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

Chapter 1. WHY STUDY THE BONE MICROSTRUCTURE OF FOSSIL TETRAPODS?

Kevin Padian

In the 19^th 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 20^th-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, any 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 Queckett, 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 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 90s, 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 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; 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 4.1). (See Figure 1 1.)

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 they modify their size, habitat, metabolic processes, growth rates, and adaptations, 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 19^th-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. 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. Ricqlès et al. (2000) noted a similar pattern in the wing bones of pterosaurs, in which 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 non-existent (Houssaye 2009; de Ricqlès and 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, and (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 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 influences on the bone, rather than biological. 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, then if we 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 noted above. In tetrapod limbs, for example, Parallel-fibered (often called lamellar-zonal) tissue, commonly found in the long bones of lizards, crocodiles, and turtles, for example, 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 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 tissues types there 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 bird or mammal also with with reticular canals, provided that factors such as the degree of vascularization are comparable. However, this science is still in its infancy, and we need a lot more ground-truthing from experimental studies of living birds and mammals.

SUMMARY

Bone histology can provide information not available from other kinds of examination. Along with some calibration methods based on teeth in many mammalian groups, it forms the basis of skeletochronology, the only currently available universal line of evidence that provides an absolute age on the skeletons of extinct vertebrates. With the calibration of age also comes a knowledge of growth rates, which in turn can provide information about relative metabolic rates, particularly when the type of bone tissues deposited in these sequences is considered. Because the skeleton erodes and reworks itself during growth, records of earlier growth are usually erased, so specimens from a spectrum of ages are needed to reconstruct a complete growth series. The type of tissue deposited in a bone at any given time is the reflection of several universal factors, including ontogeny, phylogeny, mechanics, and the environment. Reading these influences is most reliably done with the comparative data of living osteohistology. Regrettably, we still know far too little of the natural growth rates and trajectories of most living animals that would be instrumental to the interpretation of extinct ones.

ACKNOWLEDGMENTS

I thank Adam Huttenlocher, Brian Hall, Louise Roth, Laura Wilson for reviewing an earlier draft of this chapter.

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