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A. 21th {site )4 n.- I... .1 1r. . . . hi. 5‘ .ll 1&5..i113.519.vh ‘13,};19‘ 11.: 1.. 1.... 1.1!. lace» I... )1. 1.13.4}! o) it}... 0.. u) or . «its 1...... 1.1!... “will. 3" ‘3‘“. 1.1:, .19.? 1., 1.3.1.1131... 2 .tn.53llmt)|53|!5:u1.£v.. 11111.31!!!» (12...! 1.10.22.39.11... 1 X13315... .1. 1.3.1116“ 1.112111111113919;ij . . . . 3.1.1.1113}... Ill-1|. W. $art¥f1zaitii “Ki. 2 Ill... «1..» 1.11! 3)... 111 W) 1 It gléib; 1 1.1111111531111111... .. .. .. n . .1... .1231?! .1. pgu... u. 1. , I 1|...l‘rzvivsllhhaz: .0... 1| ‘ §l|t1-291L—l:> til .I’. 11!...1 . 1 i-.. 21.15.11, . 1. .1 ,. .. .. 2L. w E . . I. . 1 .5... t $5.1“ 1 .r. Milli!llllllllllllllfljlljlll Z RAF" 3 1293 02048 6 m ”at, This is to certify that the dissertation entitled ysiological Agents in Turtles (Reptilia: Testudines) presented by Kenneth Dale Andrews has been accepted towards fulfillment of the requirements for Ph .D. degree in Zoology ..~____._.__ Majorp so J. Alan Holmen Date 8/10/2000 MS U is an Affirmative Action/Equal Opportunity Institution 0-1277! PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11100 W.“ Skeletal Tissue as Physiological Agents in Turtles (Reptilia: Testudines) BY Kenneth Dale Andrews A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 2000 ABSTRACT Skeletal Tissue as Physiological Agents in Turtles (Reptilia: Testudines) By Kenneth Dale Andrews Neural bones from extant chelonians and fifteen bones from fossil chelonians were observed in the Scanning Electron Microsc0pe. The surface of the bones were examined to determine possible taxonomic differences and physiological uses of the vascular canals that interact with the surface of the shell. Different carapacial and plastral bones of one individual were examined for individual variation. Neural sections were extracted from extant turtles to make histological specimens. They were viewed to assess the compatibility of the structure of the carapace with previous explanations and to determine the Shell’s use as a metabolic unit for chelonians. Femora from extant chelonians were measured for total length and bisected at the narrowest margin. Measurements of the inside and outside diameters of the bones at both the narrow and wide sections of the bone were recorded. Calculations from these measurements were made for K (internal diameter/extemal diameter), KR (radius of marrow cavity), R (radius of outside measurement), t (thickness of bone), and Rh values. The outer surface of the carapace was found to be similar in appearance. The other parts of the carapace varied greatly in appearance and numbers of canals. Type A canals (perpendicular to surface) were found to be the most common type of vascular canal interacting with the outer surface of the carapace. Type B canals could be used as indicators for the total area of the surface occupied by vascular canals. Small vascular canals were found in the acellular outer layer of the carapace that was termed the subscute blood layer. This subscute blood layer allowed the blood to be affected quickly by the temperature of the outer surface (scutes) of the carapace. Cardiac shunting along with the blood flowing through the subscute blood layer was determined to be an active component of the thermoregulatory physiology of chelonians. Osteoclasts were observed in the histology sections of the carapace giving evidence for the carapace as an active storage area for Calcium. The K value derived from the femora showed a much smaller K value than any others reported in the literature. The lower K value indicated that the long bones had a very thick bone wall to support the weight of the shell. The thickened bone wall of the femur reflected the small amount of marrow that the long bone had and this lack of marrow was compensated for in the shell bones. The shell was found to be an active part of the metabolism of the chelonian as a thermoregulatory structure and a mineral storage area for the animal. ACKNOWLEDGMENTS I would like to thank my committee (Dr. J. Alan Homan, Dr. Thomas Burton, Dr. Karen Klomparens, and Dr. Donald Straney) for their support and guidance. There are many graduate and undergraduate students who have helped me in some way. Among them are Kenneth Ford, John Paul Zonneveld, Andrew Scwda, Maria Moscmovitz, as well as numerous other graduate students that discussed various topics with me. I would like to thank the histology department of the Michigan State University Medical School for their aid in determining the procedures needed for the decalcification process and use of their histology equipment. Finally, I would like to thank my wife, Susan, for her support in so many different ways. Without her support, I would have not been able to complete this work. TABLE OF CONTENTS PAGE Acknowledgments .......................................... i Table of Contents ....................................... ii List of Tables ......................................... iii List of Figures .......................................... v Introduction ............................................. 1 Materials and Methods ................................... 17 Results ................................................. 30 Discussion .............................................. 72 Literature Cited ....................................... 108 Appendix 1 ................... _ .......................... 112 Appendix 2 ............................................. 115 Appendix 3 ............................................. 121 LIST OF TABLES TABLE PAGE 1. Z distribution analysis data on significant differences in number of canal types for all chelonians examined. Sample variances were not significantly different ................................ 25 T distribution analysis data on significant differences in number of canal types for the family Emydidae. Sample variances were not significantly different ................................ 25 T distribution analysis data on significant differences in number of canal types for the family Testudinidae. Sample variances were not significantly different ......................... . ....... 25 Percentage area of the scanning electron microscope (SEM) images occupied by canals at both a standard threshold and a variable threshold for different surfaces of varied bony elements of a single Chrysemys picta specimen (MSU-H 2025) .................. 45 Percentage area of the scanning electron microscope (SEM) images occupied by canals at both a standard threshold and a variable threshold for fossil specimens .............................................. 45 Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for all Testudines ........ 59 Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of the family Emydidae ........................................ 59 Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of the family Testudinidae .................................... 59 Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of Chrysemys picta ........................................ 59 fl 10. 11. 12. 13. Percentage area of the scanning electron microscope (SEM) images occupied by canals at both a standard threshold and a variable threshold for fossil specimens ............................................ Linear regressions of femur measurements and calculations for all Testudines ...................... Linear regressions of femur measurements and calculations for members of the family Emydidae ...... Linear regressions of femur measurements and calculations for members of the family Testudinidae.. wi .61 .71 .71 .71 LIST OF FIGURES FIGURE PAGE 1. Side View of a turtle showing the arrangement of the carapace and plastron ................................... 6 2. Diagram of the bony elements of the carapace including 10. the nuchal (Nu), neurals (Ne), costals (C). peripherals (Pe),' suprapygals (S), and pygal (Py) .................. . ................................. 9 Diagram of the bony elements of the plastron including the epiplastron (Epi), hyoplastron (Hyo), hypoplastron (Hypo), and xiphiplastron (X) ............ 10 Conventions for the calculations of the bisected long bone measurements ................................ l4 Diagrammatic presentation of the three different canals and their arrangement with the surface of the carapace .............................................. 20 Scanning Electron Microscope (SEM) micrograph of the outer surface of a neural bone of Chrysemys picta (MSU—H 14309) showing an example of a Type A canal. 60x magnification ......................................... 21 Photograph of a hematoxylin and Eosin (H+E) prepared histological slide from Chrysemys picta (MSU-H 14309) showing an example of a Type A canal. 40X magnification ........................................ 21 Scanning Electron Microscope (SEM) micrograph of the outer surface of a neural bone of Chrysemys picta (MSU—H 14309) showing an example of a Type B canal. 60X magnification ......................................... 22 Photograph of a hematoxylin and Eosin (H+E) prepared histological slide from Chrysemys picta (MSU-H 14309) showing an example of a Type B canal. 40X magnification ......................................... 22 Scanning Electron Microscope (SEM) micrograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 14309) showing an example of a Type C canal. 60X magnification ........................................ 23 viii 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Photograph of a hematoxylin and Eosin (H+E) prepared histological slide from Chrysemys picta (MSU-H 14309) showing an example of a Type C canal. 40X magnification ....................................... 23 Scanning Electron Microscope (SEM) photograph of the bisected femur of Chrysemys picta (MSU-H 2025). BOX magnification ....................................... 28 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Trachemys scripta elegans (MSU-H 2716). 3000K magnification .......... 28 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon flavescens (MSU-H 2920). 320x magnification ........ 31 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Clemmys insculpta (MSU-H 4336). 2000K magnification .................. 31 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Trachemys scripta elegans (MSU-H 2716). 1000X magnification .......... 32 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Chrysemys picta (MSU-H 2025). 600K magnification .......................... 32 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Chelydra serpentina (MSU—H 3436). 360x magnification ................... 33 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chelydra serpentina (MSU-H 3436). 60X magnification ..................... 33 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 14309). 60X magnification .................... 35 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 14310). 60X magnification ................... 35 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta n 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. (MSU-H 14312). 60X magnification ................... 36 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon flavescens (MSU-H 2920). 60X magnification ......... 36 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon leucostomum (MSU-H 1414). 60X magnification ........ 37 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon subrubrum (MSU-H 2477). 60X magnification .......... 37 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the bisected carapace with the thin outer layer of compact bone shown between the non—staining scute and the deep spongy bone ................................. 38 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the spongy middle layer of a carapace with the marrow evident in the central areas ................................. 38 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing a close view of the interconnection of the scute, compact bone layer, and vascular canal ....................... 39 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the different paths for blood through the subscute layer ........... 39 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the subscute blood layer .......................................... 41 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the osteocytes in their lacunae ..................................... 41 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing an osteoclast dissolving bone ...................................... 