., 2009).

There is also variation of ossification of the appendicular skeleton, with the pectoral girdle in A. spinifera beginning to ossify before the forelimb elements, and well before the pelvic girdle (Sheil, 2002, 2003). In contrast, both limb girdles and associated limb bones begin to ossify at around the same time in E. imbricata (Sheil, 2002). Even within Chelydridae, there is variation in ossification of the appendicular skeleton, with the stylopodial and zeugopodial elements of the forelimb beginning to ossify before the pectoral girdle in C. serpentina, and the forelimb and pectoral girdle ossifying at essentially the same time in M. temminckii (Sheil, 2002, 2005; Sheil and Greenbaum, 2005).

The patterns of ossification of the forelimbs also show great variation between turtle taxa, especially among phalangeal elements. In the manus, the degree of ossification is greatest in Digits II and III in A. spinifera, whereas it is Digit III in E. imbricata, and Digits III and/or IV in the chelydrids (Sheil, 2002; Sheil, 2003). Additionally, ossification of the digits proceeds proximally-distally in A. spinifera, and distally-proximally in the chelydrids (Sheil, 2002; Sheil, 2005; Sheil and Greenbaum, 2005). In E. imbricata, Digits II–IV ossify proximally-distally, whereas Digit I ossifies distally-proximally (Sheil, 2002).

Consistencies in the Sequence of Ossification of Turtles: Though heterochronies are prevalent in the ossification sequence of turtles, both inter- and intraspecifically (Appendix E), there are some patterns that are relatively congruent among taxa. Sheil (2002) compared species from three phylogenetically diverse families (Chelydridae, Cheloniidae, and Trionychidae) and demonstrated consistencies in the pattern of ossification of major structural elements, such as the skull, limb and limb girdles, and postcranial axial skeletons. It was observed that ossification of the skull always commences before that of the postcranial skeleton (Greenbaum and Carr, 2002; Sheil, 2002; Sheil, 2003; Sheil, 2005; Sanchez-Villagra et al., 2009). A common pattern observed among cranial bones is earlier ossification of the dermal bones relative to the endochondral bones, particularly the dermal bones of the upper jaw, skull roof, and orbital series (Sheil, 2002). Elements of the lower jaw ossify after most of the dermal bones of the skull table, palate, and upper jaw, including the maxilla, prefrontal, parietal, and squamosal (Sheil, 2002; Sanchez-Villagra et al., 2009).

48 Within the lower jaw, the dentary is the first element to ossify (Greenbaum and Carr, 2002; Sheil, 2002, 2003, 2005; Sanchez-Villagra et al., 2009). Among the endochondral bones of the cranium, the elements of the basal plate generally ossify before the bones of the posterior braincase (Sheil, 2002).

Numerous consistent trends have also been observed for the postcranial skeleton of turtle embryos. The dorsal ribs generally ossify before the caudal and sacral ribs (Greenbaum and Carr, 2002; Sheil, 2002, 2003, 2005; Sheil and Greenbaum, 2005; Sanchez-Villagra et al., 2009) . In terms of the pelvic girdle, the ischium is generally the last element to undergo ossification. The first element of the pelvic girdle to ossify varies depending on the species: the ilium ossifies first in A. spinifera, C. serpentina, and M. temminckii, whereas in the pubis begins to ossify first in E. imbricata (Greenbaum and Carr, 2002; Sheil, 2002, 2003, 2005; Sheil and Greenbaum, 2005). In the forelimbs, the pattern of formation of elements is relatively congruent in that the stylopodial and zeugopodial elements ossify in a proximal-distal direction, the metacarpals generally ossify before the carpals and unguals, Digits I-IV ossify axially-preaxially, and ossification of Digit V is delayed (Burke and Alberch, 1985; Sheil, 2002). In the hind limbs, the stylopodial and zeugopodial elements ossify proximally-distally, and the phalanges and metatarsals ossify first in the pes, followed by the tarsals and unguals (Burke and Alberch, 1985; Sheil, 2002). Digits I–IV ossify axially-preaxially with Digit III exhibiting the greatest degree of ossification, whereas Digit V delays ossification (Sheil, 2002).

In summary, consistencies exist in the pattern and sequence of ossification in extant turtle embryos, although there is variation between and within species. The consistencies, however, in the ossification sequences among extant species allows for them to be used as a proxy for staging fossil turtle embryos.

49 3.2 Materials and Methods

3.2.1 Computed Tomography

Three eggs containing embryonic skeletal material (E1, E2, and E3) were removed from the clutch for further examination using an XRadia MicroXCT scanner at the High Resolution X-ray Computed Tomography Facility at The University of Texas at Austin. Each egg was imaged and hundreds of cross-sectional jpeg images were produced for each egg with a high resolution dataset (1024 X 1024 pixel images). The slice interval between adjacent scans is 0.04255 mm.

3.2.2 Three-Dimensional Reconstructions of Embryonic Remains

Amira v. 5.2.2, a multifaceted software program for visualising, manipulating and illustrating biomedical data, was used to produce three-dimensional virtual reconstructions of the embryonic bones in E1, E2, and E3. Prior to analysis with Amira, the CT images for each egg were converted from 16 bit to 8 bit images in Adobe Photoshop CS4 to reduce the size of each data set. The images were then cropped in ImageJ v.1.44 to further reduce the size of the data sets. The data sets were reduced to the dimensions of 937 X 292 pixels, 1024 X 359 pixles, and 986 X 328 pixels for E1, E2, and E3, respectively. The data set for each egg was opened in Amira and initial isosurface reconstructions were produced, which are three-dimensional analogs of the bone surfaces that were contoured to a selected greyscale threshold value, and are rendered as a mesh of polygons. The isosurface reconstructions removed all of the surrounding matrix so the bones embedded in matrix could be viewed. However, many of the bones are overlapping and it was often difficult to delineate individual bones. Thus, accurate three-dimensional representations of individual skeletal elements were produced by using the image segmentation tool of the label fields module to outline the extent of individual elements. Voxels within a selected greyscale range were selected in three planes (x, y, and z) for all image slices that contained the elements of interest. An element was completely segmented in one plane initially, followed by a second, and then a third. Because many of the skeletal elements are overlapping in the eggs, the extent of individual bones was sometimes interpolated in order to prevent numerous bones from being isolated as one. Rotation animations of numerous elements

50 were produced by selecting the axis of rotation and degrees of rotation in the MovieMaker tool, under the Animation/Demo tab. In total, 73 elements were isolated, although some of these skeletal remains are unidentifiable fragments.

3.2.3 Description of Embryonic Remains

Embryonic skeletal elements were described from a combination of three-dimensional Amira renderings and observations under a binocular microscope. All eggs containing embryos were examined under a Leica M80 binocular microscope and were photographed using a Leica DFC290 HD digital camera. Skeletal elements were identified based on morphology and relative position to identifiable associated bones. Once the elements were digitally isolated in Amira, the individual elements were measured and described. Descriptive terminology for cranial elements follows Gaffney (1979), Meylan and Gaffney (1989), and Sheil (2002), whereas descriptive terminology for postcranial elements follows Meylan and Gaffney (1989) and Sheil (2002). Most dimensions of elements were determined using the measurement tool in Amira, with the exception of the right prefrontal of E3, which was measured using digital callipers.

3.2.4 Comparative Extant Turtle Embryos

To aid in the identification of elements and staging of fossil embryos, extant embryos of A. spinifera and C. serpentina were borrowed from The University of Kansas Natural History Museum and Biodiversity Research Center (Table 3.1). The embryos were obtained from commercial turtle farms in 2000 by C. Sheil, and were stained with Alcian Blue and Alizarin Red to determine the presence of cartilage and bone, respectively. Embryos in late stages of development were selected for this study because the fossil specimens exhibit a relatively high degree of ossification. In addition to accessibility, these taxa were chosen because A. spinifera is a modern trionychid, and thus part of the group that represents the closest living relatives of Adocus. Chelydra serpentina, although distantly related to trionychids, was also examined because it has been studied extensively and has a more general morphology in comparison to the highly derived trionychid A. spinifera. The embryos were examined under a Leica M80 binocular microscope under both reflected and transmitted light and were photographed using a Leica DFC290 HD digital camera.

51 Table 3.1 – List of staged extant turtle embryos from The University of Kansas Natural History Museum and Biodiversity Research Center used for comparative purposes in this study.

Species Stage of Development Catalogue Number Apalone spinifera Stage 18 KU 290131 KU 290123 Apalone spinifera Stage 19 KU 290134 KU 290136 Apalone spinifera Stage 20 KU 285124 Apalone spinifera Stage 21 KU 290149 KU 290151 Apalone spinifera Stage 22 KU 290152 Apalone spinifera Stage 23 KU 285128 KU 290153 Chelydra serpentina Stage 22 KU 290221 Chelydra serpentina Stage 23 KU 290216 Chelydra serpentina Stage 24 KU 290212 Chelydra serpentina Stage 25 KU 290208 Chelydra serpentina Stage 26 KU 290201

3.2.5 Comparative Fossil Turtles

Fossil adult turtle specimens from the Royal Tyrrell Museum in Drumheller, Alberta, were also examined to aid in determining taxonomic affiliation. Species recovered from the Judith River Group of Alberta, where TMP 2008.27.1 was discovered, were examined. Numerous type specimens were examined, including Judithemys sukhanovi (TMP 1987.2.1), Aspidertoides splendidus (TMP 1990.59.01), Plesiobaena antiqua (TMP 1986.036.0049), and Adocus sp. (TMP 1999.63.2). Additionally, descriptions of fossil specimens from the literature were used when fossil material was unavailable (Meylan and

52 Gaffney, 1989; Brinkman and Nicholls, 1993; Brinkman, 1998; Parham and Hutchison, 2003).

53 3.3 Description of Embryonic Elements

The majority of the embryonic bones within the clutch are found within eggs; however, there are also elements dispersed throughout the matrix of the clutch. Three eggs known to contain embryonic material after initial preparation (E1, E2, and E3) (Plate 7) were isolated and micro-CT scanned. Further preparation of the clutch revealed that several other eggs, including E4–6, E11, E12, E14, E 22, E24–26, E29, and E30, also contain embryonic remains (Table 2.2), and that the entire clutch contains hundreds of embryonic elements. Although the additional eggs containing elements were not prepared to the extent of E1, E2, and E3, nor were they micro-CT scanned, it was possible to identify some elements based on the size and shape of the bones, as well as their association to identified neighbouring bones. The description and identification of skeletal elements in the clutch is based mainly on elements preserved within eggs that have been micro-CT scanned (E1–E3) because the small size of the elements and the nature of preservation (i.e., crushing and overlapping of bone) made it difficult to describe elements in hand sample, even using a binocular microscope. Three-dimensional reconstructions of CT data enabled virtual elements to be magnified significantly, and to be isolated from the matrix and overlapping bone.

3.3.1 Premaxilla

A single premaxilla is present in E3 and is associated with the right and left maxillae and prefrontals (Plate 8, Fig. A). The element is imbedded in matrix with only the labial surface exposed; therefore, the triturating and posterior surfaces are not visible. The premaxilla is 1.23 mm wide and 1.72 mm high. The labial surface is sub-rectangular in shape, and bears two large nutritive foramina, and several smaller foramina (Plate 8, Fig.B). Because the triturating surface is not visible, even in CT reconstructions, a labial ridge and concavity posterior to the ridge are not apparent. Additionally, it cannot be determined if the element narrows posteriorly, even in CT reconstructions. There is a slight process or thickening along the medial surface of the element that is likely part of the suture with the other premaxilla. The premaxilla bears no evidence of the shape of the aperture narium externa, or of a median dorsal process separating the nares. Additionally, the element bears no evidence of contact with the vomer.

54 Premaxillae are paired in most turtles, but fuse late in development in trionychids and carettochelyids (Gaffney, 1979; Meylan and Gaffney, 1989); trionychids also have small premaxillae (Gaffney, 1979). With the exception of a few fossil turtles, including Proganochelys quenstedti, Palaeochersis talampayensis, Kallokibotion bajazidi, and meiolaniids, most turtles, including these fossil embryos, lack a premaxillary process dividing the apertura narium externa in two (Joyce, 2007). In most turtles, the premaxillae contact the maxillae laterally and the vomer posteriorly. In trionychids and carettochelyids, there is an opening in the palate between the premaxillae and vomer, called the foramen intermaxillaris (Meylan and Gaffney, 1989; Joyce, 2007) . The dorsal surface of the premaxillae form the floor of the fossa nasalis, and the ventral surface is the anteromedial portion of the cranial triturating surface (Gaffney, 1979). In many turtles, the labial surface of the premaxilla also often bears nutritive foramina, a labial ridge, and a concavity behind the labial ridge, similar to the triturating surface of the maxilla (Meylan and Gaffney, 1989).

In embryos of C. serpentina, the paired premaxillae are well ossified by Stage 19 and articulate with each other by Stage 24 (Appendix E; Sheil, 2002). In trionychids, the premaxilla ossifies later, with the first indications of ossification at Stage 21 and 22 for P. sinensis and A. spinifera, respectively (Appendix E; Sheil, 2002; Sanchez-Villagra et al., 2009). In A. spinifera, the premaxillae do not fuse medially until post-hatching, whereas the premaxillae fuse early and appear to ossify as a single plate in P. sinensis (Sheil, 2002; Sanchez-Villagra et al., 2009).

3.3.2 Maxilla

Both maxillae are present in E2 and E3, and only the right maxilla is present in E1. In all three specimens, the maxillae are complete and preserved with the labial surface exposed. The maxillae do not articulate with any other cranial bones, although those of E3 are closely associated with the premaxilla and both prefrontals (Plate 8, Fig. A). The maxillae are well ossified and have an average length of 6.25 mm. Each maxilla consists of three processes: the alveolar process, which extends ventrally and forms the labial ridge; the palatine process, which extends in the same plane as the palatines and forms most of the triturating surface; and the prefrontal process, which forms the anterior and anteroventral

55 margin of the orbit (Plate 8, Fig. C; Gaffney, 1979; Sheil, 2002). The maxillae have the greatest height at the prefrontal process, with an average height of 4.5 mm, and are shortest along the alveolar process, with an average height of 1.5 mm. The maxillae do not taper posteriorly along the alveolar process. The labial surface of each maxilla bears three ridges that extend vertically relative to the long axis of the element, at the level of the prefrontal and alveolar ridges. The ridges are most prominent at the anterior end of the maxilla and terminate ventrally as small projections on the ventral margin of the labial ridge (Plate 8, Fig. D). The alveolar process of the maxillae is deep and sharp, and has numerous nutritive foramina that are irregular in size and shape (Plate 8, Fig. D). The nutritive foramina of the maxillae are less prominent then those seen in the dentaries; however, their presence indicates that the rhamphotheca was present at the time of death. Small foramina served as nutrient canals for the rhamphotheca, and mark the extent of the keratinous beak. While numerous nutritive foramina occur in the maxillae, the canalis alveolaris superior, which supplies blood to the maxilla (Gaffney, 1979), was not observed.

