For more than 300 years paleontologists have operated under the assumption that the information contained in fossilized bones lies only in the size and shape of the bones themselves. It was thought that when an animal dies under conditions suitable for fossilization, inert minerals from the surrounding environment eventually replace all the organic molecules—such as those that make up cells, tissues, pigments and proteins—leaving behind what is essentially a “cast” of the once living bones, now composed entirely of mineral. My first indication that this fundamental tenet of paleontology might not always apply came when I was a relatively new graduate student at Montana State University, studying the microstructure of a Tyrannosaurus rex bone. As I peered through the microscope, what I saw—small, round, red and apparently nucleated structures, restricted to the blood vessel channels coursing through the bone—had not been previously noted, to my knowledge. They looked similar to the nucleated red blood cells of nonmammalian vertebrates. But seeing dinosaur blood cells was impossible, according to the conventional wisdom that shaped my discipline. After I sought opinions on the identity of the red spheres from faculty members and other graduate students, word of the puzzle reached Jack Horner, curator of paleontology at the museum and one of the world’s foremost dinosaur authorities. He took a look for himself. Brows furrowed, he gazed through the microscope for what seemed like hours without saying a word. Then, looking up at me with a frown, he asked, “What do you think they are?” When I replied that I did not know but that they were the right size, shape, location and color to be blood cells, he grunted. “So prove to me they aren’t.” It was an irresistible challenge, and one that continues to help frame my research. Since that early discovery, my colleagues and I have recovered various types of organic remains—including blood vessels, bone cells and bits of the fingernail-like material that makes up claws—from multiple fossilized remains, indicating that although such preservation may not be common, neither is it a one-time occurrence. These findings not only diverge from textbook descriptions of the fossilization process, they are also yielding fresh insights into the biology of bygone creatures. For instance, bone from another T. rex specimen has revealed that the animal was a female that was “in lay” (preparing to lay eggs) when she died—information we could not have gleaned from the shape and size of the bones alone. And a protein detected in remnants of fibers near a small carnivorous dinosaur unearthed in Mongolia has helped establish that the dinosaur had structures that, not just morphologically but at the molecular level, were consistent with the feathers of modern birds. First Signs Extraordinary claims, as the old adage goes, require extraordinary evidence. Careful scientists make every effort to disprove their own cherished hypotheses before they accept that their ideas are correct. Thus, for the past 20 years I have been trying every experiment I can think of to disprove the hypothesis that the materials my collaborators and I have discovered are components of tissues produced by once living animals. In the case of the red microstructures I saw in the T. rex bone, I reasoned that if they were related to blood cells or their constituents (such as molecules of hemoglobin or heme that had clumped together after being released from dying blood cells), they would have persisted in some, albeit possibly very altered, form only if the bones themselves were exceptionally well preserved. At the macroscopic level, this dinosaur met that criterion. The skeleton, a nearly complete specimen from eastern Montana, known as MOR 555, includes many rarely preserved bones. Microscope examination of thin sections of the limb bones revealed similarly pristine preservation. Most of the blood vessel channels in the dense bone were empty, not filled with mineral deposits, as is usually the case with dinosaurs. Next, I turned my attention to the chemical composition of the blood cell look-alikes. Analyses showed that they were rich in iron, as red blood cells are, and that the iron was specific to them. Not only did the elemental makeup of the mysterious red things (we nicknamed them LRRTs, “little round red things”) differ from that of the bone immediately surrounding the vessel channels, it was also distinct from that of the sediments in which the dinosaur was buried. But to further test the connection between the red structures and blood cells, I wanted to examine my samples for heme, the small iron-containing molecule that gives vertebrate blood its scarlet hue and enables hemoglobin proteins to carry oxygen from the lungs to the rest of the body. Heme vibrates, or resonates, in telltale patterns when it is stimulated by tuned lasers, and because it contains a metal center, it absorbs light in a very distinct way. When we subjected extracts of whole bone samples to spectroscopy tests—which measure the light that a given material emits, absorbs or scatters—our results showed that somewhere in the dinosaur’s bone were compounds that were consistent with heme. One of the most compelling experiments we conducted took advantage of the immune response. When the body detects an invasion by foreign, potentially harmful substances, it produces defensive proteins called antibodies that can specifically recognize, and bind to, those substances. We injected extracts of the dinosaur bone into mice, causing the mice to make antibodies against the organic compounds in the extract. When we then exposed these dinosaur antibodies to hemoglobin from turkeys and rats, they bound to the hemoglobin—a sign that the dinosaur extracts that stimulated antibody production in the