43 photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing osteoblasts lining the trabeculae of spongy bone ............................ 43 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. Scanning Electron Microscope (SEM) photograph of the edge surface of a peripheral bone of Chrysemys picta (MSU-H 2025). 60X magnification ..................... 44 Scanning Electron Microscope (SEM) photograph of the inner surface of a peripheral bone of Chrysemys picta (MSU-H 2025). 60K magnification .................... 44 Scanning Electron Microscope (SEM) photograph of the outer surface of a costal bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 46 Scanning Electron Microscope (SEM) photograph of the outer surface of a peripheral bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 46 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU—H 2025). 60x magnification .................... 47 Scanning Electron Microscope (SEM) photograph of the outer surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 47 Scanning Electron Microscope (SEM) photograph of the edge surface of a costal bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 49 Scanning Electron Microscope (SEM) photograph of the edge surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 49 Scanning Electron Microscope (SEM) photograph of the edge surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 50 Scanning Electron Microscope (SEM) photograph of the inner surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 50 Scanning Electron Microscope (SEM) photograph of the inner surface of a costal bone of Chrysemys picta (MSU-H 2025). 60X magnification .................... 51 Scanning Electron Microscope (SEM) photograph of the inner surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60x magnification .................... 51 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Trionyx ferox n 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. (MSU-H 478). 60X magnification ..................... 53 Frequency distribution of average number of the three different canal types in the family Emydidae...54 Frequency distribution of average number of the three different canal types in the family Testudinidae ......................................... 55 Frequency distribution of average number of the three different canal types in Chrysemys picta ....... 57 Frequency distribution of average percentage area occupied by canals in the families of Testudines ..... 58 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chelus fimbriatus (MSU—H 2613). 60X magnification .......... 53 Scanning Electron Microscope (SEM) photograph of a “scrap" fossil of an unknown chelonian bone. 60X magnification .................................... 62 Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification .................................... 62 Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. ' 60X magnification .................................... 63 Scanning Electron Microscope (SEM) photograph of a “scrap" fossil of an unknown chelonian bone. 60X magnification .................................... 63 Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification .................................... 64 Scanning Electron Microscope (SEM) photograph of a “scrap" fossil of an unknown chelonian bone showing large foreign material on the surface of the fossil. 60X magnification ....................... 64 Scanning Electron Microscope (SEM) photograph of a “scrap" fossil of an unknown chelonian bone showing foreign material on the surface of the fossil. 60X magnification ........................... 65 Scanning Electron Microscope (SEM) photograph of mi a “scrap" fossil of an unknown chelonian bone showing erosion of the fossil material. 60X magnification ........................................ 65 Frequency distribution of average K (small) values for the families of Testudines ................ 67 Frequency distribution of average K (large) values for the families of Testudines ................ 68 Frequency distribution of average R/t (small) values for the families of Testudines ................ 69 Frequency distribution of average R/t (large) values for the families of Testudines ................ 70 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification ............... 73 Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification ............... 73 Number of Type A canals versus the number of Type B canals in all Testudines examined .................. 77 Percent Area occupied by all canals versus number of Type A canals in all Testudines examined .......... 78 Percent Area occupied by all canals versus number of Type A canals in the family Testudinidae .......... 79 Percent Area occupied by all canals versus number of Type A canals in Chrysemys picta .................. 80 Percent Area occupied by all canals versus number of Type A canals in the family Emydidae .............. 81 Percent Area occupied by all canals versus number of Type C canals in the family Testudinidae .......... 82 Percent Area occupied by all canals versus number of Type C canals in the family Emydidae .............. 83 Percent Area occupied by all canals versus number of Type C canals in Chrysemys picta .................. 84 Percent Area occupied by all canals versus number of Type C canals in the family Testudinidae .......... 85 xiii 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the subscute blood flow ........................................... 87 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing an osteoclast dissolving bone ................................................. 87 Total length of the femur versus the KR (small) value for all Testudines examined .................... 94 Total length of the femur versus the KR (large) value for all Testudines examined .................... 95 Total length of the femur versus the KR (small) value for the family Emydidae ........................ 96 Total length of the femur versus the KR (large) value for the family Emydidae ........................ 97 Total length of the femur versus the KR (small) value for the family Testudinidae .................... 98 Total length of the femur versus the KR (large) value for the family Testudinidae .................... 99 K (large) value versus the KR (large) value for all Testudines examined ............................. 100 K (small) value versus the KR (small) value for all Terstudines examined ............................ 101 K (large) value versus the R/t (large) value for all Testudines examined ............................. 102 K (small) value versus the R/t (small) value for all Testudines examined ............................. 103 Hematoxylin and Eosin (H+E) photograph of a carapace showing the single cell layer that produces the scute of the shell ........................................ 106 xw INTRODUCTION The morphology of shell and long bones in turtles has been studied in some detail, but little work has been conducted to determine if and how these elements are involved in the physiology of this animal, encumbered as it is by a massive shell. This study will examine the long bones and shells of testudines to look for mechanisms of physiological control exerted by these structures. The surface of the carapace will be examined for morphological structures used to regulate or control blood flow. 'Physiological areas of study included will be temperature control by blood flow throughout the carapace, the carapace and long bones as an agent for calcium and phosphate reserves, and weight constraints of the shell on both the morphology of the limbs as well as a factor in the size of the marrow cavity. As a prefix to the text that is to follow, a general introduction to types of bone and a review of previous turtle shell and long bone work is given as follows. TyPes of Bone The skeleton of vertebrate animals is composed of mineralized connective tissue commonly called bone. The primary step in the formation of skeletal tissue is the synthesis of collagen by fibroblasts. Collagen is a proteinaceous fibril that aggregates to form bundles. These bundles are woven into compact networks. It is on this network that the Calcium Phosphate and collagen fibers are deposited to form bone. The bony tissue of vertebrate animals has been classified into six main groups (Kent, 1987). These groups are categorized as compact, spongy, dentin, acellular, membrane, and replacement bone. Compact bone (also called Haversian bone) is characterized by lamallae of mineralized collagenous bundles arranged concentrically around a Haversian canal. These Haversian canals are the channels where the vascular tissue brings blood to the bone cells. Spongy bone (also called Cancellous bone) is characterized by bony trabeculae and marrow. Trabeculae are an assemblage of beams, bars, and rods that, like architectural trusses, form a rigid framework that provides maximum strength at areas of stress. These trabeculae are irregularly arranged lamellae without Haversian canals. Dentin is the material that covers teeth of vertebrates and scales of ganoid and elasmobranch fishes. It has the same constituents as compact and spongy bone except that the odontoblasts (dentin forming cells) are not trapped during osteogenesis. Thus, the odontoblasts are always at the inner border of the bone. These odontoblasts leave behind canaliculi which are the protoplasmic processes for the dentin. Acellular bone (also called Aspidin) is characterized by the osteoblasts (bone forming cells) that retreat as they deposit bone (as in dentin) and in addition leave no canaliculi behind (Bloom and Fawcett, 1962). Membrane and Replacement bone are characterized by their different depositional patterns. Before bone can be deposited, a preskeletal blastema must develop. A blastema is an aggregation of mesenchyme that differentiates into varied tissues. Once the preskeletal blastema is formed, some mesenchyme cells become fibroblasts and secrete collagen while others become either osteoblasts or chondroblasts (cartilage forming cells) and secrete enzymes essential for the formation of bone or cartilage. The collagenous matrix is then impregnated with hydroxyapatite crystals. Membrane bone is deposited directly within a membranous blastema without having been preceded by a cartilaginous model. Membrane bone may be compact or spongy, and lamellar or non-lamellar. Because of the arrangement of the blood vessels that participate in the deposition of the bone, membrane bone lacks Haversian canals. Dermal bone is derived either ontogenetically or phylogenetically from the dermis of the skin . The term Dermal bone denotes its history, not its histologic features. Dermal bone is a form of membrane bone. Replacement bone is deposited where hyaline cartilage already exists. In this process, the cartilage degenerates and eventually disappears. In replacement ossification, the cartilage must be removed before the hydroxyapatite crystals (bone) may be deposited. The cartilage is replaced by spongy bone which may later be eroded and replaced by compact bone, spongy bone, or a marrow cavity, depending on its location. Bones of Testudines Suzuki (1963) grouped the bones of chelonians into three major classifications. These classifications are Primary Vascular bone, Endosteal Haversian bone, and Avascular bone. Primary vascular bone was subdivided into three categories. These subdivisions were longitudinal, reticular, and prothaversian. Longitudinal primary vascular bone has vascular canals that are parallel to the long axis of the osseous tissue. Reticular primary vascular tissue has vascular canals that are patterned in a network. Prothaversian primary vascular tissue has osseous lamellae arranged concentrically around large irregularly shaped primary vascular channels or marrow cavities simulating Haversian canals. Endosteal Haversian bone has concentric lamellae of bone deposited around a rounded vascular channel and is Confined to the endosteal margin of the bone. Avascular bone has no vascular canals and the osteocytes are arranged concentrically around the marrow cavity. Carapace and Plastron The turtle shell (carapace and plastron) is morphologically arranged as a domed carapace and a flat plastron (Fig. 1). The outermost covering of shell structures is a horny covering primarily composed of Keratin called scutes. These scutes are periodically replaced as the turtle grows (Cagle, 1950). Underlying the scutes are the bony elements. The bony elements are comprised of two layers, the superficial epithecal layer and the central thecal layer. The epithecal layer is only present in marine and some freshwater turtles (Suzuki, 1963). Adult chelonians have apositional growth of the bony elements on the inner and outer surface forming layers of compact bone on both surfaces. These are called the inner and outer tables while the thecal layer has been called the intermediate diploe (Wallis, 1927). The outer table consists of a primary longitudinal vascular pattern and the inner table is a primary reticular vascular pattern. The vascular channels near the sutures of the bones are arranged .conumoam .H wnsmflm m o 3wfi> mpfim bamfimmnmwwm may mqflsosm Hawnm mabndu u one mo Ugo commando nub m.