The palatal aspects of the maxillae are embedded in matrix and thus can be viewed only via three-dimensional renderings of CT scans (Plate 8, Fig. C). The palatine process of each maxilla is fairly narrow, with a slight thickening anteromedially, at the level of the planum antorbitale. The palatal process shows no evidence of accessory lingual ridges; however, this could be an ontogenetic artefact, as the palatine process extends towards the palatines throughout development and forms later than the alveolar process (Sheil, 2002; Sheil, 2003). Additionally, the palatine process is very thin and delicate in extant turtle embryos, and its narrow width in the fossil embryos may therefore be a result of preservation.

The anterior margin of the prefrontal process of each maxilla, which is in contact with the prefrontals, likely form the lateral margins of the external nares. Though the maxillae are among the first elements to ossify (Stage 17–20) in extant turtles, they do not articulate with other bones of the dermatocranium until relatively late in development (Stage 22–25), depending on the species (Appendix E; Sheil, 2002; Sheil, 2003). The maxillae usually articulate with the palatines and prefrontals first, and then articulate with the vomer (Sheil, 2002; Sheil, 2003).

3.3.3 Prefrontal

The paired prefrontals are present and well ossified in E3 (Plate 8, Fig. A and E), with the dorsal surfaces exposed. The ventral surface of each prefrontal is embedded in matrix and can only be viewed via three-dimensional renderings. The prefrontals are slightly displaced and there is no evidence of contact between them. They are associated with the maxillae and the premaxilla, and are positioned slightly antero-posteriorly to the prefrontal process of the maxillae (Plate 8, Fig, A). They are sub-rectangular in shape, with an average width of 2.56 mm and an average length of 3.10 mm. The posterior margin of the prefrontals are irregular and have a V-shaped notch (Plate 8, Fig. E), whereas all other margins are regular. The anterior margin bears evidence of the apertura narium externa.

The prefrontals make up the anterior portion of the skull roof in most extant and many fossil turtles. The prefrontals articulate with each other along the midline, contact the prefrontal process of the maxilla anterolaterally, and contact the anterior margin of the frontal posteriorly (Gaffney, 1979; Sheil, 2002). Each prefrontal consists of two plates: a horizontal plate that contributes to the skull roof, and a descending process that articulates with the vomer and palatine (Gaffney, 1979). A slight descending process is observed in the fossil embryos that run antero-posteriorly along the lateral edge of each element, on the ventral surface (Plate 8, Fig. F). The anterior terminus of the process is longer, with a depth of 3.47 mm; the remainder of the ridge tapers posteriorly to a depth of approximately 2.84 mm, and likely represents the thickened dorsal margin of the orbit. The descending process is quite thin, or delicate, and its short length may be related to preservation or to an early stage of ontogeny. Additionally, the descending process shows some evidence of the dorsolateral margin of the fissura ethmoidalis.

In C. serpentina, the skull roof plate is robust and well ossified by Stage 18, but the descending process does not ossify until around Stage 23, after the prefrontals have contacted each other along the midline and established contact with the prefrontal process of the maxilla (Sheil, 2002). In trionychids, the descending process develops earlier than in

57 C. serpentina, at Stage 20+ in A. spinifera (Sheil, 2002), and Stage 21 in P. sinensis (Sanchez-Villagra et al., 2009).

3.3.4 Dentary

Both the right and left dentaries are present in E2 with the labial surface visible, and the lingual surface embedded in matrix. The dentaries are well ossified and have an average length of 6.5 mm, at an average height of 3.5 mm at the dorsal process (Plate 9, Fig. A). The dentaries are disassociated along the mandibular symphysis, and the right dentary is broken perpendicular to the triturating surface, approximately halfway along the length of the element.

The lower jaw of most turtles is formed by the dentary, angular, articular, prearticular, coronoid, surangular, and occasionally the splenial (Gaffney, 1979). Each element forms separately, with the dentaries ossifying first among all turtle species where the sequence of ossification has been examined (Appendix 5). The dentaries are the dominate bones in the mandible, forming nearly the entire triturating surface (Gaffney, 1979). The dentaries articulate with the other lower jaw bones posteriorly along a series of complex suture lines that interdigitate and overlap, so the internal points of articulation can often be more complex than those seen externally (Gaffney, 1979). Suture lines between individual bones are not visible in these fossil embryos; however, the size and shape of the lower jaw bones suggest that numerous elements are ossified (Plate 9, Fig. A, B, and C). A projection posterior to the dentary suggests that additional lower jaw bones were present and ossified, likely at least the coronoid and surangular. The articular, prearticular, and angular on the medial surface of the lower jaw are not readily identifiable because they are embedded in matrix, and the CT data renders the entire lower jaw ramus as one solid element, rather than a composite of multiple bones.

Each dentary curves dorsally at the anterior terminus; therefore, it appears that a dorsal projection may have formed after fusion along the symphysis. The triturating surface is not clearly developed in the fossilized embryos, but appears to be concave and lacks any apparent crenulations or cuspules. A well-developed labial ridge is present on the dorsal surface and extends to the level of the most dorsal margin of the dorsal process; the lingual ridge is absent. The sulcus cartilaginis Meckelii, a longitudinal groove along the medial

58 surface of the dentary, is well preserved in both rami (Plate 9, Fig. C). The groove is relatively narrow compared to adult specimens, therefore suggesting that the mandibles were well ossified.

The disassociation of dentaries in the fossil embryos is likely due to preservation because the mandibular symphysis synostotically fuses relatively early in development among extant taxa (Stage 20, 21, and 22, in A. spinifera, M. temminckii, and C. serpentina, respectively; Sheil, 2002; Sheil, 2003; Sheil, 2005; Sheil and Greenbaum, 2005), at around the same time that nutritive foramina become apparent. Nutritive foramina are present along the anterior portion of the lateral surface of the dentaries, indicating that the horny rhamphotheca covered these surfaces in life and that the dentaries were likely fused at the mandibular symphysis, but divided during preservation (Plate 9, Fig. A).

3.3.5 Plastron

Plastral bones are present in E1, E2, and E3, all of which are heavily ossified with no observable processes (Plate 9, Fig. D, E, and F; Plate 10, Fig. A and B). The epi- and entoplastron are not apparent in any prepared or imaged specimens. In E2, the plastral elements are in close association and are arranged in life position (Plate 9, Fig. E, Plate 10, Fig. A), and in E1 and E3 the elements are disarticulated.

In E1, both hyo-, hypo-, and xiphiplastral elements are present; however, the hyoplastral elements are displaced posteriorly relative to the hypo- and xiphiplastral elements (Plate 9, Fig. D and F). This could be due to the curvature of the developing embryo within the egg, which causes the hyo- and hypoplastral elements to be slightly angled relative to one another, rather than lying in a single plane (Sanchez-Villagra et al., 2009). The left and right hyoplastra are approximately 8.65 mm and 11.12 mm wide, respectively, with axillary buttresses of 7.61 mm and 6.71 mm long, respectively. The variation in width between the left and right elements may be a result of difficulty in delimiting the extent of individual bones from the CT data. The left hyoplastron is overlain by the right; therefore, the marginal edge of the hyoplastron was obscured and may be underestimated. The left and right hypoplastra are 11.70 mm and 11.82 mm wide by 4.75 mm and 4.22mm long at the inguinal buttresses, respectively. The xiphiplastra are 11.93

59 mm and 9.73 mm long. The anterior and posterior margins of the shorter xiphiplastron are not well preserved.

In E2, both xiphiplastra and the right hyo- and hypoplastra are present, and arranged in life position. The hyoplastron has a width of 14.09 mm and a length of 5.97 mm at the axillary buttress. The hypoplastron has a width of 15.21 mm and a length of 5.93 mm at the inguinal buttress. The xiphiplastra are 10.89 mm and 12.04 mm long, respectively. The right xiphiplastron is fragmented posteriorly in E2 (Plate 10, Fig. A). The xiphiplastra appear to broaden posteriorly and lack any evidence of an anal notch.

In E3, the plastral bones are broken, and with the exception of the right hypoplastron, it is difficult to identify individual plastral elements. The right hypoplastron is 12.45 mm wide, and the inguinal buttress is 5.54 mm long. Identification of the element as the right hypoplastron is aided by its close proximity to the right ilium, femur, tibia and fibula. Egg 3 also contains a large, flat element that is likely a fragment of either the hyo- or hypoplastron (Plate 10, Fig. B). The medial portion of the element appears to be missing, but it has an axillary or inguinal buttress, with a length of 8.28 mm.

In extant trionychids, the hyo-, hypo-, and xiphiplastra start to ossify before the epiplastra and entoplastron (Sheil, 2002; Sheil, 2003; Sanchez-Villagra et al., 2009). In contrast, the epiplastra and entoplastron ossify first in C. serpentina (Sheil, 2002) (Appendix E). The posterior plastral elements begin to ossify at Stages 18–19 in A. spinifera and P. sinensis, and the anterior plastral element begin ossification by Stages 20– 21 (Sheil, 2002; Sanchez-Villagra et al., 2009). By Stage 24, all of the elements of the plastron in A. spinifera are essentially fully ossified and resemble those found in an adult (Sheil, 2002). In comparison to the fossil embryos, the plastral bones in modern embryos are very lightly ossified, with observable processes and foramina (Plate 10, Fig. C and D).

3.3.6 Dorsal Ribs

Four associated dorsal ribs are present in E3, and what appears to be a single isolated dorsal rib is present in E2 (Plate 10, Fig. E and F). The ribs do not articulate with vertebrae in either specimen. The associated ribs in E3 are broadened dorsoventrally and vary in length. The shortest rib is broken and has a cumulative length of 13.94 mm; the longest rib

60 is approximately 17.91 mm. The dorsal ribs diminish in length anteriorly and posteriorly, and are longest in the mid-dorsal region of the body; however, as the embryonic remains are disarticulated in E3, it is not possible to determine if the shorter ribs are posterior or anterior. The single rib in E2 is relatively cylindrical (i.e., not broadened dorsoventrally) and appears to be less ossified than those in E3. Additionally, the rib is only 11.66 mm long, suggesting that it is either from an embryo that was in an earlier stage of development, or that the rib is not from the midbody. Because the element is anterior to the hyoplastron, and in close proximity to cranial bones and the right forelimb, it may be an anterior dorsal rib.

In extant turtle embryos, the dorsal ribs begin to ossify relatively late (Appendix E). They begin to ossify at Stage 20 in M. temminicki (Sheil, 2005) and at Stage 21 in A. spinifera and C. serpentina (Rieppel, 1993; Sheil, 2002; Sheil, 2003; Sheil and Greenbaum, 2005). Dorsal ribs ossify at the midbody first and then ossification progresses anteriorly and posteriorly in A. spinifera, C. serpentina, and M. temminicki (Sheil, 2002; Sheil, 2003; Sheil, 2005; Sheil and Greenbaum, 2005), whereas ossification begins anteriorly and progresses to the posterior ribs in P. sinensis (Sanchez-Villagra et al., 2009). The anterior ribs begin to ossify at Stage 21, and the posterior dorsal ribs have started to ossify by Stage 24. The broadening of the ribs and ossification of associated costal plates also progresses from the anterior to the posterior; however, they remain unarticulated until after hatching.

3.3.7 Humerus

A single humerus is present in each of E1, E2, and E3. All three humeri are fairly well ossified along the shaft, but the ends of the bones are less ossified (Plate 11, Fig. B). In E1, the left humerus is observed and is associated with the left hypoplastron. The element is 3.72 mm long and 1.48mm wide at midshaft.

In E2, the humerus, pelvic girdle elements, and plastral bones are closely associated and oriented in life position; therefore, the humerus can be identified based on relative position in addition to shape (Plate 11, Fig. A). In E2, it is the right humerus that is preserved just anterior to the right hyoplastron. The distal end of the humerus is in association with a partial ulna and radius. The humerus is 9.83 mm long, 1.57 mm wide at midshaft, and 4.26 mm at its widest point. It is a well ossified humerus, with portions of the

61 head and medial and lateral processes present at the proximal end. There is also an indication of the ectepicondyle and entepicondyle at the distal end of the element.

In E3, a single humerus is present, but the head of the element is not preserved, so it cannot be determined it is the right or the left humerus. It is associated with an ulna, and a second bone that could be a radius, although it is difficult to determine with certainty. The bone is 7.58 mm long, 1.51 mm wide at midshaft, and 3.08 mm at the widest point. The element appears to be fractured sagittally, perhaps due to compaction during burial (Plate 11, Fig. B).

In extant embryos, ossification of the long bones begins at Stage 19 for A. spinifera, C. serpentina, and P. hilarii, Stage18 for M. temminckii, and Stage 20 for P. sinensis (Greenbaum and Carr, 2002; Sheil, 2002; Sheil, 2003; Sheil, 2005; Sheil and Greenbaum, 2005; Bona and Alcalde, 2009; Sanchez-Villagra et al., 2009) (Appendix E). The ends of the shafts appear hollow, with the cortical bone present and cancellous bone absent (Plate 11, Fig. C). This is to be expected as perichondral growth proceeds at a faster rate than endochondral ossification among turtles (Carter, 1998).

3.3.8 Femur

A single femur is present in E3 and it is associated with a tibia and fibula. The femur is 4.97 mm long and 1.13 mm wide at midshaft. The head, trochanter major, trochanter minor, and intertrochanteric fossa are observed at the proximal end (Plate 11, Fig D).

In extant turtles, the femora and humeri generally begin to ossify within the same stage (Stages 18–20) (Greenbaum and Carr, 2002; Sheil, 2005; Sheil and Greenbaum, 2005; Bona and Alcalde, 2009).

3.3.9 Zeugopodial Elements

Though it is difficult to differentiate between the radius, ulna, tibia, and fibula (as only the mid shafts have ossified), zeugopodial elements can be identified in each of the three embryos. Among most tetrapods, the ulna is more robust than the radius, and the tibia is more robust than the fibula. Additionally, association with identifiable stylopodial elements aids in the identification of the zeugopodial elements.

62 In E1, a single element is present; this element is not very robust and may be either a radius or a fibula. It is elongated and cylindrical, but not large enough to be a stylopodial element with a length of 2.69 mm. It is not associated with any other limb bones, but is adjacent to the right hyoplastron; therefore, it is likely a radius.

In E2, a radius and ulna are associated with the right humerus (Plate 11, Fig. E), and the radius is 4.39 mm long and the ulna is 4.69 mm long. A prominent radial and ulnar ligament ridge for an interosseous ligament is observed. A third zeugopodial element is present in E2 that is not associated with a stylopodial element. The element is 5.03 mm and is of similar robustness to the ulna. Additionally, it located anterior to the plastral elements, thus suggesting it is the left ulna.

In E3, a forelimb zeugopodial element is associated with a humerus; however, because the humeral head is not preserved it cannot be determined if the element is a radius or ulna. Additional zeugopodial elements are associated with a femur in E3. The broader, more robust element is interpreted to be the tibia, with a length of 5.96 mm. The tibia possesses a prominent tibial crest, and its proximal end interacts with the distal end of the femur. The fibula is 5.35 mm long and is in close proximity to the humerus, running parallel to it.

