Bone Histology of Fossil Tetrapods: Advancing Methods, Analysis, and Interpretation

Bone Histology

of Fossil Tetrapods

Bone Histology

of Fossil Tetrapods

Advancing Methods, Analysis,

and Interpretation

Edited by

Kevin Padian

Ellen-Thérèse Lamm

UNIVERSITY OF CALIFORNIA PRESS

BerkeleyLos AngelesLondon

University of California Press, one of the most distinguished university presses in the United States, enriches lives around the world by advancing scholarship in the humanities, social sciences, and natural sciences. Its activities are supported by the UC Press Foundation and by philanthropic contributions from individuals and institutions. For more information, visit www.ucpress.edu.

University of California Press

Berkeley and Los Angeles, California

University of California Press, Ltd.

London, England

© 2013 by The Regents of the University of California

Library of Congress Cataloging-in-Publication Data

Bone histology of fossil tetrapods : advancing methods, analysis, and interpretation / edited by Kevin Padian, Ellen-Therese Lamm.

p. cm.

Includes bibliographical references and index.

ISBN 978-0-520-27352-8 (cloth : alk. paper)

eISBN 9780520955110

1. Vertebrates, Fossil—Microstructure. I. Padian, Kevin. II. Lamm, Ellen-Thérèse, 1966-

QE841.B674 2013

566-dc23 2012018397

Manufactured in China

22   21   20   19   18   17   16   15   14   13

10   9   8   7   6   5   4   3   2   1

The paper used in this publication meets the minimum requirements of ANSI/NISO Z39.48-1992 (R 2002) (Permanence of Paper).

Cover illustration: Tyrannosaurus rex fibula—Museum of the Rockies specimen (MOR 1152). Photographed on a Leica DMEP Microscope under polarized light with a Lambda compensator. Photo by Ellen-Thérèse Lamm.

Dedicated to three pioneers of

vertebrate paleohistology

Donald H. Enlow

Armand J. de Ricqlès

John R. Horner

with respect and affection from their

colleagues, students, and friends

CONTENTS

Preface

Authors and Contributors

1.Why Study the Bone Microstructure of Fossil Tetrapods?

Kevin Padian

2.The Biology of Bone

Adam K. Huttenlocker, Holly Woodward, and Brian K. Hall

3.Selection of Specimens

Kevin Padian, Ellen-Thérèse Lamm, and Sarah Werning

4.Preparation and Sectioning of Specimens

Ellen-Thérèse Lamm

5.Image Standardization in Paleohistology

Timothy Bromage and Sarah Werning

6.Database Standardization

Laura Wilson and Maria de Boef Miara

7.Skeletochronology

Holly N. Woodward, Kevin Padian, and Andrew H. Lee

8.Analysis of Growth Rates

Andrew H. Lee, Adam K. Huttenlocker, Kevin Padian, and Holly N. Woodward

9.Evolution of Growth Rates and Their Implications

Kevin Padian and Koen Stein

10.Research Applications and Integration

Kevin Padian, Maria de Boef Miara, Hans C.E. Larsson, Laura Wilson, and Timothy Bromage

PREFACE

This book was conceived thanks to Dr. Susan Williams, Drew Lee’s former colleague at Ohio University in Athens. We were discussing with Susan the explosion of histological studies of fossil bone that had occurred in the past two decades, and we thought that it might be helpful to provide guidance to people new to the field about how to select, process, and image specimens, and about the questions and issues in the field that had been addressed and that needed further work. Susan suggested that we look into convening a National Evolutionary Synthesis Center (NESCent) workshop on the subject, because she had found them very rewarding in her own field. We contacted Dr. Kathleen Smith, then the Director of NESCent, and she found us funds to bring a small group of people together for a short workshop in December 2009. It was so successful that we decided to use our discussions as the basis for an introductory book.

Our working group included experienced hands as well as graduate students and new faculty members who are making some of the strongest contributions and innovations in the field. We would have liked to include a great many other colleagues in our group, but unfortunately the pressures of time, previous commitments, and our own limited funding prevailed as they usually do, so we were not able to assemble every one of the stars of our field. Nevertheless they and their work were with us in spirit, and some have served as useful advisors and reviewers of these chapters.