>m.>eo.m . “NV.” «)3: or“ a. . 1... 3...... 3.»: 1.3;). 3f" . .. w p—.>.m 4 m 0 Z transversely to the plane of the sutures and gradually modified as they anastomose with the more mature parts of the carapace (Suzuki, 1963). Enlow and Brown (1957) noted differences between the vascular patterns in the outer and inner table of a dermal bone from a Cretaceous turtle, Glyptops. No mention was made if such variation existed in modern turtles. The shell is composed of modified vertebral column elements, ribs, and dermal bones except for Dermochelys (Zangerl, 1939). The turtle body plan represents a major 'exception of the structural “bauplan” (body plan) of the vertebrates. This exception is that the girdle elements of the chelonian skeleton are inside the ribs instead of outside the ribs as in all other vertebrates (Burke, 1989). The carapace of most turtles is composed of six different bony elements arranged in an ovoid shape. A central row of bones (nuchal, neurals, suprapygal, and pygal) are surrounded by rectangular bones (costals). Finally, arranged around the outer rim of the costals are the peripherals (Ernst and Barbour, 1989) (Fig. 2). The plastron of most chelonians is composed of four paired bony plates (epiplastron, hyoplastron, hypoplastron, and xiphiplastron) and a single entoplastron between the epiplastra and hyoplastra (Fig. 3). The number and arrangement of these bones has some familial and species specific variations (Hay, 1908; McDowell, 1964). There are also extra bones that are ,present in some groups. Examples of this is the presence of a mesoplastron between the hyoplastra and hypoplastra in some taxa of the Family Pelomedusidae and in Claudius of the Kinosternidae. Previous Shell work The testudine carapace has been widely used in taxonomic studies. The main characters that have been used are scute pattern, bone suture pattern, and relationship of scutes to the underlying bone (Pritchard, 1979; Ernst and Barbour, 1989). There are occasional reports of bone abnormalities in the shell. These anomalous bones are almost always reported the abnormalities with no explanation as to cause or effect of the abnormalities (Newman, 1906; Procter, 1922). Turtles have one of the largest body mass to body volume ratios of the vertebrates (Hall, 1924). This is caused mainly by the bony shell. The extra weight that chelonians carry causes major changes in the animals habits and mode of locomotion. The testudinal gait reflects the need to exert enough force to move a body along with the burden of a heavy shell (Jayes and Alexander, 1980). The shell restricts the range of motion in many turtles and forces the animals to move in short strides. This gait Figure 2. Diagram of the bony elements of the carapace including the nuchal (Nu), neurals (Ne), costals (C), peripherals (Pe), suprapygals (S), and pygal (PY). Figure 3. Diagram of the bony elements of the plastron including the epiplastron (Epi), entoplastron (En), hyoplastron (Hyo), hypoplastron (Hypo), and xiphiplastron (X). 10 allows the weight of the shell to reside almost entirely on one leg at a time (Alexander, 1982). Marvin and Lutterschmidt (1997) determined that the stride length of Terrapene carolina was affected by their body mass. The more weight that the chelonian carried, the shorter the length of the stride. The shell has generally been assumed to be metabolically inert (Hall, 1924; Benedict, 1932; Hutton, et al., 1960; Hughes, et al., 1971; Dunson, 1986). However, using regression equations, Bennett and Dawson (1976) failed to detect any significant differences in body weight versus metabolic rate in lizards, snakes, and turtles. This indicates either (1) that the shell is metabolically active or (2) that the metabolic rate of other tissues in turtles is enough higher (20-40%) than other reptiles to compensate exactly for the inert character of the shell in the total metabolism (Bennett and Dawson, 1976). The carapace has been reported to have no seasonal change in density, (Suzuki, 1963), thus supporting the hypothesis that the carapace is metabolically inert. In a study of Pseudemys and Chelydra shells, application of radiant heat to the carapace increased local blood flow; whereas cooling of the carapace decreased it, implying that local blood flow can be controlled by the animal (Avery, 1982); Changes in heart rate also aid in 11 rapid absorption or radiation of heat (Zug, 1993). Zug (1993) also stated that to retard cooling, reptiles reduce heart rate and peripheral circulation. The reduction of the heart rate and reduction of the peripheral circulation or the pulmonary circulation is commonly called cardiac shunting (Hicks and Wang, 1996). Long Bones . The limb bones of chelonians allow growth only on the inner surface of the marrow filled cavity. This leads to reduction in the size of the marrow cavity over time. Reabsorption of bone tissue acting as a calcium reserve in the long bones occurs for metabolic purposes (Suzuki, 1963). It is known that the endosteal long bones have lamellar layers of growth (Enlow, 1969). This allows the assumption that the marrow cavity of the long bones of turtles is reduced over time. Long bones are much stronger or resistant to bending if the center section (marrow cavity) is hollow or filled with a material less dense than the bone (Alexander, 1982). The strength of these long bones can be calculated if the material in the cavity is known (Currey and Alexander, 1985). .Turtle long bone cavities are filled with marrow which is about 0.44% as dense as bone. The direction from which pressure is exerted on a long bone also determines its strength or resistance to bending. 12 These "stresses" can also alter the shape of the long bone during growth (Currey, 1984). Additional pressure on chelonian limb bones is caused by the excess weight of the shell. Comparison of the size of the marrow cavity to the thickness of the bone wall (Currey and Alexander, 1985) is labeled as K (Fig. 4). K may range from 0.0 (where the bone is solid) to near 1.0 (where the bone is very thin with a large marrow cavity). This value depends on whether it is selected for yield strength, fatigue strength, ultimate strength, impact strength, or stiffness. Yield strength is described as the tissue being strong enough not to yield under the greatest bending moments likely to act on it. Fatigue strength is described as the tissue being strong enough not to fail by fatigue under the bending moments expected to act repeatedly on it. Ultimate strength is described as the tissue being strong enough not to fracture under the greatest bending moments likely to act on it. Impact strength is described as the tissue being strong enough in bending under impact loading. Stiffness is described as the tissue being stiff enough in bending. The optimum values for these were computed so that the K values would reflect the limiting factor on the limb being examined. These optimum values are 0.67 (Ziglg or Fatigue Strength); 0.56 (Ultimate Strength); 0.75 (Stiffness); and 13 .mEoEoSmmoE SEE 3835 2: mo mecca—:28 2: com $598280 .v 835 8 88m Eco mafiefi 2: 8 9: 2082882 286330 2.: Co mega 2: mo cued M $— 56830 =2:me 2: 8 88:35 BEBE 2: .8 23m n v— ocom 05 Co 36562:. n a beau 36:32 05 do 36am H xv— EoEoSmaoE 86:5 2: mo Exam n ~— 14 0.55 (Impact Strength). Currey and Alexander (1985) also compared the K values in limb bones of mammals, birds, and reptiles. They concluded that air filled bones of birds have very high K values due to the fact that bird bones require much bending strength and require a minimized mass for flight. Mammals have lower values of K as they increase in size. Aquatic animals like the alligator and marine mammals have low values of K, as this loss of marrow size is thought to aid the animal in attaining neutral buoyancy. Currey and Alexander (1985) examined the ratio of the ratio of the radius of the complete bone (R) to the thickness of the bone wall (t), which yields the value R/t. This value is used to emphasize how greatly the shape changes as K approaches one. The value (R/t) is often used as well as or instead of K. R/t equals 1/(1-K). If the marrow cavities of the long bones of testudines were the only place for the reabsorption of calcium, one would assume that the long bones would be reduced over time (ontogeny) and the strength of the bone would be greatly affected by the current calcium status of the turtle in question. Previous Long Bone work The long bones of the unique pelagic turtle Dermochelys coreacea (Rhodin, et al., 1981) were studied relative to the 15 quantity of vascular canals in their cartilaginous plates. It was found that the chondro—osseous plates of Dermochelys has many vascular canals, a characteristic that is shared among many different marine vertebrates, but that is unique among the reptiles. The osseous growth marks of long bones have been studied to indicate the age of turtles (Castanet and Cheylan, 1979) and it has been shown that the growth marks are useful in determining the age of turtles up to about twenty years. 16 MATERIALS AND METHODS . Turtle shell bones were studied under the SEM to reveal the minute canalicular vascular system within them and histological slides were made to detect cells, such as osteoclasts, that would reflect metabolic activity in the shell. Long bones (femora) were sectioned and analyzed to reflect mechanical and physiological factors related to the restraints imposed by the shell. SEMZMethods The neural plates of 44 specimens from 9 families of Testudines were viewed in the Scanning Electron Microscope (SEM) in order to view their minute canalicular vascular system within them. Carapacial bones from 15 fossil turtles were also viewed in the SEM. Varied skeletal elements (neural, peripheral, costal, and plastral) from one Chrysemys picta specimen (MSU-H 2025) were viewed. Moreover, several different areas of the outer surface of the above neural were examined to evaluate variability within it. The neural plates of the extant species were either taken as a complete neural bone from disarticulated specimens or a section of the neural bone was cut from whole specimens by using a dremel tool. 17 The fossil turtle specimens were taken as whole fragments of “scrap” fossil chelonian specimens from the MSU Museum collection. Scrap fossils are pieces that are usually identifiable only to the generic level. The neural plates were cut to sizes compatible with mounting on stubs. After cutting, the bones were mounted on the stubs with an adhesive. The mounted bones were critical point dried (Balzers Critical Point Dryer). After critical point drying, the mounted bones were sputter-coated with gold (Emscope Sputter Coater). Each mounted bone was then viewed in a Scanning Electron Microscope (SEM) (model JEOL JSM-35C). Micrographs were taken with the SEM at sixty times normal size to allow for maximal viewing of the neural with the best clarity. These micrographs also could be enlarged to many times the current magnification with little or no reduction in the clarity of the micrographs. Some neurals were examined under high magnification to assess the type of bone growth on the surface of the shell as well as the lining of the vascular canal. The different shell elements from one specimen of Chrysemys picta (MSU-H 2025) were observed to determine if variation occured in the different surfaces of each bone. SEM micrographs were taken from each elements outer, edge and inner surfaces to assess the variation of these 18 elements. The SEM micrographs were digitized by scanning into computer formats (TIFS). The computer files were then used in area analysis using Bioscan Optimas. The area analysis was collected using two different light intensity thresholds. The first threshold was set at a standard of one hundred (100) for all specimens for control purposes. The accurate method used a variable light intensity threshold that filled all of the holes allowing no bleeding of light into the bony tissue. The resulting numbers yielded percentages of the area of the micrograph that was occupied by canals. The vascular canals of the bones were divided into three categories. These were Types A, B, and C (Fig. 5). Type A vascular canals have a perpendicular arrangement with the outer surface of the carapace with no retreating vascular canal from the surface (Figs. 6 and 7). Type B 'vascular canals have a nearly perpendicular arrangement with the outer surface and an immediately adjacent retreating vascular canal (Figs. 8 and 9). Type C vascular canals have a tangential arrangement with the outer shell with a long parallel junction with the outer surface before the retreating tangential vascular canal (Figs. 10 and 11). A Z-distribution analysis of canal types compared with each other was conducted for all testudines (Table 1). T- 19 TYPE A CANAL TYPE B CANAL TYPE C CANAL Figure 5. Diagrammatic representation of the three different canals and their arrangement with the surface of the carapace. 