In extant turtle embryos, all four zeugopodial elements ossify within the same stage: Stage 20 for A. spinifera and M. temminckii (Sheil, 2002; Sheil, 2003; Sheil, 2005); Stage 21 or 22 in C. serpentina (Rieppel, 1993; Sheil, 2002; Sheil and Greenbaum, 2005); and Stage 22 in P. sinensis (Appendix E; Sanchez-Villagra et al., 2009). Additionally, zeugopodial elements begin to ossify a stage or two after stylopodial elements, or within the same stage. The zeugopodial elements start to ossify in the same stage as the stylopodial elements in A. spinifera, a stage later in C. serpentina, and two stages later in M. temminckii and P. sinensis (Rieppel, 1993; Sheil, 2002; Sheil, 2003; Sheil, 2005; Sheil and Greenbaum, 2005; Sanchez-Villagra et al., 2009).

3.3.10 Ilia

Both ilia are present in E2 and a single ilium is present in E3. In E2, the ilia are closely associated with the posterior plastral bones and are anterodorsal to the xiphiplastra (Plate

63 11, Fig. A). They appear hollow at the ventral ends in three-dimensional renderings, as only the midshafts and dorsal processes are ossified, and the ventral ends are completely absent (Plate 11, Fig. F). The ilia measure 4.41 mm and 4.59 mm long and 1.47 mm and 1.39 mm wide at mid shaft, respectively. A fairly complete ilium is present in E3 that bears the posterior ilial process on the dorsal terminus. Additionally, the medial surface bears scars for articulation with sacral vertebrae and their ribs.

In extant turtle embryos the ilia and pubes begin to ossify in the same stage. In the trionychids ossification commences in Stages 21 and 24 for A. spinifera and P. sinensis, respectively (Sheil, 2002; Sheil, 2003; Sanchez-Villagra et al., 2009). In the chelydrids, ossification occurs in Stages 21 and 22 for M. temminckii and C. serpentina, respectively (Rieppel, 1993; Sheil, 2002; Sheil 2005; Sheil and Greenbaum, 2005).

3.3.11 Ischia

The ventral surface of the left ischium is visible in E2 (Plate 12, Fig A). It is in close association with both ilia, and is adjacent to the left hypoplastron (Plate 11, Fig. A). The lateral ischial process is conical and prominent, and cross section of this process is ovoid. The central ramus of the element is broad and flat (Plate 12, Fig. B). The ischial symphysis is observed and the margins indicate the presence of cartilage at the midline. Each process of the element is ossified, although the end of each process is not preserved and likely was cartilaginous at the time of death.

In the extant turtle A. spinifera, the ischia become well ossified by early Stage 21, whereas the distal processes of these elements remain primarily cartilaginous until after hatching (Sheil, 2002). In C. serpentina and P. sinensis, the ischia begin to ossify in (Stages 22 and 24, respectively; (Rieppel, 1993; Sheil and Greenbaum, 2005; Sanchez-Villagra et al., 2009). In A. spinifera and M. temminckii, the pubes begins to ossify before the ischia (Sheil, 2002; Sheil, 2003; Sheil, 2005) (Appendix E).

3.3.12 Possible Vertebrae

In E2, a structure has been isolated that appears to be articulated dorsal and sacral vertebrae (Plate 12, Fig. D). Individual vertebrae cannot be differentiated as centra are not apparent, and the entire structure appears to be a solid element, rather than a composite of

64 various bones. The structure is 12.13 mm long, and thickens at what has been interpreted to be the posterior end. Projections suggestive of neural arches indicate the presence of approximately eight dorsal vertebrae, and three sacral vertebrae. A groove is observed on the ventral surface of the element that runs the length of the element and may represent the neural canal and there is evidence of several intervertebral foramina, particularly between those of the middle and posterior-most regions. The structure is 12.13 mm long, and thickens at what has been interpreted to be the posterior end. In turtles, the dorsal and sacral vertebrae are akinetic as the neural spines are fused to, and dorsally invest, the neural plates, whereas the cervical and caudal vertebrae are kinetic. Therefore, it is unlikely that any cervical or caudal vertebrae are incorporated into the structure. It is interpreted that the thickening or widening of the structure at the posterior end represents the sacral region, and possibly portions of neural plates or the sacral ribs, which would have articulated with the ilia.

In extant trionychids the dorsal centra begin to ossify at Stage 19 (Sheil, 2002; Sheil, 2003; Sanchez-Villagra et al., 2009). In M. temminckii and C. serpentina the dorsal centra ossify in Stages 20 and 22, respectively (Rieppel, 1993; Sheil, 2002; Sheil 2005; Sheil and Greenbaum, 2005) (Appendix E).

3.3.13 Phalanges

All three embryos have small, isolated elements that may represent autopodial elements. In E1, there are at least four elements resembling phalanges proximal to the hyoplastron that may belong to the forelimb. Additional phalangeal elements have been isolated in E1, adjacent to the right xiphiplastron. Other eggs within the clutch contain unguals, and phalanges, most notably E5.

In extant turtle embryos the phalanges of A. spinifera and M. temminckii begin to ossify at Stage 20 (Sheil, 2002; Sheil, 2003; Sheil, 2005). In C. serpentina the phalanges begin to ossify at Stage 22 (Rieppel, 1993; Sheil, 2002; Sheil and Greenbaum, 2005), and in P. sinensis they begin to ossify at Stage 23 (Sanchez-Villagra et al., 2009).

65 3.3.14 Unidentified elements.

All three embryos have small, isolated elements that likely represent unarticulated vertebrae, autopodial elements, or cranial bones.

66 3.4 Discussion

3.4.1 Ossification and Staging of Embryos

This study is the first to describe fossilized embryonic remains of a turtle, and give insight into the anatomy and development of fossil turtle embryos. The fossil clutch TMP 2008.27.1 contains 33 eggs, 15 of which contain numerous embryonic elements clustered together. The other 18 eggs of the clutch do not appear to contain embryonic elements; however, as the elements are so small (many less than a millimetre in size), the remainder of the clutch should be micro-CT scanned to verify this. Of the three eggs that were CT scanned, 21 elements were identified in E1, 30 elements were identified in E2, and 26 elements were identified in E3. Nineteen different types of elements were identified in total (Table 3.2). The postcranial elements of TMP 2008.27.1, specifically the pelvic girdle elemenst, dorsal ribs, unguals, and sacral ribs, are the most useful for determining the stages of development of the fossil embryos because they ossify relatively late in embryos of extant taxa.

Table 3.2 - Bones identified from Eggs 4–6, 11, 12, 14, 22, 24–26, 29 and 30. All eggs, except 14 and 22, contained numerous bones, many of which were unidentifiable using the binocular microscope. The range in the minimum predicted stage of development is a result of interspecies variation in extant turtle taxa (see Table 3.3). Egg Identified bones Latest bone Minimum to ossify stage of development 1 right maxilla, right and left hyo-, hypo-, xiphiplastra, phalanges 20–23 humerus, radius, phalanges, possible additional cranial elements or disarticulated cervical or caudal vertebra, other unidentifiable bone

67 Egg Identified bones Latest bone Minimum to ossify stage of development 2 both maxillae, both lower mandibles, right hyo- and sacral ribs 22–24 hypoplastron, both xiphiplastra, dorsal rib, right humerus with associated radius and ulna, left ulna, both ilia, ischium, articulated dorsal vertebrae with sacral ribs, possible additional cranial elements, phalanges or disarticulated cervical or caudal vertebra, other unidentifiable bone 3 premaxilla, both maxillae, both prefrontals, right ribs and 20–24 hypoplastron, four associated dorsoventrally phalanges broadened ribs, humerus, radius or ulna, femur, tibia and fibula associated with femur, ilium, possible additional cranial elements, platy element (likely a plastral element) phalanges or disarticulated cervical or caudal vertebra, other unidentifiable bone 4 plastral element(s), numerous rib fragments, limb ribs and 20–24 bone(s), unguals (at least 4) unguals 5 plastral element(s), limb bones, ribs with dermal ribs and 20–24 plates, phalanges (at least 10), unguals (at least 4) unguals 6 maxilla, plastral element(s), stylopodial element, zeugopodial 19–22 zeugopodial element, phalanx, quadrate (?) element 11 plastral elements(s), stylopodial element with ribs and 20–24 associated zeugopodial elements, phalanges, phalanges textured (cranial?) elements, ribs 12 plastral elements, limb bones, ribs Ribs 20–24 14 pelvis or ischium pelvic girdle 21–24 element

68 Egg Identified bones Lastest bone Minimum to ossify stage of development 22 Hypoplastron hypoplastron 19–21 24 plastral bone(s), ribs, stylopodial element, ribs and 20–24 zeugopodial element(?), phalanges phanlanges 25 both maxillae, cranial bones(possibly premaxilla, plastral and 18–22 quadrate, prootic, or opisthotic), plastral elements, limb limb bones elements 26 plastral elements, ribs, stylopodial element, ribs and 20–24 zeugopodial elements (fore- and hind limb), unguals unguals 29 plastral elements, ribs, stylopodial element, ribs, unguals, 20–24 zeugopodial element, unguals, phalanges and phalanges 30 plastral elements, ribs, stylopodial element Ribs 20–24 Note: Stage ranges are based on ossification sequences of Apalone spinifera, Pelodiscus sinensis, Chelydra serpentina, Macrochelys temminickii, and Phrynops hilarii. Eretmochelys imbricata was not included because the fossil embryos are not sea turtles.

The high degree of ossification observed in the fossil turtle embryos indicates that they were at a later stage of embryonic development, well beyond Stage 17, which is the earliest stage at which ossification commences in extant turtle species (Appendix E). Some of the bones (i.e., the plastral bones) are extensively ossified, suggestive of a late stage of development. Additionally, the presence of nutritive foramina on the maxillae, dentaries and premaxilla, indicate that the keratinized rhamphotheca had begun to develop. Each process of the ischium in E2 is ossified, and this element does not begin to ossify until relatively late in embryonic development in extant species. The ends of each process are not preserved and likely were cartilaginous at the time of death; however, this does not indicate an early stage of development because the distal processes of these elements in A. spinifera remain primarily cartilaginous until after hatching (Sheil, 2002; Sheil, 2003).

69 Ossification sequences have been documented for various species of extant turtles (Appendix E), and can be used to more precisely stage the fossil embryos. However, in extant taxa, there is variation in the timing of ossification of elements between and within species; therefore, the predicted stage of development of the fossil embryos can vary depending on the species of turtle used for comparison (Table 3.3; Appendix E). For this reason, predicted minimum stages of development of the fossil embryos are presented as ranges (Table 3.2). If the ossification sequence of A. spinifera is used as a model, then the embryo in E2 was at approximately Stage 21 or 22, based on the pelvic girdle elements preserved. The articulated vertebrae in E2 may also include sacral ribs, which would extend the stage of development to Stage 24. If C. serpentina or P. sinensis are used as model organisms then the embryo in E2 is at least Stage 24, based on the ribs, pelvic girdle elements, and possible sacral ribs. If M. temminickii is used for comparison then the embryo would be at Stage 24 of development, based on the ischium.

Table 3.3 - Stage at which various bones ossify in six species of extant turtle. Apalone Pelodiscus Chelydra Macrochelys Eretmochelys Phrynops spinifera 1,2 sinensis3 serpentina1,4,5 temminckii1,6 imbricata1 hilarii 7 Premaxilla 22 21 19 or 20 20 n/a 21 Maxilla 17 19 19 or 20 17 25 19 Prefrontal 18 20 19 or 21 18 25 19 Dentary 18 19 19 18 25 19 Surangular 18 20 21 20 25 19 Coronoid 20 21 21 n/a 25–26 22 Plastral elements 19 19 21 n/a n/a 19 Dorsal ribs 21 22–24 21 20 25 21–22 Humerus 20 20 21 18 25 19 Femur 20 20 21 18 25 19 Zeugopodial 20 22 21 or 22 20 25 19 elements Ilium 21 24 22 21 25-26 21 Ischium 22 24 22-24 24 27–28 21 Pubis 21 24 22 21 25-26 21

70 Apalone Pelodiscus Chelydra Macrochelys Eretmochelys Phrynops spinifera 1,2 sinensis3 serpentina1,4,5 temminckii1,6 imbricata1 hilarii 7 Dorsal centra 19 19 22 20 25 21–23 Sacral ribs 24 n/a 22 or 24 n/a n/a n/a Phalanges/ 20 23 22 20 25–26 23 unguals Sources: 1Sheil, 2002; 2Sheil, 2003; 3Sanchez-Villagra et al., 2009; 4Rieppel, 1993; 5Sheil and Greenbaum, 2005; 6Sheil, 2005; 7Bona and Alcalde, 2009.

The stages of development predicted for the fossil embryos may be underestimated for numerous reasons. First, the information on extant embryos is based on the commencement of ossification of an element, and many of the elements in the fossil embryos are well ossified, beyond what would be expected if they were initially beginning to ossify. Additionally, it is likely that further preparation of the clutch or micro-CT scanning of the other blocks from the clutch could reveal additional elements that ossify late in development, thus extending the predicted stages of embryonic development. Also, some elements, such as phalanges or unguals, are so small they could easily be lost, and because they often ossify late relative to larger elements (i.e., stylopodial elements, plastral bones), the loss of these elements could result in an underestimation of the stage of development of the fossil embryos.

3.4.2 Mortality of the Embryos

Turtle mortality is highest in the embryonic and hatchling stages of ontogeny for numerous reasons, including increased predation, desiccation, exposure to temperature extremes, siltation, and erosion or flooding of the nest site (Allen, 1938; Cagle, 1950; Gibbons, 1968; Moll and Legler, 1971; Wilbur, 1975; Plummer, 1976; Iverson and Ewert, 1991), with flooding being a major risk for riverine turtles (Plummer, 1976; Orenstein, 2001). As the clutch was laid near an ephemeral stream, in what is interpreted to be warm- temperate to sub-tropical environment (Dodson, 1971; Hamblin, 1997), it is unlikely that the embryos of the clutch died as a result of desiccation or temperature extremes. Also, the fossil clutch is interpreted to have been buried, based on the large clutch size (n = 33 eggs) and the closely packed arrangement of the eggs, and because buried clutches are known to

71 be quite humid (Moll and Legler, 1971; Ewert, 1979; Packard, 1980; Seymour and Ackerman, 1980), it is unlikely that the embryos expired due to desiccation. Humid environments can, however, result in increased vulnerability to predators, including ants, bacteria, mold, fungi, and other invertebrates (Moll and Legler, 1971; Ewert, 1979; Packard, 1980), and one response to increased predation is to increase eggshell thickness (Packard and Packard, 1980). Because the eggs of the clutch have thick eggshell relative to extant rigid-shelled turtle eggs (Appendix A), perhaps the thick eggshell was a response to intense predation. Additionally, rigid-shelled eggs are less likely to absorb a significant amount of water, except under extreme conditions (Packard and Packard, 1979; Packard, 1980; Booth, 2002); therefore, perhaps the extreme thickness of the eggshell from the clutch is an adaptation to laying a nest next to an ephemeral stream that is prone to flooding. However, if an egg is inundated long enough it will drown, regardless of the shell thickness. Because the clutch was laid on a levee deposit in an area suggested to have experienced periods of seasonal rainfall and tropical storms (Eberth et al., 2001), it seems likely that the clutch could have drown. Additionally, the clutch was laid in a sandy siltstone matrix, which relative to sandy media, has an increased capacity for water retention after precipitation (Plummer, 1976); therefore, the silty nature of the matrix may have increased the likelihood of drowning.