mice had included a molecule similar in some way to the hemoglobin in living animals. None of the many chemical and immunological tests we performed rose to Jack’s original challenge to “prove they aren’t” blood cells from a T. rex. Yet we could not show that the hemoglobinlike substance was specific to the red structures—the available techniques were not sufficiently sensitive to permit such differentiation at the time I was conducting these studies. Thus, we could not claim definitively that these structures were derived from blood cells. When we published our findings in 1997, we drew our conclusions conservatively, stating that hemoglobin proteins might be preserved and that the most likely source of the protein was the cells of the dinosaur. The Evidence Builds Through this initial study on T. rex, I began to realize just how much fossil molecules could reveal about extinct animals. If we could obtain proteins, we could conceivably decipher the sequence of their constituent amino acids, much as geneticists sequence the “letters” that make up DNA. And like DNA sequences, protein sequences contain information about evolutionary relationships between animals, how species change over time and how the acquisition of new genetic traits might have conferred advantages to the animals possessing them. But first I had to show that ancient proteins were present in fossils other than the wonderful T. rex we had been studying. Working with Mark Marshall, then at Indiana University, and with Seth Pincus and John Watt, both at Montana State during this time, I turned my attention to two well-preserved fossils that looked promising for recovering other organic molecules. The first was a beautiful primitive bird named Rahonavis that paleontologists from Stony Brook University and Macalester College had unearthed from deposits in Madagascar dating to the Late Cretaceous period, around 70 million to 66 million years ago. During excavation they had noticed a white, fibrous material on the skeleton’s toe bones. No other bone in the quarry seemed to have the substance, nor was it present on any of the sediments there. They wondered whether the material might be akin to the strong sheath made of keratin protein that covers the toe bones of living birds, forming their claws. Keratin proteins are good candidates for preservation because they are abundant in vertebrates, and the composition of this protein family makes them very resistant to degradation. They come in two main types: alpha and beta. All vertebrates have alpha-keratin, which in humans makes up hair and nails and helps the skin to resist abrasion and dehydration. Beta-keratin is absent from mammals and occurs only in birds and reptiles among living organisms. To test for keratins in the white material on the Rahonavis toe bones, we employed many of the same techniques I had used to study T. rex. Notably, antibody tests indicated the presence of alpha- and beta-keratin, both of which are expressed in the claws of living birds and reptiles. We also applied additional diagnostic tools. One method detected amino acids that were localized to the toe-bone covering, which was supported by the identification of nitrogen (a component of amino acids). The results of all our tests supported the notion that the cryptic white material covering the ancient bird’s toe bones was the remainder of its once lethal claws. The second specimen we probed was a spectacular Late Cretaceous fossil that researchers from the American Museum of Natural History in New York City had discovered in Mongolia. Although the scientists dubbed the animal Shuvuuia deserti, or “desert bird,” it was actually a small carnivorous dinosaur. While cleaning the fossil, Amy Davidson, a preparator at the museum, noticed small white fibers in the animal’s neck region. She asked me if I could tell if they were remnants of feathers. Birds are descended from dinosaurs, and fossil hunters have discovered a number of dinosaur fossils that preserve impressions of feathers, so in theory the suggestion that Shuvuuia had a downy coat was plausible. I did not expect that a structure as delicate as a feather could have endured the ravages of time, however. I suspected the white fibers instead came from modern plants or from fungi. But I agreed to take a closer look. To my surprise, initial tests ruled out plants or fungi as the source of the fibers. The fibers were hollow and contained tiny filaments consistent with the molecular structure of beta-keratin. Moreover, subsequent analyses of the composition of the strange white strands pointed to the presence of keratin. Mature feathers in living birds consist almost exclusively of beta-keratin. If the small fibers on Shuvuuia were related to feathers, then they should harbor beta-keratin alone, in contrast to the claw sheath of Rahonavis, which contained both alpha- and beta-keratin. That, in fact, is exactly what we found when we conducted our antibody tests—results we published in 1999. Extraordinary Finds By now I was convinced that small remnants of original proteins could survive in extremely well preserved fossils and that we had the tools to identify them. But many in the scientific community remained unconvinced. Our findings challenged everything they thought they knew about the breakdown of cells and molecules. Test-tube studies of organic molecules indicated that proteins should not persist more than a million years or so; DNA had an even shorter predicted life span. Researchers had claimed previously that they had recovered DNA millions of years old, but subsequent work failed to validate the results, enhancing the controversy surrounding claims of recovery of very old molecules. The only widely accepted claims of ancient molecules were no more than tens of thousands of years old. In response to