20 ‘Figure 6. Figure 7. Scanning Electron Microscope (SEM) micrograph of the outer surface of a neural bone of Chgysemys picta (MSU-H 14309) showing an example of a Type A canal. 60X magnification. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide from ghgyggmyg picta (MSU-H 14309) showing an example of a Type A canal. 40X magnification. 21 Figure 8. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU—H ) showing an example of a Type B canal. 60X magnification. Figure 9. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide from Chrysemys picta (MSU—H ) showing an example of a Type B canal. 40X magnification. 22 Figure 10. 23%. I ‘ -"M’ "‘ 1 . ‘- ”-5.370 8 O . . V r . . - - ~ 6‘ . s . 1 ” ‘ . .5. - ‘ . . . ‘ 6-. O -0 . "Q a. - o - ‘- . . . . I. . .' g e C 9 . D ". . 3‘0 .. ' o ' ‘ ‘- \- -‘ O Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU—H ) showing an example of a Type C canal. 60X magnification. Figure 11. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide from Chrysemys picta (MSU-H ) showing an example of a Type C canal. 40X magnification. 23 distribution analyses of the canal types compared to each other were conducted for the families Emydidae (Table 2) and Testudinidae (Table 3). Linear regressions were performed for each type of canal (A, B, and C) compared to the percent area of the canals. The tests were performed for all turtles examined, the members of the family Emydidae, the members of Chrysemys piggg, and the members of the family Testudinidae. These data were derived from the SEM photographs previously scanned into the TIF files. The level of significance was placed at .05. All statistical analyses were conducted using the Softstat program (Softstat, 1996). Histological Specimens Histological slides of turtle bones were made in order to identify cells capable of physiological activity. Neural bones (N2) of a formalin preserved specimen of Trachemys scripta elegans, Chelydra serpentina, and Terrapene carolina carolina were cut from.the complete individual using a dremel tool. The pieces were then decalcified using the formdc acid method (Sheehan and Hrapchak, 1980). The specimens were placed in a formic acid solution to allow for the tissues decalcification. The formic acid solution contained 500 ml of 88% formic acid and 500 ml of 10% neutral buffered formalin. The bones were washed in the solution for 24—48 hours. The bones were then washed with 24 Table 1. Z distribution analysis data on significant differences in number of canal types for all testudines examined. Sample variances were not significantly different. Taxa Average Variance Z—value Prob>T Type A Canals 44.02 29.57 vs. 14.26 0.00 Type B Canals 3.64 10.01 Type A Canals 44.02 350.89 vs. 1 1.19 0.00 Type C Canals 9.69 72.49 Type B Canals 3.64 10.01 vs. -4.46 0.00 Type C Canals 9.69 72.49 Table 2. T distribution analysis data on significant differences in number of canal types for the family Emydidae. Sample variances were not significantly different. Taxa N Mean T DF Prob>T Type A Canals 28 51.36 vs. 18.24 27 0.00 Type B Canals 28 3.21 Type A Canals 28 51.36 vs. 12.80 27 0.00 Type C Canals 28 10.28 Type B Canals 28 3.21 vs. 4.79 27 0.00 Type C Canals 28 10.28 Table 3. T distribution analysis data on significant differences in number of canal types for the family Testudinidae. Sample variances were not significantly different. Taxa N Mean T DF Prob>T Type A Canals 7 29.57 vs. 4.39 6 0.00 Type B Canals 7 4.00 Type A Canals 7 29.57 vs. 3.09 6 0.02 Type C Canals 7 7.43 Type B Canals 7 4.00 vs. 129 6 0.23 Type C Canals 7 7.43 25 ‘- ’1 I running water for 3 to 8 hours. After washing, the bones were mounted in paraffin and then sectioned to 4 micrometers. The sections were mounted and stained with Hematoxylin and Eosin using Sheehan and Hrapchak’s method (1980). These histological slides were compared with descriptions of the histological arrangement of carapacial bones in Suzuki (1963). The slides were viewed for the presence of cells that indicate metabolic activity in the shell such as osteoclasts and were viewed for the presence of other structures associated with physiological activities. Long Bone Specimens Long bones were-sectioned and analyzed to reflect mechanical and physiological factors related to the burden of the shell. Femora from.49 specimens from 6 families were obtained from skeletal collection specimens of the Michigan 'State University Museum (MSU-H). The femora were cut at the narrowest point of diaphyses using a dremel tool. I The greatest total length of the femur was measured before the specimens were cut with the dremel tool. The femora were measured using vernier calipers to the nearest hundredth of a millimeter. The femora of chelonians are not round as the cross section of the bone has a roughly triangular shape (Fig. 12). This triangular shape gives the 26 bone both a narrowest and widest point. This allowed for two complete sets of measurements. Sets of measurements were made of the bisected femora at the inside diameter of the marrow cavities and the outside diameter of the femora. Inside measurements were made by inserting the vernier calipers into the outermost section of marrow cavity in the long bone. The measured values of the bisected femora allowed for many other values to be calculated (Appendix 3). Both sets of data were used for each set of calculations. These calculations start with the value K, which is defined as the ratio of the outer bone diameter to the inner bone diameter. The bone has a radius R (half the measured diameter) and a marrow cavity of radius KR (half the measured inside diameter). The thickness of the bone wall (t) results from subtracting the inside diameter from the outside diameter. Finally, the ratio of the radius of the complete femur (R) to the thickness of the bone wall (t), will yield R/t. Linear regressions were performed of the different measurements and calculations from.the long bones to determine which of the measurements or calculations would be most useful in determining relationships of turtles on the basis of their long bones. The measurements and calculations used in the linear regressions were total length of the bone, K (large), K (small), KR (large), KR 27 -Figure 12. Scanning Electron Microscope (SEM) photograph of the bisected femur of Chrysemys picta (MSU- H 2025). 30X magnification. Figure 13. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Trachemys scripta elegans (MSU-H 2716). 3000X magnification. 28 (small), R/T (large), R/T (small). These regressions were performed for all turtles examined, members of the family Emydidae, and members of the family Testudinidae. The level of significance for these regressions was placed at the .05 level. Images in this dissertation are presented in color. All Hematoxylin and Eosin (H+E) prepared photographs are color photographs. 29 RESULTS This section presents the results of the SEM studies, the histology work and the analyses of the long bone data. Types of bone throughout the shell Neurals that were examined under high magnification (up to 6600X) showed that the outer layer of the shell was composed of acellular bone composed of a fibrous matrix in which the orientation of collagenous fibers are in a random arrangement (Enlow, 1969)(Figs. 13, 14, and 15). The lining of the vascular canals were also composed of acellular bone (those areas that could be seen with the SEM)(Figs. 16, 17, and 18). The surfaces of the different chelonian neurals were structurally diverse. They all had the same type of bone (acellular) on their surface although the appearance of the matrix was diverse. -Ace11ular bone is characterized by a unorganized aggregation of collagen fibril bundles (Bloom and Fawcett, 1962). The collagen fibers in some photographs were very large and gave the matrix a rugose appearance. ’ The Chelydra serpentina specimen is a good example of this rugose matrix (Fig. 19). The collagen fibers in other photographs were small and gave the matrix a smooth 30 Figure 14. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon flavescens (MSU—H 2920). 320x magnification. Figure 15. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Clemmys insculpta (MSU-H 4336). 2000X magnification. 31 Figure 16. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Trachgmys scripta elegans (MSU-H 2716). lOOOX magnification. Figure 17. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Chgysgmys picta (MSU—H 2025). 600x magnification. 32 Figure 18. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone showing the lining of one of the vascular canals of Chelydra serpentina (MSU-H 3436). 360x magnification. Figure 19. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chelydra serpentina (MSU—H 3436). 60X magnification. 33 appearance. The Chrysemys picta specimens have a smooth appearing matrix (Figs. 20, 21, and 22). A few neurals had matrix that appeared granular. This appearance comes from the lack of collagen fibers with the primary amount of matrix being the calcium phosphate. The members of the Family Kinosternidae are good examples of this type of matrix (Figs. 23, 24, and 25). The cross sectional slides of the shell in Trachemys scripta elegans, Chelydra serpentina, and Terrapene carolina carolina were similar in that they all had an outer table and an inner table (diploe) of compact bone (Fig. 26) and a middle section of spongy bone (Fig. 27). The outer and inner tables were proportionally thin as the middle spongy layer comprised most.of the shell. This spongy layer was the location of the majority of the canals that carry blood to the surface of the shell as seen in the SEM photos. The canals are lined with acellular bone unlike the trabeculae found in the spongy middle layer composed of trabecular bone and marrow. The canals do not form a direct route to the surface of the shell, but follow a tortuous winding path to the surface and back down again to the marrow. In fact, it is the type of connection of these canals to the surface that defines the structural makeup of the different canal types (see fig. 28). A feature observed here that has not been previously 34 -Figure 20. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 14309). 60X magnification. Figure 21. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 14310). 60X magnification. 35 Figure 22. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU—H 14312). 60X magnification. Figure 23. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon flavescens (MSU-H 2920). 60X magnification. 36 Figure 24. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon leucostomum (MSU—H 1414). 60X magnification. Figure 25. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Kinosternon subrubrum (MSU—H 2477). 60X magnification. 37 . v, 4': Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the bisected carapace with the thin outer layer of compact bone shown between the non—staining scute and the deep spongy bone. Figure 26. 