3.4.3 Taxonomic Affinity of TMP 2008.27.1

It can be difficult to assign fossil embryonic remains to a specific taxon because diagnostic morphological features of the adults may not be present in the embryos, due to either the condition of preservation of the fragile embryonic elements or a lack of autapomorphies in early stages of ontogeny. Therefore, the skeletal morphology of the embryos, as well as other lines of evidence, such as adult carapace length, eggshell type, and geological age of previously identified turtles from the formation, are considered here to determine the taxonomic identity of the fossil embryos and clutch. Because the fossil clutch was discovered in the Oldman Formation of the Judith River Group (JRG), it can likely be attributed to one of the fossil turtle taxa known from the group (i.e., Solemydidae, Baenidae, Macrobaenidae, Chelydridae, Adocidae, Nanhsiungchelyidae, and Trionychidae)

72 (Table 3.4). Here I discuss various lines of evidence for each turtle taxon known from the JRG in order to elucidate the taxonomic identity of the turtle embryos and clutch.

Table 3.4 – Fossil turtles of the Judith River Group, Alberta, Canada. Solemydidae Lapparent de Broin and Gen. et sp. indet. Murelaga, 1996 Baenidae Cope, 1882 Boremys pulchra (Lambe, 1902) Baenidae Cope, 1882 Plesiobaena antiqua (Lambe, 1902) Baenidae Cope, 1882 Neurankylus eximus (Lambe, 1902) Macrobaenidae Sukhanov, 1964 Judithemys sukhanovi (Parham and Hutchison, 2003) Chelydridae Grey, 1831 Gen. et sp. indet. Adocidae Cope, 1870 Adocus sp. indet. Nanhsiungchelyidae Yeh, 1966 Basilemys variolosa (Cope, 1876) Trionychidae Fitzinger, 1826 Aspideretoides foveatus (Leidy, 1856) Trionychidae Fitzinger, 1826 Aspideretoides splendidus (Hay, 1908) Trionychidae Fitzinger, 1826 Aspideretoides alleni (Gilmore, 1923) Trionychidae Fitzinger, 1826 Apalone latus (Gilmore, 1920) Trionychidae Fitzinger, 1826 ?Platomeninae gen. et sp. indet. Sources: Eberth et al., 2001; Brinkman, 2003; Brinkman, 2005

Solemydidae: It is unlikely that the fossil embryos are solemydids, because these turtles are found in older rock formations than the Oldman Formation of Alberta. Solyemydid fragments are abundant in the Milk River Formation (Santonian), with a last occurrence in Alberta at the base of the Foremost Formation (Brinkman, 2003), which is dated at approximately 79.14 Ma (Eberth and Hamblin, 1993). The fossil embryos are from the uppermost Oldman Formation, which is dated at 75.86 ± 0.54 Ma (Eberth pers. comm. 2011), and thus occur about four million years after the last known solyemydids in Alberta.

Baenidae: The embryos are not baenids, based on the morphology of the plastron. Baenids (Paracryptodira) have extra plastral bones called mesoplastra, an anatomical

73 feature present in pleurodires and primitive cryptodires, which is absent in all eucryptodires (Joyce, 2007). Mesoplastra are situated between the hyo- and hypoplastra, and contribute to the bridge of the plastron. In E2 of the fossil clutch, the plastral bones are closely associated and it evident that the embryos, unlike baenids, do not have mesoplastra (Plate 10, Fig. A; Fig. 3.3). As plastral bones ossify early in extant turtle embryos (Stages 19–21), it is highly unlikely that the lack of mesoplastra in the fossil embryos is an artefact of an early ontogenetic stage of development. With respect to the dentary, two of the three baenid species known from the group, Plesiobaena antiqua and Boremys pulchra, have wide, flat triturating surfaces (Brinkman, 2003; Brinkman, 2005). The triturating surfaces of the embryonic dentaries in TMP 2008.27.1, however, are relatively narrow and exhibit a clear labial ridge, rather than a wide flat surface, thus also suggesting the embryos do not belong to these baenid species.

The carapace length of baenids are much smaller than that predicted for the fossil turtle that laid the clutch, which had a predicted carapace length of 71.4 cm (Appendix C). The largest baenid from the Judith River Group is Neurankylus eximus, with a carapace length of 54 cm (Brinkman, 2005); thus, the baenids are too small to have laid the fossil clutch.

Figure 3.3 Ventral surface of the baenid species Boremys pulchra demonstrating the presence of mesoplastra between the hyo- (red arrow) and hypoplastra (blue arrow). Scale

74 is 5 cm (Brinkman, 2005). Compare to the plastron of the embryos (Plate 10, Fig, A) which lacks mesoplastra between they hyo- and hypoplastra.

Macrobaenidae: The embryos of TMP 2008.27.1 are probably not macrobaenids because known macrobaenids from the JRG are much smaller in terms of carapace length than that predicted of the fossil turtle that laid the clutch (Appendix C). Judithemys sukhanovi is the only known macrobaenid from the group, which has a much smaller carapace length (average, 38.83 cm; n = 5; Parham and Hutchison, 2003) than that predicted for the fossil turtle that laid the clutch.

Macrobaenids are unknown from the formation in which the fossil embryos were found, and have only been discovered in the Dinosaur Park Formation of the JRG, primarily in Dinosaur Provincial Park (Brinkman, 2003; Brinkman, 2005). Because the embryos were discovered in the Oldman Formation in the MRNA of southeastern Alberta, it is unlikely that they are macrobaenids.

Chelydridae: The fossil embryos are preserved in rigid-shelled eggs and are therefore likely not chelydrids, as these turtles have pliable-shelled eggs (Packard, 1980). The calcareous portion of the eggshell in the fossil clutch is thick and rigid, and has tall interlocking shell units, whereas cheydrid eggshell is thin and pliable, and has non- interlocking shell units (Fig. 2.1) (Packard, 1980). The differences between their eggshells indicate that the fossil embryos do not belong to Chelydridae.

Trionychidae: The embryos of TMP 2008.27.1 are likely not trionychids, based on the extent of ossification of the plastral elements in the fossil embryos. The plastral elements of extant trionychid embryos are lightly ossified and have finger-like projections that grow into neighbouring plastral elements (Cherepanov, 1995; Sheil, 2002; Sheil, 2003; Sanchez- Villagra et al., 2009), and often have a large fontanel in the middle of the plastron, even after hatching (Cherepanov, 1995). Neither of these features is evident in the heavily ossified plastral elements of the fossil embryos (Plate 9, Fig. D, E, and F; Plate 10, Fig. A and B). The greater degree of ossification observed in the fossil embryos thus suggests that they are not trionychids.

75 Trionychid species are common in the JRG, but are most diverse in the Dinosaur Park Formation, with only one species, Aspideretoides foveatus, known from the Oldman Formation (Eberth et al., 2001). This taxon, however, is small and rarely exceeds a carapace length of 30 cm (Brinkman, 2005), which is much smaller than the predicted carapace length of the fossil turtle that laid the clutch (Appendix C).

Adocusia: The only remaining fossil turtles known from the JRG of Alberta that may have laid the fossil clutch are turtles from the families Adocidae and Nanhsiungchelyidae; families recently united in the Adocusia (Danilov and Parham, 2006; Danilov and Parham, 2008; Syromyatnikova and Danilov, 2009). Within Adocusia, only Basilemys variolosa, a large and fully terrestrial turtle, and Adocus sp. an aquatic form, are known from the JRG (Eberth et al., 2001; Brinkman, 2003, 2005). In the MRNA, where the fossil clutch was found, Adocus is abundant and often represented by complete shells, whereas Basilemy is rare (Brinkman, 2005).

Similarities were found in skulls and plastral elements between Adocus, Basilemys, and the fossil embryos. Both Adocus and Basilemys possess ridges along the anterior end of the maxilla, thus forming a well-developed “maxillary tooth” (Meylan and Gaffney, 1989; Brinkman, 1998) that is not found in other fossil turtles from the group. The maxillae of the fossil embryos also exhibit prominent ridges at the anterior end of the maxilla. The maxilla of Basilemys, however, also bears a groove that borders the antero-ventral edge of the orbit on the external surface, a groove that is not present in Adocus (Brinkman, 1998) or the fossil embryos. The triturating surface of the lower jaw of Basilemys has posterior and anterior dentary pockets that are separated by a low, rounded ridge that extends from the lingual to the labial ridges (Brinkman, 1998), whereas Adocus has a single pocket or groove (Meylan and Gaffney, 1989). The fossil embryos appear to have a single groove, similar to Adocus, rather than two distinct dentary pockets as in to Basilemys. As for the plastron, both Basilemys and Adocus have extremely well developed plastrons that are broad, well ossified, and akinetic (Meylan and Gaffney, 1989; Brinkman, 2003, 2005). Whereas the plastral elements in the fossil embryos remain unfused, they are well-ossified for such an early stage in ontogenetic development.

76 Among turtles known from the JRG, various lines of evidence suggest that the fossil embryos and clutch can be ascribed to Adocusia, specifically Adocus sp. Features of the dentary, maxilla, eggs, and eggshells of TMP 2008.27.1 are similar to those of Adocus. Additionally, the predicted carapace length of the adult that laid the clutch is also consistent with the known carapace length of Adocus. Finally, skeletal remains of Adocus are abundant in the area and in the formation in which the fossil clutch was found.

77 PLATE 7

The three eggs that were isolated from the clutch and micro-CT scanned. Fig. A is E1; Fig. B is E2; and Fig. C is E3. Scale is 1 cm.

Plate 7

79 PLATE 8

Figure A. Closely associated cranial elements preserved in E3. Labial view of premaxilla (white arrow), right and left maxillae (red arrows), and dorsal surface of right and left prefrontals (yellow arrows).

Figure B. Enlargement of the premaxilla in Figure A. Note the nutritive foramina (arrows).

Figure C. Amira image of the lingual view of the left and right maxilla in E2, respectively. Note the alveolar process (blue arrow), the prefrontal process (red arrow), and palatal process (black arrow). Scale is 1 mm. A = anterior; P = posterior.

Figure D. Labial view of maxillae in Figure C. Note the anterior ridges (white arrows), as well as nutritive foramina (red arrows).

Figure E. Enlargement of prefrontals in Figure A. Note the V-shaped notch along the posterior margin of the element (arrows).

Figure F. Amira image of ventral surface of the left prefrontal in E3. Note the descending process (arrows). Scale is 1 mm. A= anterior; P = posterior.

80 A B

C D A

E F P

Plate 8

81 PLATE 9

Figure A. Labial view of left ramus of the mandible in E2. Note the nutritive foramina (arrows).

Figure B. Amira image of labial view of the left ramus of lower jaw in Figure A, illustrating the dorsal process of the dentary (black arrow); posterior projection suggestive of at least the surangular (red arrow) present. Scale is 1 mm.

Figure C. Amira image of lingual view of the left rami in Fig. A, illustrating the sulcus cartilaginis Meckelii (arrows). Scale is 1 mm.

Figure D. Ventral view of the embryo in E1. Note the hyo- (red arrow), hypo- (white arrow), and xiphiplastra (black arrow). Scale is 1 cm.

Figure E. Ventral view of the embryo in E2. Note the hyo- (red arrow), hypo- ( white arrow), and xiphiplastra (black arrow). Scale is 1 cm.

Figure F. Amira image of the ventral view of E1. The hyoplastra are red, the hypoplastra are blue, and the xiphiplastra are green. Scale is 5 mm.

82 A B

C D

E F

Plate 9

83 PLATE 10

Figure A. Amira image of the ventral view of E2. The hyoplastra are red, the hypoplastra are blue, and the xiphiplastra are green. Scale is 5 mm.

Figure B. Amira image of the ventral view of the right hypoplaston of E3. Scale is 1 mm.

Figure C. Ventral view of clear and stained Chelydra serpentina embryo at Stage 26 (Yntema, 1968), showing lightly ossified hypoplastron (white arrow) and xiphiplastron (black arrow).

Figure D. Ventral view of clear and stained Apalone spinifera embryo at Stage 23 (Yntema, 1968), showing lightly ossified hypolaston (white arrow) and xiphiplastron (black arrow).

Figure E. Amira image of four dorsoventrally-broadened associated ribs in E3. Scale is 1mm.

Figure F. Amira image of a single dorsal rib in E2. Scale is 1 mm.

84 A B

C D

E F

Plate 10

85 PLATE 11

Figure A. Amira image of ventral view of E2, illustrating the association of plastral bones with the humerus (orange), ilia (pink), and ischium (gold). Scale is 5 mm.

Figure B. Amira image of the humerus in E3, illustrating a sagittal fracture. Scale is 1 mm.

Figure C. Humerus of clear and stained Chelydra serpentina embryo at Stage 25 (Yntema, 1968), illustrating how the ends of the element have not completely ossified.

Figure D. Amira image of the femur in E3. Intertrochanteric fossa (white arrow) present at the proximal end of the element. D = distal; P = proximal. Scale is 1 mm.

Figure E. Amira image of the right radius (black arrow) and ulna (red arrow) of E2, in close association with the right humerus. Scale is 1 mm.

Figure F. Amira image the one ilium in E2. Scale is 1 mm.

86 A B

C D

D P

E F

Plate 11

87 PLATE 12

Figure A. Ventral surface of the left ischium (arrow) in E2.

Figure B. Amira image of the ventral view of the left ischium in E2. Red arrow indicates lateral ischial process, black arrow indicates central ramus. Scale is 1 mm.

Figure C. Pelvic girdle of clear and stained specimen of Chelydra serpentina at Stage 25 (Yntema, 1968). Black arrow indicates left ilium, red arrow indicates left ischium, and blue arrow indicates left pubis.

Figure D. Amira image of ventral view of potentially fused dorsal vertebrae in E2. Note the groove that runs the length of the element (arrows) may represent the neural canal. Scale is 1 mm. A = anterior, P = posterior.

A B

C D

A P 88 Plate 12

89 CHAPTER 4: CONCLUSIONS

This study represents the first description of fossilized embryos within an egg clutch of an extinct turtle. The clutch is from the uppermost Oldman Formation of southeastern Alberta and was preserved in an ancient levee deposit. Description of the clutch reveals that it contains approximately 33 spherical in situ eggs that are closely packed and arranged in two or more layers, suggesting that it was likely laid in a pit or depression. The large clutch size suggests that it was buried because many extant turtle species that lay large clutches bury their eggs. Also, the fossil turtle that laid the clutch was likely a freshwater turtle, based on the large clutch size and on the geology of the nest site.