this resistance, a colleague advised me to step back a bit and demonstrate the efficacy of our methods for identifying ancient proteins in bones that were old but not as old as dinosaur bone. Working with analytical chemist John Asara of Harvard University, we obtained proteins from mammoth fossils that were estimated to be 300,000 to 600,000 years old. Sequencing of the proteins using a technique called mass spectrometry identified them unambiguously as collagen, a key component of bone, tendons, skin and other tissues. The publication of our results in 2002 did not trigger much controversy. Indeed, the scientific community largely ignored it. But our proof of principle was about to come in very handy. The next year a crew from the Museum of the Rockies finally finished excavating another T. rex skeleton, which at 68 million years old is the oldest one to date. Like the younger T. rex described above, this one—called MOR 1125 and nicknamed “Brex,” after discoverer Bob Harmon—was recovered from the Hell Creek Formation in eastern Montana. The remote site has no vehicle access, so a helicopter ferried plaster jackets containing excavated bones from the site to the camp. The jacket containing the leg bones was too heavy for the helicopter to lift. To retrieve them, then, the team broke the jacket, separated the bones and rejacketed them. But these bones are very fragile, and when the original jacket was opened, many fragments of bone fell out and were collected and boxed up for me. Because my original T. rex studies were controversial, I was eager to repeat the work on a second T. rex, and this find presented the perfect opportunity. As soon as I laid eyes on the first piece of bone I removed from that box, a fragment of thighbone, I knew the skeleton was special. Lining the internal surface of this fragment was a thin, distinct layer of a type of bone that had never been found in dinosaurs. This layer was very fibrous, filled with blood vessel channels, and completely different in color and texture from the cortical bone that constitutes most of the skeleton. “Oh, my gosh, it’s a girl—and it’s pregnant!” I exclaimed to my then assistant, Jennifer Wittmeyer. She looked at me like I had lost my mind. But having studied bird physiology, I was nearly certain that this distinctive feature was medullary bone, a special tissue that appears for only a limited time (often for just about two weeks), when birds are in lay. The special bone tissue exists to provide an easy source of calcium to fortify the eggshells, and when the last egg is laid, the medullary bone is quickly resorbed. One of the characteristics that sets medullary bone apart from other bone types is the random orientation of its collagen fibers, a characteristic that indicates very rapid formation. (This same organization occurs in the first bone laid down when you have a fracture—that is why you feel a lump in healing bone.) The bones of any animal can be demineralized using mild acids to reveal the telltale arrangement of the collagen fibers. In living birds, removing the minerals from medullary bone should leave behind randomly oriented fibers. But this was dinosaur, not bird. I thought that almost all organics were gone from dinosaur bone, and if we let the reaction run too long, we would have nothing left, so Wittmeyer and I decided to lightly etch the surface. To our surprise, as the minerals dissolved they left a flexible and fibrous clump of tissue. I could not believe what we were seeing. I asked Wittmeyer to repeat the experiment multiple times. Each time we placed the distinctive layer of bone in the mild acid solution, fibrous stretchy material remained—just as it does when medullary bone in birds is treated in the same way. Furthermore, when we then dissolved pieces of the denser, more common cortical bone, we obtained more soft tissue. Hollow, transparent, flexible, branching tubes emerged from the dissolving matrix—and they looked exactly like blood vessels. Suspended inside the vessels were either small, round red structures, very like those in the original T. rex that started me down this path, or amorphous accumulations of red material. Additional demineralization experiments revealed distinctive-looking bone cells called osteocytes that secrete the collagen and other components making up the organic part of living bone. The whole dinosaur seemed to preserve material never seen before in any dinosaur bone! When we published our observations in Science in 2005, reporting the presence of what looked to be collagen, blood vessels and bone cells, the paper garnered a lot of attention, but the scientific community adopted a wait-and-see attitude. We claimed only that the material we found resembled these modern components—not that they were one and the same—because at that time we had no chemical data, only morphological data. And after millions of years, buried in sediments and exposed to geochemical conditions that varied over time, what was preserved in these bones might bear little chemical resemblance to what was there when the dinosaur was alive. The real value of these materials could be determined only if their composition could be discerned. Our work had just begun. Using all the techniques honed while studying MOR 555, Rahonavis, Shuvuuia and the mammoth, I began an in-depth analysis of this T. rex’s bone in collaboration with Asara, who had refined the methods we used in the mammoth study and was ready to try sequencing the dinosaur’s far older proteins. This was a much harder exercise because the concentration of organics in the dinosaur was orders of magnitude less than in the much younger mammoth and because the proteins were very degraded. Nevertheless, we were eventually able to sequence protein fragments. And gratifyingly, when our colleague Chris Organ of Harvard