4 Figure 27. Photograph of a Hematoxylin and Eosin (HPE) prepared histological slide showing the spongy middle layer of a carapace with the marrow evident in the central areas. 38 Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing a close view of the interconnection of the scute, compact bone layer, and vascular canal 1"! Figure 28. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the different paths for blood through the subscute layer. Figure 29. 39 r1“ {)1 _l p _I (I) (r discussed in the literature occurs between the outer table of bone and the scutes of the shell as a thin layer of blood. This blood moves from relatively large canalsmto canals of (as small as) a single blood cell in thickness. These subscute blood flow is immediately beneath the scutes with no bone between the capillary and the scute. This arrangement would allow the blood in the capillary to absorb or radiate heat with no interference from the thick acellular bone. The scute would be the only interference with the ambient temperature of the external environment. Bone is much less dense than Scutes. This allows the keratin of the scutes to change temperature in relationship to its surroundings much faster than bone (Monteith, 1973). The blood flowing through the capillaries was contiguous with other blood in the system except that it was not determined how its flow was directed through the subscute layer of the shell (Figs. 29 and 30). Netebolic materials observed in the shell There were no living cells detected in the thin inner and outer tables. The middle layer of spongy bone was very similar in appearance to the marrow cavity of a long bone as trabeculae were present in large quantities. Osteocytes were also observed in the trabeculae of the spongy middle layer (Fig. 31). These “mature bone cells" maintain normal bone structure by recycling the calcium salts in the bony 40 - _ a ,- ‘ Photograph of a Hematoxylin and Eosin (H+ prepared histological slide showing the subscute blood layer. v; Figure 30. Figure 31. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the osteocytes in the lacunae. ~41 matrix around themselves and assisting in repairs (Martini and Bartholomew, 2000). Osteoclasts are large .multinucleated cells that secrete acids to break down bone and release the calcium and phosphate that is bound in the tissue (Martini and Bartholomew, 2000). Osteoclasts were observed in the process of osteolysis (Fig. 32). Osteoblasts are cells that produce the fibers and matrix of bone or the production of new bone (Martini and Bartholomew, 2000). Osteoblasts were observed lining the marrow cavities of the spongy bone (Fig. 33). “variation of the shell of an individual Organimm The elements from a single specimen of Chrysemys picta (MSU-H 2025) showed large variations in the number and types of canals. The percentage of canals compared to the bony tissue ranged from 5.34% to 42.35%. The only two measurements that exceeded 15% occurred in the edge of the peripheral bone (Fig. 34) and the inner surface of the peripheral bone (Fig. 35)(Table 4). The photomicrographs of the outer surfaces of the costal, peripheral, and neural were all similar in appearance with large numbers of Type A canals and moderate numbers of Types B and C canals (Figs. 30, 31, and 32). .The outer surface of the plastron had differences in types of canals present. Only Type C canals occurred in the outer surface photograph of the plastron (Fig. 39). Cross cut edge photographs of all specimens were 42 Figure.32. 'Photograph of a' ematoxy in an; oSih’YH+E) prepared histological slide showing an osteoclast in the process of dissolving bone. Figure 33. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing osteoblasts lining the trabeculae of spongy bone. 43 Figure 34. Scanning Electron Microscope (SEM) photograph of the edge surface of a peripheral bone of Chrysemys picta (MSU-H 2025). 60X magnification Figure 35. Scanning Electron Microscope (SEM) photograph of the inner surface of a peripheral bone of Chrysemys picta (MSU—H 2025). 60X magnification. Table 4. Percentage area of the scanning electron microscope (SEM) images occupied by canals at both a standard threshold and a variable threshold for different surfaces of varied bony elements of a single casemys picta specimen (MSU-H 2025). Shell Element Surface % Area (100) % Area (Var) Var. Neural Outer 09.71 10.09 110 Neural Inner 16.86 06.83 40 Neural Edge 14.60 08.78 50 Peripheral Outer 20.71 12.34 57 Peripheral Inner 48.13 26.26 45 Peripheral Edge 66.22 42.35 45 Costa] Outer 15.00 08.99 57 Costa] Inner 26.55 12.83 46 Costa] Edge 60.19 12.88 23 Plastral Outer 23.18 10.69 42 Plastral Inner 28.68 07.84 48 Plastral Edge 76.3 1 05.34 30 Table 5. Percentage area of the scanning electron microsc0pe (SEM) images occupied by canals at both a standard threshold and a variable threshold for fossil specimens. MSU-VP # % Area (100) % Area (V ar.) (Var) 847 08.36 09.04 135 849 09.04 07.91 91 Williston, FL 10.06 06.13 70 Williston, FL 09.40 07.79 80 Keya Paha Co., NE 12.82 10.27 75 Keya Paha Co., NE 09.02 11.76 130 Keya Paha Co., NE 11.47 09.53 85 Keya Paha Co., NE 09.51 09.58 105 Keya Paha Co., NE 09.18 08.92 90 Keya Paha Co., NE 08.93 08.55 95 Keya Paha Co., NE 12.52 11.40 85 Keya Paha Co., NE 09.06 08.46 90 Keya Paha Co., NE 07.50 08.25 110 Keya Paha Co., NE 50.88 09.82 40 Keya Paha Co., NE 20.40 07.25 50 Keya Paha Co., NE 09.40 07.79 80 45 Figure 36. Scanning Electron Microscope (SEM) photograph of the outer surface of a costal bone of Chrysemys picta (MSU-H 2025). 60X magnification. Figure 37. Scanning Electron Microscope (SEM) photograph of the outer surface of a peripheral bone of Chrysemys picta (MSU-H 2025). 60X magnification. 46 __ _ 1 a9." ‘7‘ -"\_ .v-‘\ a.“ ‘ ‘A. A fl: A.“ ’\7 flax, fr 2‘! ,\l- -\.lllrlll\.r.\ll;\ll\l)\lil\ili\l \llxllllll -7 .7 M ‘e., u f w, \4.....,_’ Figure 38. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification. Figure 39. Scanning Electron Microscope (SEM) photograph of the outer surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60X magnification. 47 similar in appearance with low amounts of vascularization near the outer and inner tables and high amounts in the middle table. (Figs. 34, 40, 41, and 42). Inner surface photographs were the most diverse in appearance, as well as the size and type of canals. The inner surface of the neural (Fig. 43) had 3 Type A canals and 2 Type B canals (all small in size) within a very smooth matrix. The inner surface of the peripheral (Fig. 23) had one large Type B canal with several other smaller canals of Type A and B. The matrix was rugose with the collagen fibers easily seen in this view. The inner surface of the costal (Fig. 44) had a large number of all types of canals along with the presence of a rugose matrix. The inner surface of the plastral (Fig. 45) had 5 moderately sized Type B canals and one Type C canal. The matrix appeared intermediate between the matrix of the neural and the matrices of the peripheral and costal. variation of shells of different groups examined Several families of turtles were so structurally distinct that they could be identified from the photographs without the use of statistics. These families include the Trionychidae, Chelydridae, and Kinosternidae. The modern Trionychid specimen (Fig. 46) had very few large canals within a smooth matrix. The Chelydrid specimen (Fig. 19) had a large number of large Type C canals (the only specimen 48 »Figure,40. Scanning Electron Microscope (SEM) photograph of the edge surface of a costal bone of Chrysemys picta (MSU—H 2025). 60X magnification. Figure 41. Scanning Electron Microscope (SEM) photograph of the edge surface of a neural bone of Chrysemys picta (MSU—H 2025). 60X magnification. 49 Figure 42. Scanning Electron Microscope (SEM) photograph of the edge surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60X magnification. Figure 43. Scanning Electron Microscope (SEM) photograph of the inner surface of a neural bone of Chrysemys picta (MSU-H 2025). 60X magnification. 50 Figure 44. Scanning Electron Microscope (SEM) photograph of the inner surface of a costal bone of Chrysemys picta (MSU-H 2025). 60X magnification. Figure 45. Scanning Electron Microscope (SEM) photograph of the inner surface of a plastral bone of Chrysemys picta (MSU-H 2025). 60X magnification. with Type C canals equaling the number of Type A canals) within a rugose matrix. The Kinosternid specimens (Figs. 23, 24, and 25) had generally small canals within a primarily calcium phosphate matrix. ‘One of the Kinosternid specimens had a large number of Type C canals (the only specimen with Type C canals exceeding the number of Type A canals). The types of surface—reaching canals markedly differed between taxonomic groups (Appendix 2), although all turtles, both at the ordinal and familial level, had significantly more Type A canals than either of the other types of canals (Tables 1,2, and 3). The average number of each canal type was compared between the families Emydidae (Figure 47) and Testudinidae (Fig. 48). Although the number of canals in each family was different, the variance between the numbers of each type of canal was similar. The Type A canals were significantly larger than the other two types in both famdlies, but Type C canals were significantly larger than the Type B canals in the family Emydidae but not in the Testudinidae. All specimens of Chrysemys picta were examined for comparisons of averages of canal types (T- tests)(Table 5) with each type of canal significantly different than the other (Fig. 49). The area analysis of the SEM photographs was averaged with the percentage of area occupied by canals in the 52 Figure 46. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Trionyx ferox (MSU-H 478). 60X magnification. Figure 51. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chelus fimbriatus (MSU—H 2613). 60X magnification. 53 .mmeenAEB sansmu was an wodxu Hmcmo ucmeMMHU cause one wo muoDEsc momuw>m mo coflusnfluumflo >ocmsqmum .sv engage ow om ov om ON 9. o womb. .230 .5 959.52 omwta>< omEo>Em < 03:. m on? MVMXH>H 54 .mmaecansbmma seesaw we» as amaze Hmcmo acmHmMMHc omen» ecu mo muobEsc oomuo>m mo cofiunefluumflc xocmsvmum .mv musofim xxmxehh m_mxu>p. ”unwaec. medic. 3ch no muonEsz emato>< omoEEBme... 55 photographs. This data enabled the comparison of the different families of chelonians by their percentage of area occupied by canals (Fig. 50). Figure 50 is misleading because the other catagory contains many of the testudines that live in unique or unusual habitats. In fact, the largest area occupied by canals was in a single individual of the cheliid genus, Chelus fimbriatus (Fig. 51). The linear regressions of the types of canals compared to the area occupied by canals for all chelonia was significant for type B canals but not for type A or C canals '(Table 6). The linear regressions of the types of canals compared to the area occupied by canals for the family Emydidae was significant for types A and B canals but not for type C canals (Table 7). The linear regressions of the types of canals compared to the area occupied by canals for the family Testudinidae (Table 8) and for specimens of Chrysemys picta (Table 9) were not significant for any of the three canal types. Fossil specimens examined and techniques applied to them The fossil specimens photographed were examined for the percentage area that the canals occupied compared to the matrix of the bone (Table 10). Variable percentage area occupied by the canals ranged from 6.13 percent to 11.76 percent. The fossil specimens examined had the same general 56 .muoflm mXEmmNuno CH moa>u amcmo ucoumLMHp mouse onu mo muobfisc ommum>m wo cofiusnfiuumflp >ucmsqmum .mv gunmen om ov om ow or c T. l l l l _ < 8.»... m 8.»... o we»... manic. .230 E 23:52 ommto>< 52a .0 57 .mocflcsumoe mo moHHHEmm wee CH mamcmo >9 poemsooo mono oomucooumm womuw>m no coflusbfluumflc xocoskum .om mesmem aso 32:633.. $25235. 8935 2.230 E poi—.30 no.3. ems—Bosn— emmte>< 58 Table 6. Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for all Testudines. Independ. Var. Dependent Var. R-squared Prob. Value Percent Area Canal Type A 0.0073 0.5649 Percent Area Canal Type B 0.3027 0.0001 Percent Area Canal Type C 0.0166 0.3827 Table 7. Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of the family Emydidae. Independ. Var. Dependent Var. R-squared Prob. Value Percent Area Canal Type A 0.2269 0.0106 Percent Area Canal Type B 0.3821 ' 0.0005 Percent Area Canal Type C 0.0000 0.9928 Table 8. Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of the family Testudinidae. Independ. Var. Dependent Var. R-squared Prob. Value Percent Area Canal Type A 0.0004 0.9643 Percent Area Canal Type B 0.1040 0.4718 Percent Area Canal Type C 0.0627 0.5811 Table 9. Linear regressions of neural (N2) Scanning Electron Microscope (SEM) photograph area analysis compared to the different canal types for members of Chrysemys picta. Independ. Var. Dependent Var. R-squared Prob. Value Percent Area Canal Type A 0.0266 0.4947 Percent Area Canal Type B 0.1227 0.1247 Percent Area Canal Type C 0.1597 ‘ 0.0918 59 structure of ground substance relative to the canal types present as did the extant specimens (Fig. 52). Many different problems were detected in viewing the fossil specimens. All problems arose from foreign materials associated with the fossil or from erosion of the specimen at the microscopic level. In many specimens the canals were covered or filled in with foreign material (Figs. 53, 54, 55, and 56). Foreign material occurred on the surface of five different fossils (Figs. 57 and 58). Erosion of the carapace occurred in one fossil specimen (Fig. 59) which affected the canal area data for that specimen. Long Bone Data The long bones of the chelonian samples provided data on total length, inside diameter (small), inside diameter (large), outside diameter (small), and outside diameter (large). These data were used to make multiple calculations which include K (small), K (large), R (small), R (large), KR (small), KR (large), t (small), t (large), R/t (small), and R/t (large)[see Appendix 3]. Averages for K (small) and K (large) were compared between the different families of turtles (Figs. 60 and 61) and were larger for the families Trionychidae, Kinosternidae, and Chelydridae. The averages of R/t (small) and R/t (large) were compared between the different families of turtles (Figs. 62 and 63) and were also larger for the Table 10. Percentage area of the scanning electron microscope(SEM) images occupied by canals at both a standard threshold and a variable threshold for fossil specimens. MSU-VP # % Area (100) % Area (Var.) (Var.) 847 08.36 09.04 135 849 09.04 07.90 91 Williston, FL 10.06 06.12 70 Williston, FL 09.39 07.78 80 Keya Paha Co., NE 12.82 10.27 75 Keya Paha Co., NE 09.02 11.76 130 Keya Paha Co., NE 11.47 09.53 85 Keya Paha Co., NE 09.51 09.57 105 Keya Paha Co., NE 09.18 08.92 90 Keya Paha Co., NE 08.93 08.55 95 Keya Paha Co., NE 12.52 11.39 85 Keya Paha Co., NE 09.06 08.46 90 Keya Paha Co., NE 07.49 08.24 110 Keya Paha Co., NE 50.88 09.81 40 Keya Paha Co., NE 20.40 07.25 50 Keya Paha Co., NE 09.39 07.78 80 61 Figure 52. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification. Figure 53. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification. 62 ‘Figure 54. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification. Figure 55. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification. 63 Figure 56. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone. 60X magnification. Figure 57. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown chelonian bone showing large foreign material on the surface of the fossil. 60X magnification. Figure 58. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown testudine bone showing foreign material on the surface of the fossil. 60X magnification. Figure 59. Scanning Electron Microscope (SEM) photograph of a “scrap” fossil of an unknown testudine bone showing erosion of the fossil material. 60X magnification. 65 families Trionychidae, Kinosternidae, and Chelydridae. The linear regressions of total length of the femur compared to the K values (small and large) for all chelonians (Table 11), the famdly Emydidae (Table 12) and the family Testudinidae (Table 13) were not significant at the .05 level. The linear regressions for total length of the femur compared to the KR values (small and large) for all chelonians were significant (Table 11), the family Emydidae (Table 12), and the family Testudinidae (Table 13) were significant at the .05 level. The linear regressions for total length of the femur compared to the R/t (small and large) for all chelonians (Table 11), the family Emydidae (Table 12), and the family Testudinidae (Table 13) were not significant at the .05 level. The linear regressions for the K values (small and large) compared to the KR values (small and large) for all chelonians (Table 11), the family Emydidae (Table 12), and the family Testudinidae (Table 13) were significant at the .05 level. The linear regressions for the K values (small and large) compared to the R/t values (small and large) for all chelonians (Table 11), the famdly Emydidae (Table 12), and the famdly Testudinidae (Table 13) were significant at the .05 level. 66 Trionychidae Testudinidae Kinosternidae Emydidae Chelydridae Cheloniidae Average K (small) Values O Figure 60. Frequency distribution of average K (small) values for the families of Testudines. Average K (large) Value Testudinidae 0 01 0.2 0.3 04 05 06 Figure 61. Frequency distribution of average K (large) values for the families of Testudines. .mwcflcsumoh mo moHHHEmm one u0m monam> AHHmEmV u\m ommuo>m mo coflusnfluumflc >ocosgoum .Nm gunmen mag—5820 82.368 swung empEEonx mmuEEBme. mmpEoEot... mo:_m> 238$ LE ommte>< 69 .mmchsume mo mmHHHEww one now monam> Ammumav u\m momuo>w mo coflusbfluumfip mocmsqoum .mw onsoflm m QN N mé F no 0 omEEcho 82.320 savers 8259822 $259.32. enviable; me:_~> 39m: :1 emate>< 70 Table 11. Linear regressions of femur measurements and calculations for all testudines. Independent Variable Dependent Variable R-squared Probability value K (small) Total Length 0.0022 0.7438 K (large) Total Length 0.0006 0.8660 KR (small) Total Length 0.7626 0.0000 KR (large) Total Length 0.7035 0.0000 R/t (small) Total Length 0.0049 0.6222 R/t (large) Total Length 0.0043 0.6430 KR (small) K (small) 0.1594 0.0046 KR (large) K (large) 0.1860 0.0020 R/t (small) K (small) 0.8847 0.0000 R/t (large) K (large) 0.9508 0.0000 Table 12. Linear regressions of femur measurements and calculations for members of the R-squared Probability value family Emydidae. Independent Variable Dependent Variable K (small) Total Length K (large) Total Length KR (small) Total Length KR (large) Total Length R/t (small) Total Length R/t (large) Total Length KR (small) K (small) KR (large) K (large) R/t (small) K (small) R/t (large) K (large) 0.0008 0.0164 0.6926 0.4743 0.0060 0.0110 0.1884 0.2417 0.8679 0.9425 0.8976 0.5504 0.0000 0.0003 0.7206 0.6264 0.0393 0.0176 0.0000 0.0000 Table 13. Linear regressions of femur measurements and calculations for members of the family Testudinidae. Independent Variable Dependent Variable R-squared Probability value K (small) Total Length 0.1547 0.1697 K (large) Total Length 0.1592 0.1627 KR (small) Total Length 0.8230 0.0000 KR (large) Total Length 0.7684 0.0001 R/t (small) Total Length 0.1400 0.1943 R/t (large) Total Length 0.1242 0.2246 KR (small) K (small) 0.4877 0.0081 KR (large) K (large) 0.4697 0.0099 R/t (small) K (small) 0.9737 0.0000 R/t (large) K (large) 0.9610 0.0000 71 DISCUSSION A discussion of the results of the analysis if the SEM data, histological slides, and long bone studies of turtle skeletal tissues as physiological agents follows as well as added comments on metabolic uses of the shell and its part in thermoregulation. Role of the Three Canal Types in Thermoregulation The large differences between surfaces of the varied elements of a single shell of Chrysemys picta (MSU-H 2025) show that only certain surfaces are similar enough to use for a comparative analysis; these being the outer surfaces of the neural, costal, and peripheral bones (see Figs. 36, 37, and 64). In fact, five photographs from.the same neural of this specimen (see Figs. 38, 64, and 65) were so similar that without codes on the SEM negatives the specimens could easily have been confused. Therefore, it was determined that any outer surface of the carapace could have been used for this study. The three types of canals in the shell represent a unique method of determining the amount of blood that is exposed to the outside environment by controlling not only the amount of blood that is exposed at one time to the surface but how much change in temperature occurs over that 72 Figure 64. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chrysgmys picta (MSU-H 2025). 60X magnification. Figure 65. Scanning Electron Microscope (SEM) photograph of the outer surface of a neural bone of Chgysgmys picta (MSU-H 2025). 60X magnification. 73 time. Type A canals (Figs. 6 and 7) form a T with the surface of the bone. These canals force blood into the subscute blood capillary layers. The blood would have to flow across the surface to another canal. This small amount of blood flowing just under the surface of the scute would have a high surface area to volume ratio that would allow all of the blood to be affected by the temperature of the scute at the same rate. This would make an excellent fine adjustment to the temperature of the blood and thereby, the temperature of the ectothermic turtle. Type B canals (Figs. 8 and 9) form an inverted V with the surface of the bone. This type of canal would not provide much interaction of the blood with the temperature of the scute and would have little or no effect on the temperature of the chelonian. Type C canals (Figs. 10 and 11) come to the surface of the bone and follow along the surface for some time before plunging back deep into the bone. This type of canal would. allow large amounts of blood to come into contact with the scute at one time and keep that contact for a moderate amount of time. This type of canal would allow for large amounts of temperature change in a relatively short amount of time but with little fine control of the temperature. 74 Actions of Temperature Control in the Carapace All chelonians have significantly more type A canals than any other type of canal (see Figs. 47, 48, and 49)(Tables 1, 2, 3, and 5). This fact combined with the proposed function of type A canals would allow chelonians to maintain excellent fine control of their temperature by determdning the flow of blood to the carapace. The regulatory mechanism for this was not discovered in this study. All chelonians except members of the family Testudinidae (see Table 3) have significantly fewer type B canals than any other type of canal (Tables 1, 2, and 5). This fact when compared with the proposed lack of thermal control for this type of canal would support the supposition that this type of canal lacks a thermodynamic function. The linear regression of the type B canals compared to the total area occupied by canals of the neural was usignificant for all chelonians (0.0001)(Table 6) and the family Emydidae (0.0005)(Table 7). This suggests that this low number of type B canals can be used to predict the total area of canals in the bone (Figs. 63, 64, and 65). But, the fact that type B canals are not significant predictors of percent area occupied by all canals for members of the family Testudinidae (Table 8) and members of Chrysemys picta (Table 9) would argue against that prediction. 75 A possible explanation for Type B significance when used to predict percent area occupied by all canals could be that the type A canals are extensions or processes of the type B canal. This hypothesis is not supported by further testing. A linear regression of type A canals compared to type B canals resulted in a rejection of the hypothesis (0.0838) at the 0.05 level (Fig. 66). The most numerous type of canal in all chelonians examined were the type A canals. It would be logical to assume that this type of canal would be a good predictor of the total area occupied by canals. This assumption is incorrect in all groups (Figs. 67, 68, and 69)(Tables 6, 8, and 9) except the Family Emydidae (Figs. 70)(Tables 7). The fact that the linear regressions of the type A canals compared to the total area of canals is significant only for the family Emydidae (0.0106) does not support the hypothesis that type B canals are primary sources of type A canals. The area of anastomosis between different canal types would be an area that would benefit from.further research. Linear regressions of type C canals compared to total area were not significant for any group examined (Figs. 71, 72, 73, and 74). As type C canals are the largest type of canal, it lends evidence to the proposed function of type C canals. If a single type C canal has the ability to greatly modify the temperature of the organism, there would 76 wooeosumwu Ham CH mamcmo m oQ>H mo umoEsc on» msmuo> mamcmo m odxe mo HenEoz . UOCHEMXO om on L < one; ow .OOIIOlo O 00 O 0 0 «Search .mw manage on or o .Avllxv .19 o o o N o v o o .o m. w 8 m .oi .me .4? 