Examination of the eggs revealed numerous embryonic elements within 15 eggs, and 19 different types of elements were identified. Elements identified include: premaxilla, maxillae, prefrontals, dentaries, plastral elements, ribs, vertebrae, ilium, ischia, humeri, a femur, zeugopodial elements, unguals, and phalanges. Comparisons with modern turtle embryos indicate that the fossil specimens were in a late stage of development, based on the degree of ossification, as well as the presence of bones which ossify late in embryonic development (i.e., the ribs, pelvic girdle elements, unguals). Comparisons with extant taxa also revealed that the stage of embryonic development ranges from Stage 18–24 out of 26 possible stages, which is approximately 70-90% of the way through incubation, depending on the modern species of turtle used for comparison. However, it is likely that the embryos are in a later stage of development (around Stage 24), based on the degree of ossification observed in many of the elements, suggesting the elements had not just begun to ossify.

The fossil embryos and clutch are assigned to Adocus, one of the fossil turtles known from the Judith River Group of Alberta where the clutch was found. Comparison of skeletal elements of the embryos to specimens from Alberta eliminated many of the known turtle taxa as potential egg layers of the clutch. Features of the embryos are similar to those of Adocusia, and details of the dentary and maxilla, including a lack of dentary pockets on the dentary and the lack of a groove along the antero-ventral edge of the orbit on the maxilla, suggest that the embryos are Adocus, rather than the related taxon Basilemys. Furthermore, similarities between the eggs of the fossil clutch and those of extant trionychians, which are the closest living relatives to adocids, and similarities with a gravid Adocus specimen from

90 Alberta, also support a taxonomic affinity with Adocus. Adocus is a large freshwater turtle whose body size is within the predicted size of the fossil turtle that laid the clutch, and its freshwater habitat is consistent with the reconstructed environment of the fossil nest site. Finally, the stratigraphic position and geographic location of the nest site also supports this hypothesis because Adocus is abundant in the Oldman Formation of southeastern Alberta.

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APPENDIX A: EGGSHELL CHARACTERS OF FOSSIL AND EXTANT TAXA 104

Taxa Specimen # Egg Shape Egg Shell Ornamentation ES SU Width SU Height to Doming SU Visible on Pores Feathering Clutch Clutch Diameter Type Thickness (mm) Width ratio Outer SU Margins Size Frequency (mm) (mm) Surface fossil clutch ZEC 352, spherical 41-42.5 brittle smooth/none 0.76-0.92 0.130- 3.5 yes no rare yes 33 n/a (Albera) TMP 0.310 2008.27.1 gravid Adocus ZEC 059, spherical 35-40 brittle smooth/none 0.50-0.63 0.140- 2.3 yes no rare yes 19 n/a (Alberta) TMP 0.220 1999.63.2 gravid Adocus LBA-06-7 spherical 35 brittle smooth/none 0.25-0.28 0.100- 2.5 no n/a n/a no 25 to 30 n/a (Utah) 0.112 fossil clutch ZMNH spherical 42-45 brittle smooth/none 0.70-1.00 0.200- 2.5-3 n/a n/a rare n/a 27 n/a (China) M8713 0.350 Apalone ZEC 258 spherical 25.1-27.0 brittle smooth/none 0.20-0.24 0.120 1.5 yes yes rare yes 4 to 32 1+ spinifera Apalone ZEC 448 spherical 20-23 brittle smooth/none 0.10-0.12 0.115 1.1 yes yes rare yes 3 to 33 2 to 3 mutica (ave. 11 to 22) Amyda ZEC 307 spherical 21-33 brittle smooth/none 0.35-0.43 0.144 2.3 yes yes rare yes 5 to 30+ 3 to 4 cartilaginea Lissemys KT 101-1 spherical 26.0-28.6 brittle smooth/none n/a 0.114 2.3 yes yes rare yes 2 to 16 2 to 3 punctata Carettochelys ZEC 293 spherical 37.4-40.0 brittle dimpled/smooth 0.43-0.45 0.203 2.1 yes yes rare yes 4 to 39 1+ sp. Claudius ZEC 292 elongated 26.4-33.8 X brittle whorled/ridged 0.21-0.23 0.263 1.1 no no rare no 1 to 6 (ave. several angustatus 16.2-19.7 2 to 3)

Kinosternon ZEC 308 elongated 30 X 16 brittle whorled/ridged 0.27-0.32 0.281 0.8 no no rare no n/a n/a intergrum Kinosternon ZEC 227 elongated 24.2-33.2 X brittle whorled/ridged 0.14-0.15 n/a n/a no no rare no 1 to 7 2 to 4 hirtipes 14.6-18.6

Kinosternon ZEC 259 elongated 24-28.5 X brittle whorled/ridged 0.13-0.15 n/a n/a no no rare no 1 to 6 (ave. 1 flavescens 13.3-16.5 2-4) Kinosternon ZEC 289 elongated n/a brittle whorled/ridged n/a 0.209 1.2 no no rare no n/a n/a odoratum APPENDIX A: EGGSHELL CHARACTERS OF FOSSIL AND EXTANT TAXA 105

Staurotypus ZEC 354 elongated 35-44 X 21- brittle whorled/ridged 0.30-0.42 0.172 1.7 no no rare no 1 to 16 up to 4 triporcatus 26 (ave. 11.07)

Sternotherus ZEC 255 elongated 28.5 X 17.2 brittle whorled/ridged 0.30-0.33 0.199 1.5 no no rare no 1 to 5 2 to 5 minor Sternotherus ZEC 254 elongated 24-32 X 14- brittle whorled/ridged 0.21-0.25 0.221 1 no no rare no 1 to 9 (ave. up to 4 odoratus 17 2-5) Dermatemys ZEC 294 elongated 54.1-72 X brittle smooth/none 0.42-0.45 0.332 1.3 no no rare no 2 to 20 up to 4 mawii 32.4-49.8 Chelydra ZEC 235 spherical 23-33 parchme smooth/none 0.20-0.21 0.158 0.7 yes yes numerous no 6-100 (ave. 1 serpentina nt 20-40)

References: Hirsch, 1983; Iverson and Ewert, 1991; Packard, 1980; Packard et al., 1984a; Packard and Packard, 1979; Webb et al., 1986; Zelenitsky et al., 2008 APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES 106

Species Family Wet Body Ave Carapace Egg Length Egg Width Egg Mass Egg Clutch Size Clutch Mass Clutch Volume Number of Mass (g) Length (mm) (mm) (mm) (g) Volume (g) (cc) Nests (cc) Carettochelys insculpta Carettochelidae 10050 421 41 41 39 n/a 13.1 517.03 n/a n/a Acanthochelys pallidipectoris Chelidae 369 144 n/a n/a 10 n/a n/a n/a n/a n/a Chelodina expansa Chelidae 4744 305 37 25 18 n/a 18.8 329.00 n/a n/a Chelodina longicollis Chelidae 1293 245 30 20 7 6 13.6 100.59 81.78 1 Chelodina novaeguineae Chelidae 719 231 29 20 9 n/a 10.2 87.38 n/a n/a Chelodina oblonga Chelidae 1133 198 35 21 10 n/a 9.0 89.10 n/a n/a Chelonina parkeri Chelidae 1065 220 n/a n/a 12 n/a n/a n/a n/a n/a Chelodina siebenrocki Chelidae 1217 244 35 28 16 15 11.7 190.92 177.38 n/a Chelys fimbriata Chelidae 5000 310 38 38 29 n/a 16.0 460.96 n/a 1 Elseya dentata Chelidae 4295 347 n/a n/a 18 n/a 8.5 151.73 n/a n/a Emydura australis Chelidae n/a 254 34 18 n/a n/a n/a n/a n/a n/a Elseya latisternum Chelidae 1377 232 n/a n/a 12 n/a 17.0 204.85 n/a n/a Emydura krefftii Chelidae 1433 235 33 19 8 n/a 12.2 97.91 n/a 3 Emydura macquarii Chelidae 1740 289 33 23 11 n/a 19.6 206.00 n/a n/a Emydura subglobosa Chelidae 1030 255 34 18 10 n/a 10.3 99.84 n/a n/a Phrynops dahli Chelidae 777 198 30 25 15 n/a 4.0 60.64 n/a n/a Phrynops geoffroanus Chelidae n/a 325 34 32 19 n/a 15.0 287.55 n/a 1 Phrynops gibbus Chelidae 707 195 43 31 26 n/a 3.0 76.70 n/a n/a Phrynops hilarii Chelidae 2380 280 n/a n/a 19 n/a 18.0 334.80 n/a n/a Phrynops zuliae Chelidae 1983 263 n/a n/a 18 n/a 7.0 128.52 n/a n/a Platemys platycephala Chelidae 325 177 51 28 27 n/a 3.5 94.50 n/a n/a Pseudoemydura umbrina Chelidae 324 128 35 20 9 n/a 4.3 37.10 n/a 1 Rheodytes leukops Chelidae 1691 249 30 21 75 n/a 15.9 1192.50 n/a 4 Caretta caretta Cheloniidae 92400 903 46 46 35 37 121.0 4291.57 4525.40 4 Chelonia mydas Cheloniidae 128100 1007 48 48 50 57 120.7 6078.76 6857.46 4.5 Eretmochelys imbricata Cheloniidae 59000 805 36 36 28 29 143.7 4029.94 4123.33 3 Lepidochelys kempi Cheloniidae 42100 654 28 27 32 31 107.3 3421.68 3305.76 2.5 Lepidochelys olivacea Cheloniidae 39255 658 n/a n/a 34 32 110.0 3787.23 3553.97 2.5 Natator depressa Cheloniidae 74400 921 50 50 72 74 50.1 3582.79 3684.93 4 Chelydra serpentina Chelydridae 3689 274 31 29 12 10 29.2 340.27 294.75 2 Macroclemys temminckii Chelydridae 11700 357 32 32 27 27 26.3 717.00 703.96 1 Dermatemys mawii Dermatemydidae 9320 503 58 33 41 36 15.0 612.00 532.50 1 Dermochelys coriacea Dermochelydidae 372500 1529 50 50 74 74 85.2 6334.95 6270.20 5 Chrysemys picta Emydidae 454 152 32 17 5 5 6.6 35.79 30.52 2.4 Clemmys guttata Emydidae 165 117 n/a n/a 6 5 3.1 18.41 14.69 n/a Clemmys insculpta Emydidae 968 184 33 23 12 14 8.4 101.56 119.28 1 APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES 107

Species Family Wet Body Ave Carapace Egg Length Egg Width Egg Mass Egg Clutch Size Clutch Mass Clutch Volume Number of Mass (g) Length (mm) (mm) (mm) (g) Volume (g) (cc) Nests (cc) Clemmys marmorata Emydidae 282 160 38 20 8 9 4.5 37.38 41.18 1 Clemmys muhlenbergi Emydidae 132 85 30 15 5 4 3.8 17.81 14.94 1 Deirochelys reticularia Emydidae 867 190 36 22 9 9 7.9 74.79 69.52 3 Emydoidea blandingi Emydidae 1162 199 39 24 13 12 10.5 135.38 128.41 2 Emys orbicularis Emydidae n/a 148 30 17 6 8 10.0 60.00 75.00 n/a Graptemys barbouri Emydidae 1256 242 36 26 16 18 9.3 153.15 167.01 2.5 Graptemys geographica Emydidae 1138 202 n/a n/a 11 7 12.0 132.93 89.02 2.5 Graptemys nigrinoda Emydidae 416 155 n/a n/a 12 n/a 5.5 66.17 n/a n/a Graptemys oculifera Emydidae 359 168 41 20 11 9 3.0 33.69 27.30 1.5 Graptemys ouachitensisys Emydidae 1136 205 n/a n/a 10 n/a 10.5 102.48 n/a n/a Graptemys pseudogeographica Emydidae 1477 220 35 22 10 9 10.9 108.67 94.54 3

Graptemys pulchra Emydidae 1519 242 43 30 13 14 8.4 104.63 114.63 4 Malaclemys terrapin Emydidae 886 201 31 21 10 11 8.4 84.76 94.08 n/a Pseudemys concinna Emydidae 2992 329 40 26 14 13 17.5 251.56 234.50 n/a Pseudemys nelsoni Emydidae 4020 298 n/a n/a 10 n/a 14.3 146.29 n/a n/a Pseudemys rubriventris Emydidae 3477 267 n/a n/a 12 6 14.0 166.60 82.60 n/a Pseudemys scripta Emydidae 1895 257 38 24 11 9 10.2 108.73 95.88 2 Terrapene carolina Emydidae 372 140 37 22 10 7 4.3 41.75 28.55 2.75 Terrapene coahuila Emydidae 260 122 33 17 6 5 2.2 12.45 11.00 2.5 Terrapene nelsoni Emydidae 372 138 42 27 18 18 2.8 51.69 50.12 1 Terrapene ornata Emydidae 391 124 n/a n/a 10 9 4.5 46.94 40.05 1.5 Trachemys decorata Emydidae 2522 261 n/a n/a 17 n/a 12.0 201.60 n/a n/a Trachemys decussata Emydidae 630 171 n/a n/a 13 n/a 5.0 66.50 n/a n/a Trachemys dorbigni Emydidae 2497 260 n/a n/a 18 n/a 4.0 73.60 n/a n/a Trachemys scripta Emydidae 1923 n/a n/a n/a 11 n/a 8.4 90.34 n/a n/a Batagur baska Geoemydidae 17900 497 76 40 65 n/a 24.3 1585.10 n/a 3 Callagur borneoensis Geoemydidae 16900 453 n/a n/a 70 60 11.7 823.68 700.83 2 Chinemys kwangtungensis Geoemydidae n/a 200 51 27 n/a n/a 2.0 n/a n/a n/a Chinemys reevesii Geoemydidae 858 195 n/a n/a 11 n/a 6.7 75.04 n/a n/a Cistoclemmys flavomarginata Geoemydidae 499 152 n/a n/a 14 n/a 2.0 27.60 n/a n/a

Cuora amboinensis Geoemydidae 1000 188 43 32 20 n/a 1.0 19.50 n/a n/a Cuora flavomarginata Geoemydidae n/a 183 49 26 n/a n/a n/a n/a n/a n/a Cuora mccordi Geoemydidae 375 137 n/a n/a 14 n/a 1.5 20.25 n/a n/a Cuora trifasciata Geoemydidae n/a 200 50 27 n/a n/a 2.0 n/a n/a n/a APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES 108