compared the T. rex sequences with those of a multitude of other organisms, he found that they grouped most closely with birds, followed by crocodiles—the two groups that are the closest living relatives of dinosaurs. Controversy and Its Aftermath Our papers detailing the sequencing work, published in 2007 and 2008, generated a firestorm of controversy, most of which focused on our interpretations of the sequence data. Some dissenters charged that we had not produced enough sequences to make our case; others argued that the structures we interpreted as primeval soft tissues were actually biofilm—“slime” produced by microbes that had invaded the fossilized bone. I had mixed feelings about their feedback. On one hand, scientists are paid to be skeptical. On the other hand, science operates on the principle of parsimony—the simplest explanation for all the data is assumed to be the correct one. And we had supported our hypothesis with multiple lines of evidence. Still, I knew that a single gee-whiz discovery does not have any long-term meaning in science. We had to sequence proteins from other dinosaur bones. When a volunteer accompanying us on a summer expedition found bones from an 80-million-year-old plant-eating duck-billed dinosaur called Brachylophosaurus canadensis (“Brachy” for short), we suspected the duckbill might be a good source of ancient proteins even before we got its bones out of the ground. Hoping that it might contain organics, we did everything we could to free it from the surrounding sandstone quickly while minimizing its exposure to chemicals, contaminants and the elements. Air pollutants, humidity fluctuations and the like would be very harmful to fragile molecules, and the longer the bone was exposed to any of these factors, the more likely contamination and degradation would occur. Perhaps because of this extra care—and prompt analyses—both the chemistry and the morphology of this second dinosaur were less altered than Brex’s. As we had hoped, we found cells embedded in a matrix of white collagen fibers in the animal’s bone. The cells exhibited long, thin, branchlike extensions that are characteristic of osteocytes, which we could trace from the cell body to where they connected to other cells. A few of them even contained what appeared to be internal structures, including possible nuclei. Furthermore, extracts of the duckbill’s bone reacted with antibodies that target collagen and other proteins that bacteria do not manufacture, refuting the suggestion that our soft-tissue structures were merely biofilms. In addition, the protein sequences we obtained from the bone most closely resembled those of modern birds, just as Brex’s did. We reported these findings in Science in 2009. We have since demineralized bone from organisms ranging from modern chickens to Triassic material, collected in different environments and on different continents, to show that we can recover at least three of the four elements preserved in more than one dinosaur: blood vessels, vascular contents, osteocytes and collagen-based matrix. Also, we now have many independent lines of evidence indicating that those elongated, branching microstructures in the samples taken from Brex and Brachy are indeed the bone cells of once living dinosaurs. In one of the most convincing experiments, we exposed these microstructures to monoclonal antibodies that bind with a protein expressed in the osteocytes of living birds but not in the alligator osteocytes that we used as controls. We found that the antibodies did bind to the microstructures from both dinosaurs, in the same pattern that we saw in ostrich bone cells. Although we have data hinting at the presence of DNA inside these cells, our findings will not be conclusive unless we can obtain DNA sequences consistent with those predicted for dinosaurs—but the DNA may be too fragmented and altered to be sequenced. One frequent criticism of our work is that it is inconsistent with models of cellular, tissue and molecular degradation—all of which predict life spans far shorter than 80 million years. So we conducted another set of experiments to address how, under naturally occurring conditions, these components might be preserved for such long time periods. We have noticed that in all cases where we are able to recover blood vessels and cells, we also see iron, embedded as tiny particles that are only visible under high-magnification transmission electron microscopy. We reason that this iron may have come from the breakdown of iron-rich hemoglobin, released by dying red blood cells. As the hemoglobin molecules break down, the unstable biological iron they release undergoes chemical reactions that unleash highly reactive free hydroxyl radicals. These hungry molecules attack tissues, stealing electrons to make themselves more stable and in the process causing cross-links to form between molecules of the tissue. This reaction, which is lethal in living organisms because the cross-linked molecules cannot function normally, acts much like placing tissues in formaldehyde—a preservative that also causes cross-links to form. In experiments we have observed that blood vessels recovered from modern bone can remain stable in water at room temperature for more than two years if they are first briefly soaked in a lysate of red blood cells, whereas untreated blood vessels degrade in days to weeks. These results suggest that free radicals in the blood lysate may have stabilized the tissues against early degradation. Our results have met with a lot of skepticism. They are, after all, extremely surprising, but they are also full of promise. The study of ancient organic molecules from dinosaurs could yield insights into how these great beasts evolved, how they responded to major environmental changes and ultimately what did them in.