77 .oocHmeo mmcflosummu Ham CH mamcmo < onxa we quEsc mzmuo> mamcmo Ham xo omflasooo mono unmouom a o .. < .\. an an ON 2. 2. all ill i . . . i I . — ...... iii . 1 il ».l lllliil .il Pill.) lll||iolilllnllllllllllilllPll (lllll. lid.ll[itll y. I o o o o o o o o o o o o o o o 8 o o o o o o o o o % o O 0 0 0 0 o o o o o o o 0 35.532. .se masons .mv -mN mm .mv mm .m0 .2. rm» vodkr 78 one CH mamcmo < md>e mo Hanan: msmum> mamcmo Ham >o omflmoooo mmum ucmoumm .mmeecensumme seesaw .me masses ON or p me » hr or _ mp b er - e e a < 23.3.. «r «v . we _ b F onuIZunumoh Or ..oF -mv -me .om .mm 79 m p 52m massage .muoflm mNEomNubo cH mamcmu m md>e mo noose: m5muo> mamcmo Ham >9 coedsooo moum ucoouom .mm enemas _ . mm .mm .mv vedAL jmm .mw ms. 80 .mmeAUAEm seesaw one Ce maocoo m od>9 mo woman: msouo> oaocoo Ham >o ooHQsooo mono ucoouom ON » ill 'i: av » .os mesons 2. p e o c < «neocon— t. or me 3. 9. «P E. F .rlll. Elixir: i. bl b p » oau.u>Em 2. m o h m or .mu .9... .. me I mm .mo .mh mm vadkr Ill. 81 . .mmeeceesnmme sesame on» ca maocoo o odxe mo uooEsc momuo> maocoo Ham >o oofldoooo mono ucoouom .Hb ousmem ooL< S on mm cm or o? m villi l lli l lllui- ii -lr ll imi lLl .l.li¢lllll$ o s a. 2. o . W O O .m 0 M 0 O 00 WV 0 l 0 .0 o o 8 0 o m .ow l .A o o o w .mm 9 .om 0 mm o -9. .5. ooEoBmo... .oooeo>Em >HflEom one CH maocoo o odxfi mo noose: msmuo> maocoo Ham >9 coedsooo mono ucoouom .mb ousmem no.3 Booted on or or up or we er up up we or o a o o -l -l- < c O 0 O 0 . 0 . m 0 00 0 0 or O O o o o m«.M w 9 .ou 0 o o .mu .on 0 .mn oau_u>Em 83 .ouoflm mNEomxueo CH maocoo o odxe mo noose: msouo> mascoo Ham >2 coedsooo mono ucoouom .m> ousoflm alllllll:l.-- -ll; - . ll :lll-.l-.lll--s. , l l - lllllz--l:ll:- . .. ,l- ,l. ill-. ll no.3 «:3an 3. 2 NF 3 o_. m w a. o F p .l l. lll- .:|lr,illllllllll p l . p r . p p o 0 0 O 0 o -m o O 0 0 0 - or o o o 0 r me I o m a - cm 0 o 0 -mm .om 0 -mm 503 9583:”. 84 .mmeecflesnmme sansmo one ca mHocoo U oQ>B mo Hogan: msmwo> maocoo Ham >9 ooflasooo mono ucoouom .vb ousmem oo..< aroused cm or ms t. or m? 3. 9 we 3 or m w r|l ulll l.l»lllli-ll ll-,.P.lllll. .lrl ll ll». ill I r. -l p — L p b o - N 0 o o . v 0 . o O . m m. o .oew 9 - we see - or o .m. 82528:. 8 85 be little need for large numbers of them in the shell. The subscute blood layer is as small as one cell layer thick in some places which could enhance the ability of the outside environment to affect the blood, and could become the exact temperature of the scute in a short amount of time. Anatomical structures for the control of blood flow through the carapace (Avery, 1982) were not detected either in the SEM specimens or in the histology specimens. The extent of ability of the blood to fan out from the type A canal and thus gain or lose even more heat is unknown because the histological slides were sagittally sectioned at this level. My work shows that it flows across'the shell (Fig. 75), but the ability of the blood to flow in more than two directions is unknown. Shallower transverse sections may have answered this question, but unfortunately, the transverse sections were made deeper in this study. The shape of the shell optimizes the amount of heat that can be obtained from the environment. The shape allows the sun to contact at least part of the carapace no matter what direction the light is coming from. This allows the subscute thin layer of blood to obtain heat from that area of the shell that is currently under direct sunlight. Bartholomew (1982) found that peripheral vasomotor activity was locally controlled independent of the core temperature and heart beat, although he did not discuss how this control 86 .a g l._— -.r, I“ ., , . . Figure 75. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing the subscute blood flow. . . A A Figure 76. Photograph of a Hematoxylin and Eosin (H+E) prepared histological slide showing an osteoclast dissolving bone. 87 occurred. This mechanism.would allow the organism to control where on the shell blood would flow and thereby where the subscute blood would be optimally heated by the radiant heat. The hemispherical shape of the carapace gives chelonians a relatively small surface to volume ratio when compared with other reptiles of the same size (Bartholomew, 1982). Some chelonians are large enough that their mass endows them.with an inertial homeothermy similar to crocodilians (Bartholomew, 1982). This small surface to volume ratio allows these very successful reptiles to retain more heat in their systems due to smaller amounts of heat lost through surface radiation. This retention of heat means that even small amounts of heat gain are kept for long periods of time. This would mean that a method of forcing or enhancing temperature control of the shell would be of great value to this animal’s natural ability to retain heat. Actions of Temperature Control in the Plastron The large Type C canals on the plastral outer surface photograph (see Fig. 39) are exceptionally noteworthy. The proposed function of type C canals is to allow large quantities of blood over thethermodynamic surface in a relatively short amount of time. The arrangement of Type C canals next to the surface upon which this basking chelonian rests his weight, would allow the turtle to gain or lose 88 large amounts of heat rapidly. Obviously, the control of blood to this area would be of great importance. Though no mode of blood flow control was detected in this study, Hicks and Wang (1996) discuss the role cardiac shunting in reptiles. This ability to shunt most of the blood to either the pulmonary or systemic circulation is a perfect way to control large amounts of blood flow with relatively little physiological effort. Cardiac shunting in conjunction with the large numbers of Type C canals in the plastron would allow the chelonian to control temperature rapidly. Carapace as a Reservoir for Calcium and Phosphate The presence of osteoclasts in the spongy bone of carapace (Fig. 76) is solid evidence that the shell is a storage reservoir for calcium.and phosphate. This reserve is in addition to any that the long bones provide. This. leads to the supposition that osteoblasts are also present in the spongy bone of the carapace; leading to the constant Iremodeling of the shell as dictated by the needs of the organism. The process of remodeling of the shell is primarily a metabolic function and not a structural one, as the strength of the shell is determined primarily by the structure of the bridge that connects the carapace with the plastron (Currey, 1967). Red eared sliders need a mdnimum of two percent dietary calcium during growth for the carapace to develop properly 89 (Kass, et al, 1982). This calcium can be taken directly from the aquatic medium and used in carapace structure (Jeffree, 1991). The ability to extract calcium from the environment and lay it down as Calcium Phosphate in the carapace facilitated by the presence of osteoclasts in the spongy bone of the carapace suggests a process of gaining calcium from the surrounding environment. Other metabolic uses of the Shell The shell has been determined to be an agent for hematopoesis in the spongy middle layer of the shell (Vasse and Beaupain, 1981). It has also been suggested that chelonians are capable of increasing bone marrow production in cases of certain diseases (Garner, et. al., 1996). These factors give additional evidence for the metabolic activities of the chelonian shell. Reduced Size of Long Bone Marrow Cavities for Support Currey and Alexander (1985) state that the median R/t for terrestrial mammals is 2.0. This value (even though it is for an endothermic animal) is of value for comparison with the testudine data. The terrestrial mammals have a R/t value that is optimal for impact strength but lower than the expected value for static strength and stiffness. The Average value for the family Testudinidae is 2.0784 for the smaller width and 2.1299 for the larger width, which is similar to the terrestrial mammals (Appendix 3). The 90 average R/t (2.0298 for the large and 2.0163 for the small) for the family Emydidae was also similar to terrestrial mammals even though they are primarily aquatic. It should be noted that they use their limbs extensively to climb out of the water frequently for basking purposes as well as excursions out of water for multiple purposes. The marine turtle Eretmochelys imbricata had a R/t of 1.7826 for the smaller width and 1.6847 for the large width, which is much different than the R/t of 1.0 that Currey and Alexander (1985) found in the endothermic manatee '(Trichechus manatus). Excluding the sole marine turtle examined, important correlation occurs between chelonian shell size and the R/t of their long bones. The Emydidae and Testudinidae both have extensive shells and their R/t values are the lowest of the chelonians. The Kinosternidae have a somewhat reduced shell and their R/t value is larger. The Chelydridae have an even more reduced shell and their R/t value is higher yet. Finally, the Trionychidae have no external shell, only a much reduced internal shell, and their R/t value is the largest of all. This inverse correlation between the amount of shell that a chelonian has and its R/t value generally indicates the amount of metabolic activity that occurs in the shell. The higher the R/t value, the more marrow the long bone possesses. Therefore, the smaller the shell (and 91 consequently, the amount of bone in the shell), the larger the marrow cavity of the long bone. The K value, which is considered to be at optimum strength at 0.67 (Currey and Alexander, 1985), is lower than the optimum strength value in almost every turtle examined (Appendix 3). Exceptions occur in two measurements of the smaller K value (one Kinosternid and one Trionychid) and three measurements of the larger K value (one Chelydrid, one Testudinid, and one Emydid). The ultimate strength value of K was calculated as 0.55 and a large number of the turtles examined have K numbers lower than this ultimate strength number. The terrestrial turtles (Terrapene and family Testudinidae) have K(1arge) values of 0.3146 to 0.6966 with an average of 0.5122. The Marine turtle Eretmochelys has a K (large) value of 0.4064. This very low value for the rear leg (which is not used in propelling the turtle through the water) is not surprising considering that this organism is marine and propels itself through the ocean with its anterior limbs. The rear limbs trail the body and may act as a rudder. Females also use them to dig the hole when nesting, but even when on land, the rear limbs are not used to propel the animal. The linear regressions of the total length of the femora compared to the KR values (small and large) were the most informative of all the regressions performed on the 92 long bones (Tables 11, 12, and 13)(Figs. 77, 78, 79, 80, 81, and 82). The KR value is the radius of the marrow cavity in the femur. The fact that it can be used to determine the length of the bone is of great interest. This would suggest that the marrow cavity is affected by the length of the bone. This correlation should be examined more closely to determine if this is a true or a chance (stochastic) correlation. I The linear regressions of the K values (small and large) compared to the KR values (large and small)(Figs. 83 and 84), and compared to the R/t values (large and small)(Figs. 85 and 86) were primarily run as controls. The K value is the radius of the femur. The KR value is the radius of the marrow cavity. The R/t value is the ratio of the radius of the complete bone to the thickness of the bone wall. These are all derivatives of the same measurements and should therefore be highly correlated. ‘ Comments on metabolic Uses of the Shell Without mention of the shell, Edgren (1960) stated that the long bones are used as a calcium reserve for the egg shell production in Sternotherus odoratus. Another well known use of calcium in animals is to bind to troponin, [activating the movement of tropomyosin and the exposure of active sites on the thin (actin) filaments] resulting in the contraction of muscles (Martini and Bartholomew, 2000). 