Species Family Wet Body Ave Carapace Egg Length Egg Width Egg Mass Egg Clutch Size Clutch Mass Clutch Volume Number of Mass (g) Length (mm) (mm) (mm) (g) Volume (g) (cc) Nests (cc) Cyclemys dentata Geoemydidae 1250 220 57 35 30 n/a 3.0 89.10 n/a n/a Cyclemys mouhoti Geoemydidae n/a 170 40 25 n/a n/a n/a n/a n/a n/a Geoemyda silvatica Geoemydidae 230 121 n/a n/a 15 n/a 2.0 30.50 n/a n/a Geoemyda spengleri Geoemydidae 196 116 n/a n/a 8 n/a 1.0 8.20 n/a n/a Heosemys spinosa Geoemydidae 950 203 n/a n/a 49 n/a 1.0 48.70 n/a n/a Kachuga dhongoka Geoemydidae 7270 327 56 33 35 n/a 30.0 1056.00 n/a n/a Kachuga kachuga Geoemydidae 21100 560 n/a n/a 55 n/a 17.0 940.10 n/a n/a Kachuga smithi Geoemydidae 919 203 44 23 12 12 6.6 81.43 80.12 n/a Kachuga tentoria Geoemydidae 1184 218 n/a n/a 20 n/a 6.3 125.00 n/a n/a Malayemys subtrijuga Geoemydidae n/a 240 44 22 n/a n/a n/a n/a n/a n/a Mauremys caspica Geoemydidae n/a 223 35 20 n/a 15 5.5 n/a 79.75 n/a Mauremys japanica Geoemydidae 494 150 n/a n/a 8 n/a 6.0 49.98 n/a n/a Mauremys leprosa Geoemydidae 608 149 36 20 13 n/a 6.4 84.48 n/a n/a Mauremys mutica Geoemydidae 320 163 38 21 11 n/a n/a n/a n/a n/a Melanochelys trijuga Geoemydidae 760 179 47 27 13 n/a 4.0 50.80 n/a 2 Ocadia philippeni Geoemydidae 1241 214 n/a n/a 15 n/a 6.0 88.20 n/a n/a Ocadia sinensis Geoemydidae n/a 225 40 25 n/a n/a 3.0 n/a n/a n/a Orlitia borneensis Geoemydidae 12400 475 n/a n/a 94 n/a 12.0 1131.60 n/a n/a Rhinoclemmys annulata Geoemydidae n/a 190 71 37 n/a n/a 1.0 n/a n/a n/a Rhinoclemmys areolata Geoemydidae 732 169 60 31 35 n/a 1.0 34.54 n/a n/a Rhinoclemmys funerea Geoemydidae 1946 271 68 35 47 44 3.1 147.74 136.47 3.25 Rhinoclemmys melanosterna Geoemydidae 2700 243 n/a n/a 37 n/a 5.0 187.00 n/a n/a Rhinoclemmys nasuta Geoemydidae 1284 218 n/a n/a 53 n/a 1.0 52.70 n/a n/a Rhinoclemmys pulcherrima Geoemydidae n/a 196 52 31 n/a n/a 3.0 n/a n/a n/a Rhinoclemmys punctularia Geoemydidae n/a 241 71 37 n/a 49 2.3 n/a 111.15 n/a Rhinoclemmys rubida Geoemydidae n/a 183 62 25 n/a n/a 1.0 n/a n/a n/a Siebenrockiella crassicollis Geoemydidae 940 222 45 19 29 n/a 1.3 37.05 n/a n/a Claudius angustatus Kinosternidae 200 123 32 18 4 n/a 5.0 17.50 n/a 2 Staurotypus salvini Kinosternidae 900 200 41 20 11 n/a 5.7 59.81 n/a 2 Staurotypus tripocatus Kinosternidae 4200 295 38 23 16 n/a 7.1 115.63 n/a n/a Kinosternon alamosae Kinosternidae 145 105 n/a n/a 4 n/a 4.0 14.64 n/a n/a Kinosternon angustipons Kinosternidae 201 115 40 22 12 10 2.0 23.88 20.20 2 Kinosternon bauri Kinosternidae 143 94 n/a n/a 5 4 2.2 10.37 8.73 2.5 Kinosternon creaseri Kinosternidae 207 116 n/a n/a 9 n/a 1.0 8.90 n/a n/a Kinosternon dunni Kinosternidae 496 159 45 25 16 15 2.3 37.23 34.02 3 Kinosternon flavescens Kinosternidae 295 111 n/a n/a 5 4 5.0 24.83 22.05 1 APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES 109

Species Family Wet Body Ave Carapace Egg Length Egg Width Egg Mass Egg Clutch Size Clutch Mass Clutch Volume Number of Mass (g) Length (mm) (mm) (mm) (g) Volume (g) (cc) Nests (cc) Kinosternon herrerai Kinosternidae 486 143 n/a n/a 7 n/a 2.0 13.90 n/a n/a Kinosternon hirtipes Kinosternidae 203 131 28 17 5 4 4.5 21.69 18.90 n/a Kinosternon intergrum Kinosternidae 474 156 30 16 7 n/a 5.0 33.20 n/a n/a Kinosternon leucostomum Kinosternidae 373 135 37 20 8 7 1.1 8.65 7.79 2 Kinosternon scorpioides Kinosternidae 266 146 35 19 7 7 5.1 34.19 34.17 n/a Kinosternon sonoriense Kinosternidae 340 138 33 19 5 n/a 4.3 23.11 n/a n/a Kinosternon spurrelli Kinosternidae n/a 102 n/a n/a n/a 7 3.0 n/a 22.20 2 Kinosternon subrubrum Kinosternidae 148 92 26 15 4 4 3.6 15.31 13.78 3 Sternotherus carinatus Kinosternidae 248 116 29 17 5 5 3.8 20.79 17.10 2 Sternotherus depressus Kinosternidae 113 104 n/a n/a 6 n/a 3.2 17.55 n/a 2 Sternotherus minor Kinosternidae 155 104 28 17 5 n/a 3.9 19.89 n/a 3 Sternotherus odoratus Kinosternidae 152 105 29 16 4 3 3.8 13.91 12.95 2 Pelomedusa subrufa Pelomedusidae 2273 234 38 22 12 6 27.5 340.45 162.25 1 Peltocephalus dumerilianus Pelomedusidae 3600 324 n/a n/a 47 n/a 10.7 498.62 n/a n/a Peltocephalus tracaxa Pelomedusidae n/a 457 55 36 n/a n/a 15.0 n/a n/a n/a Pelusios castanoides Pelomedusidae 800 220 n/a n/a 9 n/a 25.0 220.75 n/a n/a Pelusios rhodesianus Pelomedusidae 900 230 n/a n/a 9 n/a 12.5 118.50 n/a n/a Pelusios subniger Pelomedusidae n/a 295 36 21 10 18 10.0 97.80 177.00 n/a Podocnemis expansa Pelomedusidae 25800 737 n/a n/a 33 n/a 88.5 2885.10 n/a 1 Podocnemis lewyana Pelomedusidae 9599 475 n/a n/a 23 n/a 16.5 381.15 n/a n/a Podocnemis unifilis Pelomedusidae 6380 419 41 28 24 23 22.7 537.73 516.88 2 Podocnemis vogli Pelomedusidae 2013 288 n/a n/a 17 n/a 12.8 215.73 n/a 2 Platysternon megacephalum Platysternidae n/a 127 n/a n/a n/a n/a 2.0 n/a n/a n/a Chersina angulata Testudinidae 715 157 39 34 29 n/a 1.3 36.25 n/a 1 Geochelone carbonaria Testudinidae 3356 289 n/a n/a 50 n/a 4.0 200.80 n/a n/a Geochelone chilensis Testudinidae 3181 262 46 36 34 26 2.7 91.58 70.49 1.5 Geochelone denticulata Testudinidae n/a 330 n/a n/a n/a 37 3.0 n/a 110.10 n/a Geochelone elegans Testudinidae n/a 214 46 35 27 26 4.3 115.60 112.20 2.25 Geochelone elephantopus Testudinidae n/a 914 n/a n/a 107 91 9.8 1042.08 882.38 1 Geochelone gigantea Testudinidae 33000 756 n/a n/a 86 n/a 9.3 799.78 n/a 2.75 Geochelone pardalis Testudinidae 20000 390 n/a n/a 60 28 10.3 615.76 293.51 6 Geochelone radiata Testudinidae 7955 345 39 35 36 28 4.2 149.74 116.20 3 Geochelone sulcata Testudinidae 22400 514 n/a n/a 40 39 17.0 685.10 657.90 n/a Gopherus agassizi Testudinidae 2290 245 46 38 39 35 4.8 187.89 167.60 1 Gopherus berlandieri Testudinidae 755 167 50 36 27 n/a 1.8 48.42 n/a n/a Gopherus polyphemus Testudinidae 2784 247 44 44 42 38 5.4 225.65 205.07 1 APPENDIX B: REPRODUCTIVE CHARACTERS OF EXTANT TURTLE SPECIES 110

Species Family Wet Body Ave Carapace Egg Length Egg Width Egg Mass Egg Clutch Size Clutch Mass Clutch Volume Number of Mass (g) Length (mm) (mm) (mm) (g) Volume (g) (cc) Nests (cc) Homopus areolatus Testudinidae 289 107 30 22 8 8 3.0 24.45 23.10 n/a Homopus boulengeri Testudinidae 213 98 38 22 10 10 1.0 10.20 9.90 1 Homopus femoralis Testudinidae 599 140 n/a n/a 13 n/a 1.5 19.49 n/a n/a Indotestudo elongata Testudinidae 2551 260 n/a n/a 43 n/a 4.0 171.20 n/a n/a Indotestudo forstenii Testudinidae 1680 139 n/a n/a 16 n/a 3.0 47.10 n/a n/a Kinixys belliana Testudinidae 1202 172 n/a n/a 36 27 2.5 90.20 67.25 n/a Kinixys erosa Testudinidae n/a 232 40 35 n/a 27 5.0 n/a 135.50 n/a Kinixys homeana Testudinidae n/a 159 n/a n/a n/a 22 4.0 n/a 89.60 n/a Malacochersus tornieri Testudinidae 400 158 47 29 22 20 1.5 31.98 29.63 2 Psammabates geometricus Testudinidae 414 125 n/a n/a 11 n/a 8.3 92.40 n/a n/a Psammobates oculifera Testudinidae n/a 133 n/a n/a n/a 20 n/a n/a n/a n/a Psammabates tentorius Testudinidae 423 124 30 23 8 8 2.3 18.83 17.71 n/a Testudo graeca Testudinidae 1430 197 33 28 18 12 2.8 49.53 32.55 1 Testudo hermanni Testudinidae 1203 181 n/a n/a 16 n/a 4.2 67.26 n/a 2 Testudo horsfieldii Testudinidae n/a 197 n/a n/a n/a 17 n/a n/a n/a n/a Testudo kleinmanni Testudinidae n/a 126 30 23 n/a 8 2.0 n/a 15.60 n/a Testudo marginata Testudinidae 2080 253 n/a n/a 17 n/a 5.6 96.88 n/a n/a Apalone ferox Trionychidae 3326 331 26 26 13 8 16.7 210.29 136.61 n/a Apalone muticus Trionychidae 819 195 22 22 8 6 15.2 122.42 97.02 2.5 Apalone spinifera Trionychidae 4765 328 30 30 11 12 22.5 249.47 268.11 1 Aspideretes gangeticus Trionychidae 19000 583 34 34 25 17 30.1 761.53 517.72 n/a Aspideretes nigricans Trionychidae 6971 436 n/a n/a 26 n/a 20.2 525.20 n/a n/a Chitra indica Trionychidae 108000 1110 n/a n/a 20 n/a 102.0 2040.00 n/a n/a Cyclanorbis sengalensis Trionychidae n/a 225 n/a n/a n/a 24 6.0 n/a 146.40 n/a Cycloderma frenatum Trionychidae 14591 543 35 30 18 21 18.7 335.13 384.60 n/a Lissemys punctata Trionychidae 1149 268 28 28 11 16 6.3 71.96 100.17 2 Pelodiscus sinensis Trionychidae 2328 248 25 20 5 4 19.0 102.60 79.80 3 Rafetus euphraticus Trionychidae n/a n/a n/a n/a 8 n/a n/a n/a n/a n/a Trionyx triunguis Trionychidae 10818 603 n/a n/a 18 20 30.2 537.27 612.05 n/a

References: Congdon and Gibbons, 1985; Doody et al., 2003; Elgar and Heaphy, 1989; Ewert, 1979; Iverson et al., 1993; Henry M. Wilbur and Morin, 1988

111 APPENDIX C: BODY SIZE OF THE FOSSIL TURTLE

Introduction

Numerous comparative studies have been conducted that examine the relationship between various life history characters within, and across, turtle taxa (Moll, 1979; Ewert, 1979; Congdon and Gibbons, 1985; Elgar and Heaphy, 1989; Iverson, 1992; Iverson et al., 1993; Janzen and Warner, 2009). Comparative studies have examined the effects of body size, latitude, diet, and habitat (marine, freshwater, and terrestrial), on reproductive output, including: clutch size, clutch mass, clutch volume, clutch frequency, and egg size (Moll, 1979; Ewert, 1979; Congdon and Gibbons, 1985; Elgar and Heaphy, 1989; Iverson, 1992; Iverson et al., 1993; Janzen and Warner, 2009). There is a general tendency to examine life history variables in relation to body size (body mass or carapace length), as they tend to covary strongly with body size (Moll, 1979; Ewert, 1979; Congdon and Gibbons, 1985; Elgar and Heaphy, 1989; Iverson, 1992; Iverson et al., 1993; Janzen and Warner, 2009).

One major issue with previous studies that examined the relationship of life history traits in turtles is that the data were not phylogenetically corrected for non-independence. If using individual species as data points in regressions, statistically significant associations may result due to shared ancestry, rather than convergent or parallel evolution (Elgar and Heaphy, 1989). This is especially pertinent in turtles as many families of turtles (Dermochelyidae, Cheloniidae, Testudindae) form ecological groups (Elgar and Heaphy, 1989). Of the few studies that have considered the effects of phylogenetic independence, the sample size of taxa examined has either been small (Congdon and Gibbons, 1985; Iverson, 1992), or the study only looked at traits within a single turtle family (Iverson et al., 1993).

In this thesis, taxa from all 14 families of extant turtle were phylogenetically- corrected and regressions of carapace length versus clutch mass and body mass versus clutch mass were performed in order to estimate the size of the fossil turtle that laid the clutch (TMP 2008.27.1). Clutch mass was used rather than clutch size, as there is a well established inverse relationship between clutch size and egg size in turtles (Moll, 1979; Ewert, 1979; Congdon and Gibbons, 1985; Iverson, 1992; Iverson et al., 1993), and clutch mass takes both of these variables into consideration.

112 Materials and Methods

A table of characters relating to reproductive output (e.g. clutch size, egg volume, egg length and width, clutch mass, clutch volume) was compiled from published literature for 171 taxa (Appendix B and references therein) of extant turtle species from all 14 families (Pelomedusidae, Podocnemidae, Chelidae, Cheloniidae, Dermochelydidae, Chelydridae, Kinosternidae, Dermatemydidae, Trionychidae, Carettochelydidae, Geoemydidae, Testudinidae, Emydidae, Platysternidae) in order to assess relationships between body size and clutch mass via regression analyses.