My first indication that this fundamental tenet of paleontology might not always apply came when I was a relatively new graduate student at Montana State University, studying the microstructure of a Tyrannosaurus rex bone. As I peered through the microscope, what I saw—small, round, red and apparently nucleated structures, restricted to the blood vessel channels coursing through the bone—had not been previously noted, to my knowledge. They looked similar to the nucleated red blood cells of nonmammalian vertebrates. But seeing dinosaur blood cells was impossible, according to the conventional wisdom that shaped my discipline. After I sought opinions on the identity of the red spheres from faculty members and other graduate students, word of the puzzle reached Jack Horner, curator of paleontology at the museum and one of the world’s foremost dinosaur authorities. He took a look for himself. Brows furrowed, he gazed through the microscope for what seemed like hours without saying a word. Then, looking up at me with a frown, he asked, “What do you think they are?” When I replied that I did not know but that they were the right size, shape, location and color to be blood cells, he grunted. “So prove to me they aren’t.” It was an irresistible challenge, and one that continues to help frame my research.
Since that early discovery, my colleagues and I have recovered various types of organic remains—including blood vessels, bone cells and bits of the fingernail-like material that makes up claws—from multiple fossilized remains, indicating that although such preservation may not be common, neither is it a one-time occurrence. These findings not only diverge from textbook descriptions of the fossilization process, they are also yielding fresh insights into the biology of bygone creatures. For instance, bone from another T. rex specimen has revealed that the animal was a female that was “in lay” (preparing to lay eggs) when she died—information we could not have gleaned from the shape and size of the bones alone. And a protein detected in remnants of fibers near a small carnivorous dinosaur unearthed in Mongolia has helped establish that the dinosaur had structures that, not just morphologically but at the molecular level, were consistent with the feathers of modern birds.
First Signs Extraordinary claims, as the old adage goes, require extraordinary evidence. Careful scientists make every effort to disprove their own cherished hypotheses before they accept that their ideas are correct. Thus, for the past 20 years I have been trying every experiment I can think of to disprove the hypothesis that the materials my collaborators and I have discovered are components of tissues produced by once living animals.
In the case of the red microstructures I saw in the T. rex bone, I reasoned that if they were related to blood cells or their constituents (such as molecules of hemoglobin or heme that had clumped together after being released from dying blood cells), they would have persisted in some, albeit possibly very altered, form only if the bones themselves were exceptionally well preserved. At the macroscopic level, this dinosaur met that criterion. The skeleton, a nearly complete specimen from eastern Montana, known as MOR 555, includes many rarely preserved bones. Microscope examination of thin sections of the limb bones revealed similarly pristine preservation. Most of the blood vessel channels in the dense bone were empty, not filled with mineral deposits, as is usually the case with dinosaurs.
Next, I turned my attention to the chemical composition of the blood cell look-alikes. Analyses showed that they were rich in iron, as red blood cells are, and that the iron was specific to them. Not only did the elemental makeup of the mysterious red things (we nicknamed them LRRTs, “little round red things”) differ from that of the bone immediately surrounding the vessel channels, it was also distinct from that of the sediments in which the dinosaur was buried. But to further test the connection between the red structures and blood cells, I wanted to examine my samples for heme, the small iron-containing molecule that gives vertebrate blood its scarlet hue and enables hemoglobin proteins to carry oxygen from the lungs to the rest of the body. Heme vibrates, or resonates, in telltale patterns when it is stimulated by tuned lasers, and because it contains a metal center, it absorbs light in a very distinct way. When we subjected extracts of whole bone samples to spectroscopy tests—which measure the light that a given material emits, absorbs or scatters—our results showed that somewhere in the dinosaur’s bone were compounds that were consistent with heme.
One of the most compelling experiments we conducted took advantage of the immune response. When the body detects an invasion by foreign, potentially harmful substances, it produces defensive proteins called antibodies that can specifically recognize, and bind to, those substances. We injected extracts of the dinosaur bone into mice, causing the mice to make antibodies against the organic compounds in the extract. When we then exposed these dinosaur antibodies to hemoglobin from turkeys and rats, they bound to the hemoglobin—a sign that the dinosaur extracts that stimulated antibody production in the mice had included a molecule similar in some way to the hemoglobin in living animals.