We do not intend this book to be an exhaustive, encyclopedic treatment of all subjects related to fossil bone histology or to the biology of bone. We wanted to provide an introduction to the problems and methods of the field. Why study the bone histology of fossil tetrapods? What questions can it illuminate? How do we explain variations in the expression of bone tissue in fossil tetrapods? How do you decide when sectioning a fossil bone might be a good idea? How do you select the best candidate bones? How do you prepare them for sectioning, and how do you make the sections and slides? How do you ensure the best quality photographs? How do you scale and archive your images? What data should accompany them? How do you know if your observations are showing some really important new insights?

Because many of these questions are interrelated, and because many studies have bearing on more than one aspect of these questions, readers will note that there is some overlap in coverage from one chapter to another. We felt that it would be preferable to remind readers of connections to other subjects within the book than to leave them wondering sometimes about what those connections might be.

We don’t guarantee that all questions will be fully answered, but we intend to provide a start, as well as food for thought for more experienced researchers. We also would like colleagues in related fields, such as human biology and medicine, forensic anthropology, bone mechanics, and vertebrate functional morphology to see what kinds of insights and advances have been made in our field in the past two decades.

The dissemination of information is changing very quickly these days. We wanted our efforts to be as comprehensively available as possible. A traditional book is one manifestation of this, and we hope that it will be useful to many colleagues. Others may wish to download parts of the book only, and for this reason the University of California Press was our preferred publisher, because they were able to make this option available through eBooks. But we also wanted to include more images, more details, and updates to our treatments of methods and issues in the field. For this reason, we have established an independent companion website that will eventually include all the information in this book and more. It can be found at www.histoworkshop.org. Keep checking it for updates, and if you have things to contribute or subjects that you would like covered, please contact the webmaster.

We thank Kathleen Smith, Danielle Wilson, and the staff of NESCent for their hospitality and support. And we thank Chuck Crumly and the staff at the University of California Press for enabling us to produce this work in the formats that will make it most useful for our community of scholars. We are indebted to Charles E. Wilson III and Frank S. Benjamin of McKenna, Long & Aldridge LLP for their advice and counsel on copyright issues related to histological image data. Among all our participants and contributors, as a group we have to single out for thanks Ellen-Thérèse Lamm, the Lapidaire Extraordinaire of the Museum of the Rockies. Ellen has been conducting workshops on bone histology for many years and intending to put all her carefully crafted advice, handouts, and guides into print one day. We are delighted that she let this be the day.

Kevin Padian

December 2011

AUTHORS AND CONTRIBUTORS

The NESCent Paleohistology Working Group

TIMOTHY G. BROMAGE

New York University

AMANDA CURTIN

Duke University

BRIAN HALL

Dalhousie University

ADAM HUTTENLOCHER

University of Washington

ELLEN-THÉRÈSE LAMM

Museum of the Rockies

HANS C.E. LARSSON

McGill University

ANDREW H. LEE

Midwestern Medical College

MARIA DE BOEF MIARA

Harvard University

KEVIN PADIAN

University of California, Berkeley

V. LOUISE ROTH

Duke University

MARTIN SANDER

University of Bonn

KOEN STEIN

University of Bonn

SARAH WERNING

University of California, Berkeley

LAURA WILSON

University of Colorado, Boulder

Present address: Fort Hays State

University, Kansas

HOLLY WOODWARD

Montana State University

1

Why Study the Bone Microstructure of Fossil Tetrapods?

Kevin Padian

In the nineteenth century, when morphology was the queen of the biological sciences, every student of the living world had to know the intimate details of plant and animal anatomy, including microscopic anatomy, as well as the theories of the generation and determination of form and structure that underpinned the science of morphology (Sloan 1992; Desmond 1982, 1989). As a matter of routine, Richard Owen took a thin section of a bone of Scelidosaurus when he described the dinosaur in 1861. However, with the twentieth-century advent of genetics, and later the great advances in cellular and molecular biology, histology—the study of tissues—was eclipsed by these many new fields of study. There simply was not enough time for everything. Besides, in this new world order, histology was regarded as old-fashioned; it wasn’t telling us anything new or anything more than human anatomy was. People had to learn these subjects in order to do medicine or surgery or functional morphology, and then they needed to move on to more active fields. In fact, the analysis of the microscopic structure of fossil bone was never a huge field. We think of pioneers such as Quekett, Gross, Foote, Ørvig, Enlow, Peabody, de Ricqlès, and Reid in the field of fossil tetrapod bone histology, but we do not always realize that they were usually rather lonely.