Before the regressions analyses were performed the issue of phylogenetic non- independence was corrected for using the PDAP module, version 1.15, of the software MESQUITE v. 2.74. Thus, a phylogenetic tree off all taxa used in the regression analyses was constructed in Mesquite using the results of previously published phylogenetic studies (Fig. A1). Since there has been much contention over the phylogenetic relationship of turtles, I provide a review of the literature on the topic that is relevant to the tree constructed for this study.

Whereas the monophyly of most families and the two suborders (Pleurodira and Cryptodira) are relatively well established (Gaffney, 1975; Bickham and Carr, 1983; Shaffer et al., 1997; Cervelli et al., 2003; Fujita et al., 2004; Joyce et al., 2004; Joyce, 2004, 2007; Krenz et al., 2005; Parham et al., 2006; Bickham et al., 2007; Barley et al., 2010; Thomson and Shaffer, 2010; Knauss et al., 2011), there is much debate about the interrelationship between families within the suborders, especially the more diverse Cryptodira. The most recent phylogenies tend to use molecular data, or a combination of both morphological and molecular characters (Gaffney, 1975; Bickham and Carr, 1983; Shaffer et al., 1997; Cervelli et al., 2003; Fujita et al., 2004; Joyce et al., 2004; Joyce, 2004, 2007; Krenz et al., 2005; Parham et al., 2006; Bickham et al., 2007; Barley et al., 2010; Thomson and Shaffer, 2010; Knauss et al., 2011), and therefore molecular characters were used to build the tree for this study. For example, morphological studies traditionally unite Kinosternoidea and Trionychia as sister taxa (Gaffney, 1975; Meylan and Gaffney, 1989; Joyce, 2007) , whereas all molecular studies from the last thirty years do not recognize the group as a monophyletic clade, placing Trionychia as sister taxa to all other

Pelomedusa subrufa Pelusios subniger Pelusios castanoides Pelusios rhodesianus

Pe Peltocephalus tracaxa

lomedu Podocnemis expansa Podocnemis lewyana Podocnemis vogli

sidae Peltocephalus dumerilianus Chelodina oblonga Chelodina longicollis Chelodina expansa P

leur Chelodina novaeguineae Chelodina siebenrocki

odira Chelonina parkeri Pseudoemydura umbrina Elseya dentata Elseya latisternum Rheodytes leukops Emydura australis Ch Emydura macquarii eli

dae Emydura subglobosa Platemys platycephala Acanthochelys pallidipectoris Phrynops dahli Phrynops zuliae Phrynops gibbus

Trionychinae Phrynops hilarii Rafetus euphraticus Triony Apalone ferox Apalone spinifera Apalone muticus chidae Trionychia Pelodiscus sinensis Aspideretes gangeticus Aspideretes nigricans Trionyx triunguis Chitra indica Cycloderma frenatum Cyclanorbis sengalensis 1 Lissemys punctata Carettochelys insculpta Claudius angustatus Staurotypus salvini Staurotypus tripocatus Kinosternon creaseri Kinosternon dunni Kinosternon alamosae Kinosternon angustipons Kinosternon herrerai Kinosternon hirtipes Kinosternon intergrum Kinosternon leucostomum Kinosternon scorpioides Kinosternon bauri Kinosternon subrubrum Kinoste Kinosternon sonoriense Kinosternon spurrelli Sternotherus minor

rnoidea Sternotherus odoratus Sternotherus carinatus Sternotherus depressus Dermatemys mawii Macroclemys temminckii 2 Chelydra serpentina Eretmochelys imbricata Caretta caretta C

rypto Lepidochelys olivacea Lepidochelys kempi Chelonia mydas Ch dira Natator depressa

elonioidea Dermochelys coriacea Cuora amboinensis Cuora mccordi

Ocadia philippeni Cuora trifasciata Mauremys mutica Mauremys caspica Chinemys kwangtungensis Chinemys reevesii Ocadia sinensis Mauremys japanica Mauremys leprosa Heosemys spinosa Cyclemys mouhoti Cyclemys dentata Melanochelys trijuga Siebenrockiella crassicollis Geoemyda spengleri Geoemyda silvatica Malayemys subtrijuga Orlitia borneensis Callagur borneoensis Kachuga dhongoka Geoemydidae Kachuga smithi Kachuga tentoria Kachuga kachuga Batagur baska Rhinoclemmys rubida Rhinoclemmys nasuta Rhinoclemmys pulcherrima Rhinoclemmys annulata Rhinoclemmys areolata Rhinoclemmys punctularia Rhinoclemmys funerea Rhinoclemmys melanosterna Gopherus polyphemus Gopherus agassizi Gopherus berlandieri Geochelone elephantopus Geochelone gigantea Geochelone pardalis Geochelone radiata Chersina angulata Homopus boulengeri Homopus areolatus Homopus femoralis Testu Psammabates tentorius Psammabates geometricus Psammobates oculifera dinidae Geochelone carbonaria Geochelone denticulata Geochelone chilensis Kinixys homeana Kinixys belliana Kinixys erosa Geochelone sulcata Geochelone elegans Malacochersus tornieri Testudo marginata Testudo kleinmanni Testudo graeca Indotestudo elongata Indotestudo forstenii Testudo hermanni Platysternon megacephalum Clemmys muhlenbergi Clemmys insculpta Emys orbicularis Emydoidea blandingi Clemmys marmorata Clemmys guttata Terrapene nelsoni Terrapene ornata Terrapene coahuila Terrapene carolina Emydidae Deirochelys reticularia Chrysemys picta Trachemys dorbigni 2. Chelydridae 1. Cyclanorbinae Malaclemys terrapin Graptemys pseudogeographica Graptemys ouachitensisys ouachitensis Graptemys nigrinoda Graptemys oculifera Graptemys barbouri Graptemys pulchra Graptemys geographica Trachemys decussata Trachemys decorata Trachemys scripta Pseudemys rubriventris

Pseudemys nelsoni 113 Pseudemys scripta Pseudemys concinna

114 cryptodirans (Bickham and Carr, 1983; Shaffer et al., 1997; Cervelli et al., 2003; Fujita et al., 2004; Krenz et al., 2005; Near et al., 2005; Parham et al., 2006; Chandler and Janzen, 2009; Thomson and Shaffer, 2010; Barley et al., 2010). Additionally, morphological support for Trionychoidea (Trionychia + Kinosternoidea) is decreasing, and previously cited synapomorphies are likely a result of a priori assumptions, or are suggested to be homoplasies (Joyce, 2007).

The interrelationship of taxa within Testudinoidea (Kinosternoidea, Chelydridae, and Chelonioidea) has also been widely contested and, in some studies, the aforementioned taxa have been grouped as a polytomy (Chandler and Janzen, 2009; Thomson and Shaffer, 2010), whereas others have suggested different combinations of the sister groups (Shaffer et al., 1997; Krenz et al., 2005; Danilov and Parham, 2006; Chandler and Janzen, 2009; Barley et al., 2010; Knauss et al., 2011). For this study Kinosternoidea and Chelydridae are grouped as sister taxa, as the most recent studies link the two clades, and because this union is supported by both molecular (Barley et al., 2010) and morphological data (Knauss et al., 2011). The confusion over the interrelationships of these families is likely due to the fact that they evolved rapidly. The fossil record indicates that the major groups of cryptodires appeared over a relatively short period of time, between 120-90 Ma, which is approximately 14% of the total history of turtles (Shaffer et al., 1997). Another area of contention involves the closest relative to the monospecific family Platysternidae. In this study, Platysternon is treated as the sister taxon to Emydidae, which is suggested by most recent phylogenetic studies (Parham et al., 2006; Barley et al., 2010; Thomson and Shaffer, 2010)

In addition to discrepancies in the phylogenetic relationships between taxa, there is also a lot of confusion resulting from inconsistent use of terminology. A coherent nomenclatural system was proposed by Joyce (2004), in order to promote consistent use of terminology and reduce confusion. The term Trionychoidea originally referred to soft- shelled turtles only; however, it has since been used to refer to more inclusive clades, uniting soft-shelled turtles with Carettochelys insculpta, as well as with Kinosternoidea (Joyce, 2004; Joyce et al., 2004). According to Joyce’s (2004) nomenclatural system, the term Trionychidae includes the soft-shelled turtles only, Carettochelyidae includes the pig-

115 nosed turtle, Kinosternidae refers to the mud turtles, Dermatemydidae refers to the Central American river turtle, Trionychia includes Trionychidae and Carettochelyidae, and Kinosternoidea includes Kinosternidae and Dermatemydidae.

The phylogenetic tree (Fig. A1) that was built for this study is a combination of a recent high-order phylogenetic supermatrix that demonstrates the interrelationships of families (Thomson and Shaffer, 2010) and several low-order phylogenetic hypotheses at the family or genus level (Table A1). The supermatrix is based on molecular data collected from GenBank, and is in agreement with most other molecular phylogenetic studies in terms of the placement of families. The lower order phylogenies used for the tree in this study are based on molecular data, or a combination of molecular and morphological data (Table A1). Despite the numerous low-order phylogenies that were used to create the tree, many speciose genera were unresolved and numerous polytomies persist. For the PDAP analysis, branch lengths of one were used for the tree as it was not possible to determine the true branch lengths due to the uncertainty of divergence times between many taxa, and due to the large number of taxa included. With branch lengths of one, it is assumed that all evolutionary change took place during speciation events (Garland et al., 1993). The resultant tree contained 171 taxa; however, depending on the regression, species were eliminated due to lack of data for certain variables.

Table A1 – Sources used to determine the lower level relationships among turtles for tree construction in Mesquite. Taxon Author Analysis type Pelomedusidae Fritz et al., 2010 Molecular Trionychidae Engstrom et al., 2004 Molecular and morphological Geoemydidae Spinks et al., 2004 Molecular Testudinidae Le et al., 2006 Molecular Emydidae Stephens and Wiens, 2003 Molecular and morphological Testudinoidea Barley et al., 2010; Knauss et Molecular and morphological al., 2011

116 Log-transformed regressions were produced using the PDAP module v. 1.15, of the software MESQUITE v. 2.74 in order to assess the relationships between body size and clutch mass among living species, so that the body size of the turtle that laid the fossil clutch could be determined. As there is an inverse relationship between clutch size and egg size (Ewert, 1979; Moll, 1979; Congdon and Gibbons, 1985; Iverson and Ewert, 1991; Iverson, 1992), clutch size should not be used in bivariate analysis with body size, as it is dependent on an additional variable (i.e., egg size). As such, clutch mass is a better variable, as it accounts for egg size as well. Variables for body mass, carapace length, and clutch mass were transformed to their natural logarithms to address allometric biases and the influence of body size on clutch size and mass. An ordinary least squares (OLS) regression was performed, instead of a reduced major axis (RMA) regression, because the relationship between the independent variables (i.e., carapace length or body mass) and dependent variable (i.e., clutch mass) are asymmetric. The relationship between variables in OLS regressions is asymmetric because the independent variable restricts, limits, or determines the dependent variable. In RMA regressions, however, the relationship is symmetric and the assignment of the two variables is arbitrary and there is no cause-effect relationship between the two variables (i.e. body mass and body length) (Clarke, 1980; McArdle, 2003; Smith, 2009).

Determination of statistical significance of each of the regressions was ascertained using p-values and r2 values. The p-value, or observed significance level, allows for rejection of the null hypothesis if the value is 0.05 or smaller, the null hypothesis being that there is no relationship between the two variables (Peck et al., 2005). The coefficient of determination (r2) gives the proportion of variance in the dependent variable that can be explained by the independent variable. The coefficient ranges from 0 to 1, with increasing values indicating that a large percentage of the variation in the dependent variable can be explained by the linear relationship with the dependent variable (Peck et al., 2005).

Results

Regressions of body size versus clutch mass were preformed on two different groups of taxa. The first set of regressions included extant taxa from all 14 turtle families and the second set excluded the marine turtles. Marine turtles were excluded as we can be certain

117 the clutch does not belong to a marine turtle, based on clutch size and the paleoenvironment as inferred from the geology of the nest site.

The log-transformed regression of carapace length to clutch mass inclusive to all turtle families (n=143) was statistically significant with values of r2=0.635 and p<0.05. The equation for the regression is y = 2.0649x – 2.7195. The second log-transformed regression of carapace length to clutch mass that excluded marine turtles (n=136), was also statistically significant, although it had a slightly lower r2 value (r2=0.615, p<0.05). The equation for the regression is y = 2.0791x – 2.7544.

The log-transformed regression of body mass to clutch mass that included taxa from all turtle families (n=136) also showed a positive, statistically significant correlation (r2=0.670, p<0.05). The equation for the regression is y = 0.7455x – 0.1969. The log- transformed regression of body mass to clutch mass which excluded marine turtles (n=129), was also statistically significant, although the r2 value was slightly lower (r2=0.652, p<0.05) than the regression that included taxa from all turtle families. The equation for the regression is y = 0.7529x - 0.2221.

The calculated egg mass from the fossil clutch (TMP 2008.27.1) is 45.42 g; therefore, as the clutch contains 33 eggs, its mass is approximately 1498.85 g. Using this value, the turtle that laid the clutch had a carapace length of 71.6 cm or 71.2cm, based on regressions inclusive to all turtle families and exclusive to marine turtles, respectively. Additionally, the turtle that laid the clutch had a body mass of 33.4 kg or 32.6 kg, based on regressions inclusive to all turtle families or exclusive to marine turtles, respectively.