None of the many chemical and immunological tests we performed rose to Jack’s original challenge to “prove they aren’t” blood cells from a T. rex. Yet we could not show that the hemoglobinlike substance was specific to the red structures—the available techniques were not sufficiently sensitive to permit such differentiation at the time I was conducting these studies. Thus, we could not claim definitively that these structures were derived from blood cells. When we published our findings in 1997, we drew our conclusions conservatively, stating that hemoglobin proteins might be preserved and that the most likely source of the protein was the cells of the dinosaur.
The Evidence Builds Through this initial study on T. rex, I began to realize just how much fossil molecules could reveal about extinct animals. If we could obtain proteins, we could conceivably decipher the sequence of their constituent amino acids, much as geneticists sequence the “letters” that make up DNA. And like DNA sequences, protein sequences contain information about evolutionary relationships between animals, how species change over time and how the acquisition of new genetic traits might have conferred advantages to the animals possessing them. But first I had to show that ancient proteins were present in fossils other than the wonderful T. rex we had been studying. Working with Mark Marshall, then at Indiana University, and with Seth Pincus and John Watt, both at Montana State during this time, I turned my attention to two well-preserved fossils that looked promising for recovering other organic molecules.
The first was a beautiful primitive bird named Rahonavis that paleontologists from Stony Brook University and Macalester College had unearthed from deposits in Madagascar dating to the Late Cretaceous period, around 70 million to 66 million years ago. During excavation they had noticed a white, fibrous material on the skeleton’s toe bones. No other bone in the quarry seemed to have the substance, nor was it present on any of the sediments there. They wondered whether the material might be akin to the strong sheath made of keratin protein that covers the toe bones of living birds, forming their claws.
Keratin proteins are good candidates for preservation because they are abundant in vertebrates, and the composition of this protein family makes them very resistant to degradation. They come in two main types: alpha and beta. All vertebrates have alpha-keratin, which in humans makes up hair and nails and helps the skin to resist abrasion and dehydration. Beta-keratin is absent from mammals and occurs only in birds and reptiles among living organisms.
To test for keratins in the white material on the Rahonavis toe bones, we employed many of the same techniques I had used to study T. rex. Notably, antibody tests indicated the presence of alpha- and beta-keratin, both of which are expressed in the claws of living birds and reptiles. We also applied additional diagnostic tools. One method detected amino acids that were localized to the toe-bone covering, which was supported by the identification of nitrogen (a component of amino acids). The results of all our tests supported the notion that the cryptic white material covering the ancient bird’s toe bones was the remainder of its once lethal claws.
The second specimen we probed was a spectacular Late Cretaceous fossil that researchers from the American Museum of Natural History in New York City had discovered in Mongolia. Although the scientists dubbed the animal Shuvuuia deserti, or “desert bird,” it was actually a small carnivorous dinosaur. While cleaning the fossil, Amy Davidson, a preparator at the museum, noticed small white fibers in the animal’s neck region. She asked me if I could tell if they were remnants of feathers. Birds are descended from dinosaurs, and fossil hunters have discovered a number of dinosaur fossils that preserve impressions of feathers, so in theory the suggestion that Shuvuuia had a downy coat was plausible. I did not expect that a structure as delicate as a feather could have endured the ravages of time, however. I suspected the white fibers instead came from modern plants or from fungi. But I agreed to take a closer look.
To my surprise, initial tests ruled out plants or fungi as the source of the fibers. The fibers were hollow and contained tiny filaments consistent with the molecular structure of beta-keratin. Moreover, subsequent analyses of the composition of the strange white strands pointed to the presence of keratin. Mature feathers in living birds consist almost exclusively of beta-keratin. If the small fibers on Shuvuuia were related to feathers, then they should harbor beta-keratin alone, in contrast to the claw sheath of Rahonavis, which contained both alpha- and beta-keratin. That, in fact, is exactly what we found when we conducted our antibody tests—results we published in 1999.
Extraordinary Finds By now I was convinced that small remnants of original proteins could survive in extremely well preserved fossils and that we had the tools to identify them. But many in the scientific community remained unconvinced. Our findings challenged everything they thought they knew about the breakdown of cells and molecules. Test-tube studies of organic molecules indicated that proteins should not persist more than a million years or so; DNA had an even shorter predicted life span. Researchers had claimed previously that they had recovered DNA millions of years old, but subsequent work failed to validate the results, enhancing the controversy surrounding claims of recovery of very old molecules. The only widely accepted claims of ancient molecules were no more than tens of thousands of years old.