Although fossil bones have been sectioned for centuries, they have never been systematically sampled until recently. By systematically, we mean not only phylogenetically but also in the sense of having an explicit, standard approach to sampling with the goal of identifying and controlling as many variables in your sample as possible. A simple illustration will show how much and how quickly things have changed. In the 1960s, when Armand de Ricqlès was assembling his samples for his dissertation, which ultimately became the great series of works that he published in Annales de Paléontologie in the 1960s and 1970s, curators of museum collections were reticent to give him perfectly good bones to sacrifice to the saw. Generally, he was able to work with incomplete or fragmentary bones, occasionally with dubious identifications of element and taxon. So it was very difficult for him to standardize his samples and control his variables. Nevertheless, he managed to induce a series of generalizations about patterns of bone growth strategies in tetrapods that in its general outlines and many of its details holds up very well nearly half a century later (Padian 2011).

Paleohistologists have historically faced this main problem: an adventitious, capricious sample set. It was only in the 1980s and 1990s, when John R. (Jack) Horner and contemporaries became interested in what paleohistology could tell us about the ages and growth rates of dinosaurs, that samples could be adequately standardized. The reason was that Jack collected his own dinosaurs, mostly from the Cretaceous of Montana. As Curator of Paleontology at the Museum of the Rockies, he was able not only to cut up whatever he wanted but also to standardize his sections so that he could get a range of sizes and ages of specimens, always sampling the same bone in the same place. Along with pioneering studies by Anusuya Chinsamy and others, Jack, Armand, and their students and colleagues were soon applying the same approaches to a great many extinct amphibians, reptiles, and birds. Jack employed a full-time technician to process thin sections of fossil and recent bones. The burgeoning of this field and the great insights that it has given us into the paleobiology of dinosaurs and other fossil vertebrates are the main reasons for this book.

THE FOUR SIGNALS OF FOSSIL BONE HISTOLOGY

The microscopic structure of fossil bone generally reveals four influences or signals (Horner et al. 1999, 2000; de Ricqlès et al. 2001; Padian et al. 2001). These are ontogeny, phylogeny, mechanics, and environment. These signals hold sway to different degrees in different bones in different taxa at different times of life, as well as in different environments. Reading these signals requires comparative information; without it, inferences about age, growth rate, and other factors can be mistaken. And it is important to note from the start that more than one of these signals can be manifested synergistically in any given section of bone tissue.

Ontogeny

As a bone grows, it changes its histological features in several ways. Cartilaginous or fibrous precursors are replaced by bone. Internal medullary cavities expand, eroding away previous bone, while new periosteal bone is deposited on the outer surfaces. The growth rate of bone tissue drops as the bone develops, along with the number and size of blood vessels and (often) the number of bone cells. Older vascular canals fill with deposited bone, and new (secondary) generations of canals may riddle the older cortex. Remodeling of the original cortical bone by trabecular bone, endosteal bone, or some combination of tissues may take place, especially in the perimedullary region. After longitudinal growth of the bone has effectively ceased, the bone may continue to accumulate periosteal tissue slowly, and this may give the surface of the bone shaft a smooth appearance, while some trochanters and joint ends may become gnarly and rugose, with a quite different appearance than they had while actively growing.