93 .oocHono monflosuoou Ham MOM osam> AHHoEmV mm one msmuo> HsEow onu mo neocoa Houoe .bs ouooflm ll» llllll..lltlll . ll. ill .illlln 593.. Each o_. 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IL ‘nl 'ILII -LI ||i|1iolilllLl Ililfli alt . iLl . _ .I _ .......... 3L: fizllilel: lil,-i-[i .Lr F o . o o oo o m r 8 08° 0 o oo % 0 0 ov a» w 00 - AWN S a» w. 6 hu 0 o - m 00 Y, m.» 0 - v mos—53a... 103 These are both areas of metabolic activity that require high amounts of calcium. If turtles did not use the shell as a source of calcium, the animals would not only be stressed by having to acquire enough calcium to maintain metabolic activities, but also by having to lay down the calcium of the shell’s bone as well. Although the amount of marrow of most vertebrate long bones is quite large when compared to the total skeletal mass (Alexander, 1982), it is generally very small when compared to the overall skeletal size of testudines. The reason for the relatively small amount of turtle long bone marrow reflects the fact that their shells act as an additional large hemopoetic source. This extra hemopoetic source allows the animals to remodel their long bones for maximum stress resistance to carry the extra mass of the shell. Some blood that flows throughout the carapace must feed the epithelial cells that produce the keratin the comprises the scutes. An alternative theory for the blood canals would be as a nutrient and gas source for these stem cells. The amount of blood that flows through the type C canals is much more than is needed to feed this single layer of cells under the scutes (Fig. 87). Conclusions/Summation Citations and references of the shell as a metabolically inert structure has been continuously stated 104 in the chelonian literature as recently as December of 1999 (Stone and Iverson, 1999; Dunson, 1986). My work found that the carapace was used as a calcium reserve as indicated by the presence of osteoclasts that reabsorb bone for metabolic purposes. Previous studies (Vasse and Beaupain, 1981) found that the chelonian shell is used in hematopoeisis. The presence of the vascular canals with their different types of interactions with the surface of the shell affect the thermodynamics of the organism. The subscute blood layer between the carapace and the plastron 'have rapid affects on the temperature of the blood that flows through those areas. All outer surfaces of the carapace were similar in appearance when these diverse areas were compared. Moreover, photographs of different outer surface areas of the same neural showed no significant differences between the numbers and types of canals present in them. This uniformity of the outer surface of the carapace and the significant numbers of Type A canals suggests that they are highly used in thermoregulation. The marrow cavity size of the long bones in chelonians is primarily restricted by the weight of the carapace and the mode of locomotion of the chelonian in question. The fact that the K value of most chelonians falls below the optimum strength values of Currey and Alexander (1985) means that the long bones of chelonians have much thicker walls than 105 Figure 87. Hematoxylin and Eosin (H+E) photograph of a carapace showing the single cell layer that produces the scute of the shell. other vertebrate animals. These thicker walls are needed to carry the weight of the heavy bony shell along with the soft tissues of the chelonian. The much reduced marrow cavities of the long bones of chelonians requires that they must gain the calcium through their other bony structures, primarily the carapace and plastron. The calcium could not be lost from the long bones or risk reduced ability of the bones to support the animal. The carapace and plastron of chelonians has long been known as a source for thermoregulation although mechanisms for this thermal control were not known. The interaction of the ability of the chelonian for blood flow control to the shell and the surface arrangement of the vascular canals allows the chelonian to thermoregulate with little energy expenditure and fine control of the temperature change. 107 LITERATURE CITED .Alexander, R. McN. 1982. Optima for Animals. Edward Arnold (Publishers) Limited, London. 112 pp. Avery, R.A. 1982. Field studies of body temperatures and thermoregulation. 93-166. IN: Biology of the Reptilia vol. 12. C. Gans and F. H. Pough [Eds.] Academic Press, London. 536 pps. Bartholomew, G.A. 1982. Physiological control of body temperature. 167—211. IN: Biology of the Reptilia Vol. 12. C. Gans and F.H. Pough [Eds.] Academic Press, New York. 574 pps. Benedict, F.G. 1932. The physiology of large reptiles. Carnegie Inst. washington Publ. 425 pps. Bennett, A.F., and W.R. Dawson. 1976. Metabolism. 127-223. IN: Biology of the Reptilia Vol. 5. C. Gans and W.R. Dawson [Eds.] Academic Press, London. 556 pps. Bloom, W., and D.W. Fawcett. 1962. A Textbook of Histology. Eighth Edition. W.B. Saunders Company. Philadelphia. 720 pp. Burke, A.C. 1989. DevelOpment of the turtle carapace: Implications for the evolution of a novel bauplan. J. Morph. 199:363—378. Cagle, F.R. 1950. The life history of the slider turtle Pseudemys scripta troostii (Holbrook). Ecological Monographs 20:31-54. Castanet, J., and M. Cheylan. 1979. Les marques de croissance des os et des ecailles comme indicateur de l’age chez Testudo hermanni et Testudo graeca (Reptilia, Chelonia, Testudinidae). Can. J. Zool. 57:1649-1665. Currey, J.D. 1967. The failure of exoskeletons and endoskeletons. J. Morph. 123:1-16. Currey, J.D. 1984. The mechanical adaptations of bones. Princeton, University Press. Currey , J.D., and R. McN. Alexander 1985. The thickness of the walls of tubular bones. J. Zool. Lond. A 206:453- 468. 108 Dunson, W.A. 1986. Estuarine populations of the snapping turtle (Chelydra) as a model for the evolution of marine adaptations in reptiles. Copeia 1986:741—756. Edgren, R.A. 1960. A seasonal change in bone density in female musk turtles, Sternothaerus odoratus (Latreille). Comp. Biochem. Physiol. 1:213—217. Enlow , D.H. 1969. The Bone of Reptiles. 45-80. IN: Biology of the Reptilia, Vol. 1, Morphology A. Carl Gans [Ed.] Academic Press, London. 373 pp. Enlow, D.H., and 5.0. Brown. 1957. A comparative histological study of fossil and recent bone tissues. Part 2. Texas Journal of Science 9:186-214. Ernst, C.H., and R.W. Barbour. 1989. Turtles of the World. Smithsonian Institution Press, Washington, D.C. 313pp. Garner, M.M., B.L. Homer, E.R. Jacobson, R.E. Raskin, B.J. Hall, W.A. Weis, and K.H. Berry. 1996. Staining and morphologic features of bone marrow hematopoietic cells in desert tortoises (Gopherus agassizii). AJVR 57(11):1608-1615. Hall, F.G. 1924. The respiratory exchange in turtles. J. Metab. Res. 6:393—401. Hay, O.P. 1908. The fossil turtles of North America. Carnegie Inst. Washington Publ. 75:1-568. Hicks, J.W., and T. Wang. 1996. Functional role of cardiac shunts in reptiles. J. Exp. Zool. 275:204-216. Hughes, G.M., R. Gaymer, M. Moore, and A.J. Weakes. 1971. Respiratory exchange and body size in the Aldabra giant tortoise. J. Exp. Biol. 55:651-665. Hutton, K.E., D.R. Boyer, J.C. Williams, and P.M. Campbell. 1960. Effects of temperature and body size upon heart rate and oxygen consumption in turtles. J. Cell. Comp. Physiol. 55:87-93. Jayes, A.S., and R. McN. Alexander. 1980. The gaits of chelonians: walking techniques for very low speeds. J. Zool. Lond. 191:353—378. Jeffree, R.A. 1991. An experimental study of 226Ra and‘”Ca accumulation from the aquatic medium by freshwater 109 turtles (Fam. Chelidae) under varying Ca and Mg water concentrations. Hydrobiologia 218:205-231. Kass, R.E., D.E. Ullrey, and A.L. Trapp. 1982. A study of calcium requirements of the red—eared slider turtle (Pseudemys scripta elegans). J. Zoo. An. Med. 13:62-65. Kent, G.C. 1987. Comparative Anatomy of the Vertebrates. Times Mirror/Mosby College Publishing, St. Louis. 646pp. Martini, F.H., and E.F. Bartholomew. 2000. Essentials of Anatomy and Physiology. Prentice Hall, Upper Saddle River, New Jersey. 603 pp. Marvin, G.A., and W.I. Lutterschmidt. 1997. Locomotor performance in juvenile and adult box turtles (Terrapene carolina): A reanalysis for effects of body size and extrinsic load using a terrestrial species. J. Herp. 31(4):582-586. McDowell, S.B. 1964. Partition of the genus Clemmys and related problems in the taxonomy of the aquatic Testudinidae. Proc. Zool. Soc. London 143:239-279. Monteith, J.L. 1973. Principles of Environmental Physics. Arnold Publishing, London. 572 pp. Newman, H.H. 1906. The significance of scute and plate ‘abnormalities’ in chelonia. Biol. Bull. 10:86—114. Pritchard, P.C.H. 1979. Encyclopedia of Turtles. T.F.H. Publishing, Neptune, New Jersey. 895 pp. 'Procter, J.B. 1922. A study of the remarkable tortoise, Testudo loveridgii B1gr., and the morphogeny of the chelonian carapace. Proc. Zool. Soc. London 1922:483- 526. Rhodin, A.C.J., J.A. Ogden, and G.J. Conlogue. 1981. Chondro—osseous morphology of Dermochelys coriacea, a marine reptile with mammalian skeletal features. Nature 290:244-246. Sheehan, and Hrapchak. 1980. Theory and Practice of Histotechnology, Second Edition. Battelle Press, Detroit , Michigan. 481 pp. Softstat Business Statistics Software. Version 2.0. 1996. Hagar, Inc. Tempe, AZ. M0 Stone, P.A., and J.B. Iverson. 1999. Cutaneous surface area in freshwater turtles. Chelonian Conserv. Biol. 3(3):512-515. Suzuki, H.K. 1963. Studies on the osseous system of the slider turtle. Annals New York Acad. Sci. 109:351-410. Vasse, J., and D. Beaupain. 1981. Erythropoiesis and haemoglobin ontogeny in the turtle Emys orbicularis L. J. Embryol. Exp. Morphol. 62:129-138. Wallis, K. 1927. Zur Knochenhistologie und Kallusbildung beim Reptil (Clemmys leprosa Schweigg). Z. Zellforsch. Mikroskop. Anat. 6:1-26. Zangerl, R. 1939. The homology of the shell elements in turtles. J. Morph. 65(3):383-409. Zug, G.R. 1993. Herpetology An Introductory Biology of Amphibians and Reptiles. Academic Press, Inc., San Diego. 527 pp. 111 APP'IX A SPECIMENS mm All Michigan State University Museum specimens have locality data on file in the museum data base. Family Chelidae Chelodina longicollis (MSU-H 12987). Chelus fimbriatus (MSU—H 2613). Family Cheloniidae Eretmochelys imbricata (MSU—H 2117). Family Chelydridae -Chelydra serpentina (MSU—H 3010);(MSU-H 3436);(MSU—H 3773). Family Dermatemydidae Dermatemys mawei (MSU—H 2330). Family Emydidae, Subfamily Batagurinae Siebenrockiella crassicollis (MSU-H 3054). Family Emydidae, Subfamdly Emydinae. Chrysemys picta (MSU-14309);(MSU-H 14310); (MSU-H 14312); (MSU-H 14314);(MSU-H 14316);(MSU-H 14325); (MSU-H 14326); (MSU-H 14335); (MSU-H 14344); (MSU-H 14345); (MSU-H 14346); (MSU-H 14351); (MSU-H 13406); (MSU-H 1109); (MSU-H 2930); (MSU-H 3247); (MSU-H 967); (MSU-H 2025);(MSU-H 3306). Clemmys insculpta (MSU—H 598); (MSU—H 3257); (MSU-H 4324); (MSU-H 4336). Emydoidea blandingii (MSU-H 2231); (MSU-H 3955); (MSU-H 13021). 112 Graptemys geographica (MSU-H 2911); (MSU-H 3303). Graptemys pseudogeographica. USA. IL. Jackson County. (KDA 117). Pseudemys floridana (MSU-H 522); (MSU—H 3183). Pseudemys floridana peninsularis (MSU-H 3927). Terrapene carolina bauri (MSU—H 629); (MSU-H 1306). Terrapene carolina carolina (MSU—H 1696); (MSU-H 2379); (MSU-H 4053); (MSU—H 13019). Terrapene carolina triunguis (MSU-H 4349); (MSU-H 12977). Trachemys scripta (MSU—H 1730); (MSU-H 2929); (MSU-H 14307). Trachemys scripta elegans (MSU—H 2716); (MSU-H 2721). Famdly Kinosternidae Kinosternon flavescens (MSU-H 2918); (MSU-H 2920); (MSU-H 2924). Kinosternon leucostomum (MSU-H 1414). Kinosternon subrubrum (MSU-H 2477) ,- (MSU—H 2768); (MSU-H 4337). Family Pelomedusidae Pelusios derbianus (MSU-H 2100). Family Testudinidae Geochelone agassizi (MSU-H 1707). Geochelone carbonaria (MSU-H3526). Geochelone elegans (MSU—H 3238). Geochelone elongata (MSU-H 3123). Geochelone pardalis (MSU—H 2931); (MSU-H 3216). 113 Gopherus berlandieri (MSU—H 1400); (MSU-H 2220); (MSU-H 2221). Gopherus polyphemus (MSU-H 497); (MSU—H 2102); (MSU—H 3221). Kinixys belliana (MSU-H 4350). Kinixys erosa (MSU—H 2077). Malacochersus tornieri (MSU-H 4150). Testudo graeca (MSU—H 4143); (MSU—H 4152); (MSU-H 4156); (MSU-H 4158). Family Trionychidae Apalone mutica (MSU-H 1442).. Apalone spinifera (MSU-H 1443); (MSU-H 2767). 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