In response to this resistance, a colleague advised me to step back a bit and demonstrate the efficacy of our methods for identifying ancient proteins in bones that were old but not as old as dinosaur bone. Working with analytical chemist John Asara of Harvard University, we obtained proteins from mammoth fossils that were estimated to be 300,000 to 600,000 years old. Sequencing of the proteins using a technique called mass spectrometry identified them unambiguously as collagen, a key component of bone, tendons, skin and other tissues. The publication of our results in 2002 did not trigger much controversy. Indeed, the scientific community largely ignored it. But our proof of principle was about to come in very handy.
The next year a crew from the Museum of the Rockies finally finished excavating another T. rex skeleton, which at 68 million years old is the oldest one to date. Like the younger T. rex described above, this one—called MOR 1125 and nicknamed “Brex,” after discoverer Bob Harmon—was recovered from the Hell Creek Formation in eastern Montana. The remote site has no vehicle access, so a helicopter ferried plaster jackets containing excavated bones from the site to the camp. The jacket containing the leg bones was too heavy for the helicopter to lift. To retrieve them, then, the team broke the jacket, separated the bones and rejacketed them. But these bones are very fragile, and when the original jacket was opened, many fragments of bone fell out and were collected and boxed up for me. Because my original T. rex studies were controversial, I was eager to repeat the work on a second T. rex, and this find presented the perfect opportunity.
As soon as I laid eyes on the first piece of bone I removed from that box, a fragment of thighbone, I knew the skeleton was special. Lining the internal surface of this fragment was a thin, distinct layer of a type of bone that had never been found in dinosaurs. This layer was very fibrous, filled with blood vessel channels, and completely different in color and texture from the cortical bone that constitutes most of the skeleton. “Oh, my gosh, it’s a girl—and it’s pregnant!” I exclaimed to my then assistant, Jennifer Wittmeyer. She looked at me like I had lost my mind. But having studied bird physiology, I was nearly certain that this distinctive feature was medullary bone, a special tissue that appears for only a limited time (often for just about two weeks), when birds are in lay. The special bone tissue exists to provide an easy source of calcium to fortify the eggshells, and when the last egg is laid, the medullary bone is quickly resorbed.
One of the characteristics that sets medullary bone apart from other bone types is the random orientation of its collagen fibers, a characteristic that indicates very rapid formation. (This same organization occurs in the first bone laid down when you have a fracture—that is why you feel a lump in healing bone.) The bones of any animal can be demineralized using mild acids to reveal the telltale arrangement of the collagen fibers. In living birds, removing the minerals from medullary bone should leave behind randomly oriented fibers. But this was dinosaur, not bird. I thought that almost all organics were gone from dinosaur bone, and if we let the reaction run too long, we would have nothing left, so Wittmeyer and I decided to lightly etch the surface. To our surprise, as the minerals dissolved they left a flexible and fibrous clump of tissue. I could not believe what we were seeing. I asked Wittmeyer to repeat the experiment multiple times. Each time we placed the distinctive layer of bone in the mild acid solution, fibrous stretchy material remained—just as it does when medullary bone in birds is treated in the same way.
Furthermore, when we then dissolved pieces of the denser, more common cortical bone, we obtained more soft tissue. Hollow, transparent, flexible, branching tubes emerged from the dissolving matrix—and they looked exactly like blood vessels. Suspended inside the vessels were either small, round red structures, very like those in the original T. rex that started me down this path, or amorphous accumulations of red material. Additional demineralization experiments revealed distinctive-looking bone cells called osteocytes that secrete the collagen and other components making up the organic part of living bone. The whole dinosaur seemed to preserve material never seen before in any dinosaur bone!
When we published our observations in Science in 2005, reporting the presence of what looked to be collagen, blood vessels and bone cells, the paper garnered a lot of attention, but the scientific community adopted a wait-and-see attitude. We claimed only that the material we found resembled these modern components—not that they were one and the same—because at that time we had no chemical data, only morphological data. And after millions of years, buried in sediments and exposed to geochemical conditions that varied over time, what was preserved in these bones might bear little chemical resemblance to what was there when the dinosaur was alive. The real value of these materials could be determined only if their composition could be discerned. Our work had just begun.
Using all the techniques honed while studying MOR 555, Rahonavis, Shuvuuia and the mammoth, I began an in-depth analysis of this T. rex’s bone in collaboration with Asara, who had refined the methods we used in the mammoth study and was ready to try sequencing the dinosaur’s far older proteins. This was a much harder exercise because the concentration of organics in the dinosaur was orders of magnitude less than in the much younger mammoth and because the proteins were very degraded. Nevertheless, we were eventually able to sequence protein fragments. And gratifyingly, when our colleague Chris Organ of Harvard compared the T. rex sequences with those of a multitude of other organisms, he found that they grouped most closely with birds, followed by crocodiles—the two groups that are the closest living relatives of dinosaurs.