The rate at which a bone is growing at any given time is usually the principal determinant of many of these other histological features. This generalization is called Amprino’s Rule, after the insight of Rodolfo Amprino (1947). He recognized that the form of the bone tissue itself—notably, the number, size, and orientation of its vascular canals—changed throughout growth. Because the vascular canals are responsible for so much of the nutrition of a bone, it follows that greater vascularization permits higher growth rates. These change in predictable ways through ontogeny. Soon after a tetrapod is born or hatched, its growth rate begins to increase. This manifests itself in two ways: Linear growth follows an asymptotic curve in most tetrapods (the misleading term determinate growth; see Chapters 7–9), while mass increase follows one of a family of logistic growth curves. Generally speaking, growth rate is highest when an individual is young, and it tapers gradually toward the adult stage, where it may plateau entirely. Here we make a distinction between a sexually mature adult and a skeletally mature adult in which growth has effectively reached its near-maximum, or asymptote. These events are emphatically not coincident in most tetrapods (Figure 7.6). (See Figure 11.)

FIGURE 1.1. The concepts of determinate and indeterminate growth curves, the contrast between linear and mass curves, and where sexual maturity can occur on these curves. (A) Animals are often divided into those that grow determinately and indeterminately, but we maintain that these curves are identical in their shapes; only their slopes differ. In other words, every animal ceases growth at some point (the plateau at the top of the determinate curve), but animals classified as indeterminate actually grow at rates too slow for the plateau to be perceived. It is also likely that most individuals in a very slow-growing population will die before they reach a growth plateau. For these reasons, we avoid the term indeterminate growth. (B) Contrast between linear growth curves (those based on the growth of a single bone in length or a total body length (or snout-vent, etc.) and mass growth curves (which vary with the third power of any linear measurement, hence the difference in the shape of the curves). Note that linear growth will plateau before mass growth does. As a result, reproductive maturity can correspond to a growth point of (e.g.) 50% of ultimate mass but (e.g.) 67–75% or more of linear growth. Figure drafted by Sarah Werning.

Phylogeny

It is a truism that you are what you inherit, and in this sense organisms inherit the vast majority of features from their ancestors. Yet "descent with modification is the most basic definition of evolution. As organisms evolve from their ancestors, their sizes, habitats, metabolic processes, growth rates, and adaptations are all modified—and bone tissues reflect all of these changes. However, it is also true that the types of bone tissues expressed in skeletons, though primarily reflecting the processes and rates of ontogeny, generally hang together along the lines of phylogenetic groups. That is to say, small lizard species tend to grow more in the manner of larger lizard species than in the manner of small dinosaur species, because they inherit the rates of metabolism and growth that control the expression of bone tissue from common ancestors. As a result, we can generally tell lizard bone from dinosaur bone, given homologous elements. But there is a phylogenetic gradation, as much as there is an ontogenetic gradation, in the types of bone tissues expressed in the skeleton. Through the branches of a phylogeny, various features of bone—including vascular density, osteocyte density, the types of vascular canals, the compactness of tissue, and the rate of deposition—may change phylogenetically. The phylogenetic signal" in bone is persistent, but it is never the strongest signal. It is mainly a reminder that related animals do not vary capriciously in the expression of their bone tissues, and the reason is that inherited patterns are persistent.

Mechanics

There are various definitions of Wolff’s Law, named for the nineteenth-century German anatomist Julius Wolff, but they generally denote the generalization that the mechanical load on a bone will shape its form, including its internal structure. We call this the mechanical signal in bone histology. This can be studied in the course of an individual’s lifetime, among individuals in a population, or among species that are phylogenetically related or ecologically similar in their mechanical needs. Forces of torsion, which are especially experienced by flying animals, and forces of compression, which are heightened in diving aquatic animals, cause significant departures from the histological features of more typical terrestrial relatives. Increased loading may cause a bone tissue to grow with a more compact form or to remodel itself more extensively, which can be indicated by an influx of secondary osteons. Emmanuel de Margerie (2002, 2006) found that the wings of birds tend to produce a predominance of the laminar form of fibro-lamellar bone tissue. This configuration presumably helps the bone to resist the torsion that the forces of flapping place on it. Armand de Ricqlès et al. (2000) noted a similar pattern in the wing bones of pterosaurs: The tissues grow in a sort of plywood pattern so that the bone fibers of successive layers are oriented at angles to each other, presumably also for strengthening purposes.

In the same way, aquatic tetrapods tend to have denser, more compact bones with thicker walls in which the medullary cavity is often filled with trabecular bone or is virtually nonexistent (Houssaye 2009; de Ricqlès & de Buffrénil 2001). Such manifestations are usually labeled pachyostosis, but as Houssaye (2009) has shown, this term has been used to describe several different phenomena. They are, however, mechanical adaptations of the bones that help them to reduce different kinds of stresses. Through time they have become part of the genetic expression of the bone.