APPENDIX D: REVIEW OF TURTLE EMBRYOLOGY STUDIES *Asterisks indicates studies that examined ossification in turtle embryos. 118

Source Taxon Region of Embryo Examined Staging Criteria/Method Number of Stages Described

Rathke (1848) Emys orbicularis entire embryo days from ovipostion n/a Agassiz (1857) A combination of 31 different entire embryo days from ovipostion 31 (1-31) taxa Kunkel (1912) Emys lutaria skull sequential changes in morphology n/a *Shaner (1926) Chrysemys marginata skull sequential changes in morphology and size of n/a embryo Pasteel (1937) Clemmys leprosa entire embryo sequential changes in morphology 31 (1-31) Deraniyagala (1939) Eretmochelys imbricata entire embryo sequential changes in mophology and size of 6 (A-F) embryo Deraniyagala (1939) Dermochelys coriacea entire embryo sequential changes in mophology and size of 11 (A-K) embryo Milaire (1957) Pseudemys floridana entire embryo sequential changes in morphology 5 (16, 18-21) Milaire (1957) Emys orbicularis entire embryo sequential changes in morphology 11 (18-28) Pasteel (1957) Clemmys leprosa entire embryo sequential changes in mophology and size of n/a embryo Pasteel (1957) Pseudemys virginica entire embryo sequential changes in mophology and size of n/a embryo Pasteel (1957) Chrysemys picta entire embryo sequential changes in mophology and size of n/a embryo Penyapol (1958) Chelonia mydas entire embryo days from oviposition 7 (1-7) Domantay (1968) Chelonia mydas entire embryo days from oviposition 24 (1-24) Yntema (1968) Chelydra serpentina entire embryo duration of incubation and temperature 27 (0-26) Mahmoud et al. (1973) Chrysemys picta bellii entire embryo sequential changes in morphology (size duration 23 (1-23) of incubation also taken into consideration)

Burke and Alberch (1985) Chrysemys picta carpus and tarsus Yntema (1968) methodology 12 (14-25)

Burke and Alberch (1985) Chelydra serpentina carpus and tarsus Yntema (1968) methodology 12 (14-25)

Crantz (1982) Lepidochelys olivacea entire embryo sequential changes in morphology (duration of 31 (1-31) incubation also taken into consideration)

Miller (1985) Caretta caretta entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods Miller (1985) Chelonia mydas entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods Miller (1985) Chelonia depressa entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods APPENDIX D: REVIEW OF TURTLE EMBRYOLOGY STUDIES *Asterisks indicates studies that examined ossification in turtle embryos. 119

Source Taxon Region of Embryo Examined Staging Criteria/Method Number of Stages Described

Miller (1985) Eretmochelys imbricata entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods Miller (1985) Lepidochelys olivacea entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods Miller (1985) Dermochelys coriacea entire embryo combines both Yntema (1968) and Mahmoud 31 (1-31) (1973) methods Webb et al. (1986) Carettochelys insculpta entire embryo Yntema (1968) methodology 27 (0-26) Kuratani (1987) Caretta caretta orbital region carapace length 4 (I-IV) Kuratani (1989) Caretta caretta orbital region carapace length 5 (A-E) Burke (1989) Chelydra serpentina carapace Yntema (1968) methodology 4 (12-15) Renous et al. (1989) Dermochelys coriacea entire embryo Miller (1985) methodology 21 (10-31) Billet et al. (1992) Caretta caretta entire embryo Miller (1985) methodology 11 (14-25) *Rieppel (1993) Chelydra serpentina entire embryo Yntema (1968) methodology 10 (17-26) Guyot et al. (1994) Testudo hermanni entire embryo Yntema (1968) methodology 27(0-26) *Cherepanov (1995) Trionyx sinensis carapace Yntema (1968) methodology 16 -26 in 3 ranges Kuratani (1999) Caretta caretta chondrocranium carapace length 8 (I-VIII) Beggs et al. (2000) Carettochelys insculpta entire embryo Yntema (1968) methodology 15 (12-26) *Gilbert et al. (2001) Trachemys scripta carapace and plastron carapace length n/a *Gilbert et al. (2001) Chelydra serpentina carapace and plastron Yntema (1968) methodology n/a Tokita and Kuratani Pelodiscus sinensis entire embryo Yntema (1968) methodology 23 (5 to 27) (2001) Greenbaum and Carr Apalone spinifera entire embryo Yntema (1968) methodology, later stages (16+) 10 (17-26) (2002) based on morphology Greenbaum (2002) Trachemys scripta entire embryo Yntema (1968) methodology 15 (11-26) *Sheil (2003) Apalone spinifera entire embryo Greenbaum and Carr (2002) - modified from 10 (17-26) Yntema (1968) *Sheil and Greenbaum Chelydra serpentina entire embryo Yntema (1968) methodology 12 (14-26) (2005) *Sheil (2005) Macrochelys temminckii entire embryo Yntema (1968) methodology 10 (17-26) *Sanchez-Villagra et al. Caretta caretta autopodials Kuratani (1999) methodology same specimens as Kuratani (2007) (1999) *Sanchez-Villagra et al. Chelonia mydas autopodials Yntema (1968) methodology 6 (17-22) (2007) *Sheil and Portik (2008) Trachemys scripta limbs Greenbaum (2002) methodology 11 (15-25)

*Sanchez-Villagra et al. Pelodiscus sinensis entire embryo Tokita and Kuratani (2001) methodology 13 (15-27) (2009) *Bona and Alcalde (2009) Phrynops hilarii entire embryo Greenbaum and Carr (2002) - modified from 6 (19, 21, 22, 23, 26, hatchling) Yntema (1968) APPENDIX D: REVIEW OF TURTLE EMBRYOLOGY STUDIES *Asterisks indicates studies that examined ossification in turtle embryos. 120

Source Taxon Region of Embryo Examined Staging Criteria/Method Number of Stages Described

Werneburg et al. (2009) Emydura subglobosa entire embryo Standard Event System (Werneberg, 2009) *is a no numerical stages checklist of SES-characters APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 121

Stage Apalone spinifera (Sheil, Pelodiscus sinensis Chelydra Chelydra serpentina Macrochelys temminckii Pharynop hilarii (Bona Eretmochelys imbricata 2002; 2003) - Stages (Sanchez-Villagra et al. serpentina (Rieppel, 1993) (Sheil, 2002; Sheil and (Sheil, 2002; 2005) - and Alcalde, 2009) - (Sheil, 2002) - Stages determined from G+C, (2009)) - Staged based on - Stages determined by Greenbaum, 2005) - Stages determined by Staged based on G+C developed after Miller (2002) Tokita and Kuratani Yntema (1968) Stages determined by Yntema (1968) (2002) (1985) (2001) Yntema (1968)

17 Maxilla, Squamosal Postorbital Postorbital, Maxilla 18 Dentary, Prefrontal, Parietal, Prearticular, Prefrontal, Pterygoid, Surangular Pterygoid, Squamosal Squamosal, Dentary, Humerus, Femur 19 Frontal, Palantine, Parietal, Maxilla, Dentary, cervical Dentary Frontal, Parietal, No data angular, dentary, frontal, Postorbial, Pterygoid, and dorsal centra, hyo-, Prefrontal, Postorbital, jugal, maxilla, nasal, Dorsal Centra (1-10), hypo-, xiphiplastra Premaxilla, Maxilla, palatine, postorbital, Entoplastron, Hyoplastron, Vomer, Palantine, prefrontal, pterygoid, Hypoplastron, Pterygoid, Squamosal, vomer, splenial, Xiphiplastron, Dentary, Angular surangular, squamosal, Ventromedial Process of humerus, radius, ulna, Scapula, Coracoid femur, fibula, tibia, ento-, epi-, hyo-, hypo-, xiphiplastron

20 Angular, Coronoid, Jugal, Prefrontal, Parietal, Maxilla, Premaxilla, Jugal, Femur Coronoid, Prearticular, no data Prearticular, Quadrate, Squamosal, Surangular, Palantine, Vomer Premaxilla, Dorsal Centra, Quadratojugal, Epiplaston, entoplastron, epiplastra, Dorsal Ribs, Radius, Ulna, Nuchal Plate, Humerus, humerus, femur Dorsal Process Scapula, Radius, Ulna, MC II-IV, Tibia, Fibula, MT II-IV, Femur, Fibula, Tibia, MT Proximal Phalanx, Digit II-IV, Proximal Phalanx IV (pes) Digit I (pes) APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 122

21 Articular, Basiooccipital, Jugal, Palantine, Pterygoid, Basisphenoid, Dorsal Ribs, Quadratojugal, Surangular, Frontal, Parietal, Quadrate, parietal, premaxilla, Basisphenoid, Exoccipital, Frontal, Coronoid, Entoplastron, Epiplastron, Coronoid, Prearticular, Quadratojugal, Jugal, basisphenoid, exocipital, Prootic, Vomer, Axis Angular, Nuchal bone Hyoplastron, Ceratobrachial I, Axis and Vomer, Palatine, columella, cornu brachiale Centrum, Cervical Centra Hypoplastron, Atlas Centrum, All Columella, Basisphenoid, I, dorsal process of 2-8, Cervical Neural Xiphiplastron, Humerus, Cervical Centra, All Dorsal Cervical Centra, Sacrel scapula, ilium, ishium, Arches Pedicels, Dorsal Femur Centra, Dorsal Ribs II-IX, Vertebra, Caudal Centra, pubis, metatarsal I and IV, Centra, Dorsal Ribs 1-10, Sacral Centra I-II, Caudal Nuchal Osteoderm, Costal cervical centra, dorsal ribs Caudal Ribs, Sacral Neural Centra, Nuchal Osteoderms, Ventromedial Arches, MC I and V, Osteoderms, Epiplastron, Process of Scapula, All Phalanges Digits I-III Entoplastron, Hyoplastron, Phalanges Digits II-IV (manus), Proximal Hypoplastron, (manus), Ilium, Pubis Phalanges Digit Xiphiplastron, Humerus, IV(manus), Scapula, Ilium, Radius, Ulna, Tibia, Fibula Pubis, All Phalanges Digit III(pes) APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 123

22 Nuchal Plate, Sacral Quadrate, Basisphenoid, Exoccipitals, Exoccipital, Corocoid, Proximal coronoid, prearticular, Centra, All Phalanges Columella, dorsal ribs (I- Basioccipitals, Quadrate, Parabasisphenoid, Phalanges Digit IV (pes), basioccipital, opisthotic, Digits I, II, III (pes), IV), radius and ulna, fibula Stapes, Supraoccipital, Columella, Axis and Atlas Proximal and Medial parasphenoid, prootic, Ischium and tibia Prootic, Quadratojugal, Neural Arch, All Cervical Phalanx Digit III (pes), All quadrate, cornu branchiale Cervical Vertebrae, Dorsal Neural Arches, Costal Phalanges Digit II (pes), II, acromion, coracoid, MC Vertebrae, Costal Plates, Osteoderms, Dorsal and Distal and Medial Phalanx II to IV, MT II and III, Sacral Vertebrae, Caudal Anteriomedial Process of Digit I (pes) cervical neural arches, Vertebrae, Nuchal Plate, Scapula, Corocoid, MC III- dorsal centra, sacral centra Sacral Rib I, Dorsal Blade IV, Distal and Penultimate and neural arches, caudal and Ventromedial Process Phalanges Digits I-IV centra and neural arches, of Scapula, Radius, Ulna, (manus), Ilium, MT II-IV, costal plates Corocoid, Unguals Digit I- Distal Phalanx Digit IV (manus), Phalanges I(pes), Distal and Medial (manus), MC I-IV Phalanges Digit II(pes), (manus), Fibula, Tibia, All Phalanges Digit III Dorsal Process of Ilium, (pes), Distal and Medial Unguals Digit I-IV (pes), Phalanges Digit IV (pes), Pubis, Ischium, Proximal Proximal Phalanx Digit Phalanges and MT V(pes) APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 124

23 Opisthotic, Supraoccipital, Premaxilla (fuse before Opisthotic, Sacral Rib 2, Supraoccipital, Prootic, MC III and IV, Medial scleral ossicles, Atlas Centrum, Atlas staining is retained?), Neural Plates, MC V, Basioccipital, Phalanx Digit IV (pes), All supraoccipitals, epiothic, Neural Arch, Caudal Vomer, scapula and Phalanges Digit V (manus Epipterygoid, Phalanges Digits I and III corpus hyoidis, MC I and Neural Arches, Cervical acromium, phalanges and and pes) Ceratobrachial II, Pedicels (pes) V, phalanges in digits 1-3 Centrum, Cervical Neural MC (or stage 24) Dorsal and Sacral Neural (manus and pes), MT V, Arch, Costal Plates, Neural Arches, All Caudal Neural dorsal neural arches, Plates, Phalanges Digit V Arches, Metacarpal II-III nuchal plate, neural plates (manus), Terminal (manus) Phalanges Digit IV(manus)

24 Sacral Ribs, Caudal Postorbital, Quadratojugal, DC 2 (manus), MT V, All Cervical Intercentra, Prootic, Supraoccipital, no data Centra, Centrale 3 Basioccipital, Exoccipital, Astragalus, Calcanium Sacral Ribs I-II, MC I-II, Basioccipital, Opisthotic, (manus), DC 1-3, Prootic, Opisthotic, All remaining MC and Dorsal and Sacral Intermedium(manus and Prearticular, cervical and phalanges (manus), All Vertebrae, Sacral Ribs I, pes), DT 1-4, MT V, dorsal arches, caudal Phalanges Digit V (pes), Caudal Vertebrae, MC I, II Phalanges Digit V (pes) centra and arches, dorsal Ischium and V, All Phalanges Digit ribs (V-IX), costals (don't I (manus), Ischium, articulate until after Proximal and Medial hatching), coracoid, pubis, Phalanx Digit V (pes) ischium, ilium, phalanges and MT APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 125

25 Atlantal Intercentrum, Supraoccipital, Articular, Caudal Ribs, Atlas Epipterygoid, All no data 25 - Parietal, Prefrotal, Centrale 4 (manus), DC 4, Intercentrum, All Caudal Phalanges Digit V (manus Postorbital, Jugal, Maxilla, 5, MC V, Ulnare, Pisiform, Intercentra, Anteriormost and pes) Palatine, Pterygoid, Centrale 4 (pes), Fibulare Caudal Ribs Squamosal, Dentary, Surangular, Prearticular, Angular, Cervical Centrum, Dorsal Intercentra, Dorsal Ribs, Sacral Centra I-II, Nuchal Osteoderm, Humerus, Radius, Ulna, Femur, Tibia, Fibula, MT III APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 126

26 Epipterygoid Centrale 2 (manus), articular, intermedium, 25/26 - Frontal, Metatarsal V(pes), ulnare, distal tarsal V, Quadratojugal, Vomer, Intermedium(pes), DT 5 phalanges Basisphenoid, Coronoid, Premaxilla, Columella, Atlas and Axis Neural Arches, Cervical Neural Arches, Caudal Centra, Costal Osteoderms, Dorsal and Ventromedial Processes of Scapula, Coracoid, MC II-V, Distal Phalanx Digit I (manus), Proximal and Medial Phalanx Digit III and IV (manus), Proximal Phalanx Digit V (manus), Pubis, MT II and IV, Distal Phalanx Digit I (pes), Proximal and Medial Phalanx Digit III (pes), Proximal Phalanx Digit IV (pes)26 - Exoccipital, Ilium APPENDIX E: SEQUENCE OF OSSIFICATION OF EXTANT TURTLES 127

27 (after hatching) centrale 1 27/28 - Supraoccipital, and 2, pisiform, distal Basioccipital, Cervical carpals and tarsals,fibulare, Neural Arches, All Caudal intermedium, phalanges, Centra, All Caudal Neural sacral and caudal ribs, Arches, All Phalanges peripheral and suprapygal Digits I-IV (manus), plates. Ishium, MT I, All Phalanges Digits I-III (pes), Distal and Medial Phalanges Digits IV-V (pes)

28 Quadrate, Supraoccipital Median, All Dorsal Neural Arches, Sacral Neural Arches

29 Prootic, Opisthotic, Atlantal Intercentrum, Sacral Ribs I-II, All Caudal Centra, All Caudal Ribs, Ulnare, Intermedium, Pisiform, Distal Carpals 4- 5