Our papers detailing the sequencing work, published in 2007 and 2008, generated a firestorm of controversy, most of which focused on our interpretations of the sequence data. Some dissenters charged that we had not produced enough sequences to make our case; others argued that the structures we interpreted as primeval soft tissues were actually biofilm—“slime” produced by microbes that had invaded the fossilized bone. I had mixed feelings about their feedback. On one hand, scientists are paid to be skeptical. On the other hand, science operates on the principle of parsimony—the simplest explanation for all the data is assumed to be the correct one. And we had supported our hypothesis with multiple lines of evidence.
Still, I knew that a single gee-whiz discovery does not have any long-term meaning in science. We had to sequence proteins from other dinosaur bones. When a volunteer accompanying us on a summer expedition found bones from an 80-million-year-old plant-eating duck-billed dinosaur called Brachylophosaurus canadensis (“Brachy” for short), we suspected the duckbill might be a good source of ancient proteins even before we got its bones out of the ground. Hoping that it might contain organics, we did everything we could to free it from the surrounding sandstone quickly while minimizing its exposure to chemicals, contaminants and the elements. Air pollutants, humidity fluctuations and the like would be very harmful to fragile molecules, and the longer the bone was exposed to any of these factors, the more likely contamination and degradation would occur.
Perhaps because of this extra care—and prompt analyses—both the chemistry and the morphology of this second dinosaur were less altered than Brex’s. As we had hoped, we found cells embedded in a matrix of white collagen fibers in the animal’s bone. The cells exhibited long, thin, branchlike extensions that are characteristic of osteocytes, which we could trace from the cell body to where they connected to other cells. A few of them even contained what appeared to be internal structures, including possible nuclei.
Furthermore, extracts of the duckbill’s bone reacted with antibodies that target collagen and other proteins that bacteria do not manufacture, refuting the suggestion that our soft-tissue structures were merely biofilms. In addition, the protein sequences we obtained from the bone most closely resembled those of modern birds, just as Brex’s did. We reported these findings in Science in 2009.
We have since demineralized bone from organisms ranging from modern chickens to Triassic material, collected in different environments and on different continents, to show that we can recover at least three of the four elements preserved in more than one dinosaur: blood vessels, vascular contents, osteocytes and collagen-based matrix. Also, we now have many independent lines of evidence indicating that those elongated, branching microstructures in the samples taken from Brex and Brachy are indeed the bone cells of once living dinosaurs. In one of the most convincing experiments, we exposed these microstructures to monoclonal antibodies that bind with a protein expressed in the osteocytes of living birds but not in the alligator osteocytes that we used as controls. We found that the antibodies did bind to the microstructures from both dinosaurs, in the same pattern that we saw in ostrich bone cells. Although we have data hinting at the presence of DNA inside these cells, our findings will not be conclusive unless we can obtain DNA sequences consistent with those predicted for dinosaurs—but the DNA may be too fragmented and altered to be sequenced.
One frequent criticism of our work is that it is inconsistent with models of cellular, tissue and molecular degradation—all of which predict life spans far shorter than 80 million years. So we conducted another set of experiments to address how, under naturally occurring conditions, these components might be preserved for such long time periods. We have noticed that in all cases where we are able to recover blood vessels and cells, we also see iron, embedded as tiny particles that are only visible under high-magnification transmission electron microscopy. We reason that this iron may have come from the breakdown of iron-rich hemoglobin, released by dying red blood cells. As the hemoglobin molecules break down, the unstable biological iron they release undergoes chemical reactions that unleash highly reactive free hydroxyl radicals. These hungry molecules attack tissues, stealing electrons to make themselves more stable and in the process causing cross-links to form between molecules of the tissue. This reaction, which is lethal in living organisms because the cross-linked molecules cannot function normally, acts much like placing tissues in formaldehyde—a preservative that also causes cross-links to form.
In experiments we have observed that blood vessels recovered from modern bone can remain stable in water at room temperature for more than two years if they are first briefly soaked in a lysate of red blood cells, whereas untreated blood vessels degrade in days to weeks. These results suggest that free radicals in the blood lysate may have stabilized the tissues against early degradation.
Our results have met with a lot of skepticism. They are, after all, extremely surprising, but they are also full of promise. The study of ancient organic molecules from dinosaurs could yield insights into how these great beasts evolved, how they responded to major environmental changes and ultimately what did them in.