Environment

Although the environment of an organism obviously figures largely in almost every biological process in one sense or another, here we use a restricted sense of environment—namely, the direct influence that the ambient environment has on the form of bone tissues. Here are some examples. When temperatures are relatively cooler, the metabolic reactions of many tetrapods (those commonly and misleadingly called ectothermic or cold-blooded) tend to work at slower rates than when temperatures are relatively higher. The effect is that bone grows more slowly as well, and this rate difference shows up in how the tissue is expressed. Generally, one may see more pronounced lines of arrested growth and thinner zones of cortical bone between the periodic growth lines. The tissue may also become more compact, with less vascularization and fewer bone cells. Calcium deficiency, starvation, and dehydration may exercise similar effects. These effects, of course, are being expressed developmentally, like all other features of bone, but here the difference is that as opposed simply to mechanical or ontogenetic influences, the environmental conditions for bone growth and formation are less than ideal, and these trigger the effects on bone formation.

In this mix one could also list diseases of bone such as osteoporosis; scarring that results from wounds, breaks, or infections; and various other lesions. All of these, however, also reflect developmental anomalies, because they describe the response by the tissue to some external insult to it, and this usually takes the form of replacement tissue. Osteoporosis is in a slightly different category because it generally describes what happens when the rate of resorption of calcium minerals in the skeleton, which is a normal part of growth, outstrips the growth and replacement of this tissue, which is just as normal. The result is that the bones become thinner in their walls and more brittle. So it is a mechanical result, but a developmental process, that is caused by direct environmental factors. It can be seen that these four categories are not mutually exclusive; after all, one inherits a developmental program from ancestors, so the ontogeny component is in a sense part of the phylogeny component. However, these qualifications are easily recognized, and do not impair us from identifying and controlling causal variables in the expression of bone tissue.

DIAGENESIS: A POST-MORTEM SIGNAL IN FOSSIL BONE

When a bone is extracted for histological analysis from an extant animal, it suffers no loss of information except as occasioned by the removal of soft tissues and the staining and embedding of the bone before sectioning. A fossilized bone, in contrast, carries with it the history of the post-mortem processes that befell it. The matrix in

FIGURE 1.2. Reconstruction of the dorso-ventrally crushed bones of Confuciusornis sanctus. (A) Section showing adjacent left fibula and tibia with pygostyle amid matrix (brown color). (B) Reconstructions of the bones by computer enhancement of photographs of thin sections, by E. de Margerie. ICD is the index of the ratio between the cortical thickness and the diameter of the bone. From de Ricqlès et al. (2003).

which it was preserved, and the minerals brought by the groundwater that bathed it for eons, stained it and filled its micro-crevasses—which is why fossil bones feel so much heavier (actually denser) than bones of recently deceased animals.

During fossilization, bones are often crushed and abraded. Photographs of cross-sections of crushed bones can be imaged and their fragments rearranged to restore the outline of the bone in life (e.g., Confuciusornis: de Ricqlès et al. 2003; Figure 1.2). Sometimes the minerals preserved inside the bones are different than those in the surrounding sediments, and this fact attests to differential accumulation through groundwater or the transport of the bone from its original resting place.

Post-mortem invasion of bacteria and fungi can destroy internal bone tissue, making it useless for histological analysis. Unfortunately, the external features of the bone usually provide no indication of this decay.

All of these changes are environmental rather than biological influences on the bone. They are post-mortem rather than in vivo. The other four signals that we discuss above are biological, and they manifest themselves during the life of the animal.

THE EXPLANATION OF VARIATION

All four of these signals help us to make sense of the kinds of variation that we see in the bone tissues of extinct tetrapods. They have been shown empirically to be reliable guides to the interpretation of the most basic question in the paleohistology of bone: Why is this tissue formed the way it is, and why does it differ from the tissue of this other animal? These variations, after all, are what we are trying to explain in the first place.

The answers form a range of possible hypotheses. One individual is older than the other, larger than the other, of a different species, of a different sex, or growing more quickly (or more slowly); or it has suffered from disease or injury, or it is just expressing the range of normal populational variation—or more than one of these hypotheses may apply. The opportunity of the bone histologist is to make sense of this variation by testing these hypotheses and so apply a line of evidence that is not available to gross anatomy alone. Without bone histology, for example, there can be endless arguments about whether a particular character difference between a small and a large skeleton is merely ontogenetic or is diagnostic of different species. We will discuss these questions later in the book.

THE ACTUALISTIC RATIONALE

Actualism is the principle by which we assume that if we identify a structure in a living creature that has a particular function or behavior and then find that same structure with the same form in a related extinct creature, we can presume that it had a similar function or behavior. (The word actualism has a root in the Romance languages that means current or right now.) So, for example, if we find a bone tissue that has a certain character to its matrix and a certain degree of vascularity, we can presume that a fossil bone with the same features was growing at approximately the same rate.

The growth rates of the bones of a number of living tetrapods have been measured experimentally, initially by feeding animals the madder plant (Rubia tinctorum L.), which contains the calcium-binding dye alizarin, sometimes by injecting dyes at various stages of growth (e.g., Duhamel de Monceau 1742; Castanet et al. 1996; see Chapter 34 in Hall 2005 for a discussion). Because we know the time intervals at which these dyes were injected, we know how much bone growth took place in those intervals and hence how many microns of bone thickness were deposited periosteally per day on average. For lizards and other slowly growing tetrapods, it may be only a few μ/day; for young birds, it may be dozens of μ/day. Penguin chick limb bones have been recorded at over 200 μ/day (de Margerie et al. 2004). As a result, specific histological characters of bone can be associated with specific ranges of growth rates.

Various names have been given to different types of tetrapod bone tissues (see Chapter 2). Even within limb bones, there are different names that reflect the texture of bone, its compactness, and its mode of formation (Hall 2005). These reflect above all the conditions and rates at which the bone is developing, as previously noted. In tetrapod limbs, for example, parallel-fibered (often called lamellar-zonal) tissue (commonly found in the long bones of lizards, crocodiles, and turtles) is a compact bone with relatively few bone cells (osteocytes) and blood vessels (vascular canals). It forms by the deposition of successive lamellae of bone, and it is sometimes interrupted by a zone of nearly avascular and acellular bone that reflects very slow growth, as during annual periods of hiatus in normal growth. Fibro-lamellar bone, in contrast, forms as the vascular canals bring nutrients that form a scaffolding of organic tissue that is progressively mineralized by osteoblasts (bone-forming cells). Its texture is fibrous and woven, and it tends to have more blood vessels and osteocytes than lamellar-zonal bone.

Fibro-lamellar bone will be separately described by a combination of both its matrix and vascular patterns. Dinosaurs and pterosaurs, for example, typically produce this tissue in their long bones, and we infer from this that their growth rates were relatively higher than for most other reptiles.

Within the complex of bone tissue types are several sub-categories that vary continuously, mainly in the density and orientation (and sometimes size) of their vascular canals. Castanet et al. (1996) arranged these along a spectrum (Figure 1.3). Longitudinal tissue has vascular canals that are mostly oriented parallel to the long axis of the shaft; laminar or sub-plexiform tissue tends to have mostly circumferential canals with regular centrifugal anastomoses, typical of juvenile to sub-adult bone; plexiform bone has vascular canals that grow in all directions, notably with a high component of centrifugal canals that are often found in young bone; reticular tissue has common to frequent anastomoses among these canals in circumferential and centrifugal directions; and radial vascularization is characterized by canals that run mostly centrifugally and reflect very rapid growth. These vascular patterns do not indicate set rates, as Castanet and his colleagues found (2000); the absolute rates are set by other factors, including the metabolic rates of the animal, which is often a factor of the body size of the taxon (Case 1978). However, the five vascular types seem to grow in this same degree of rapidity within a single taxon. This allows us to estimate a range of rates of tissue growth among these types for particular taxa. So, for example, the growth rate of a bone with reticular vascular canals in a hadrosaur may be estimated from the growth rate of a living