Of Pregnancy Tests, Misleading Headlines, and the Dream of Dinosaur Clones

Our recent paper “Chemistry supports the identification of gender-specific reproductive tissue in Tyrannosaurus rex” (Mary Higby Schweitzer Wenxia Zheng, Lindsay Zanno, Sarah Werning & Toshie Sugiyama), sparked a little bit of controversy when a co-author was asked in a press interview if there was a possibility that these tissues might contain DNA, a question she rightly answered with a “Yes”. A scientist knows that, until something is eliminated with data, usually from multiple lines of evidence, it remains a possibility, however small. Regardless, in our paper, there is no mention of DNA. It is not mentioned because looking for DNA was not a goal of this study, nor did we conduct any tests to identify its presence in these tissues. Unfortunately, the untested possibility of DNA remaining in these tissues became confused with evidence in the mind of the interviewer, and misleading headlines followed.

Because of the importance of DNA in the study of living organisms, and the role of DNA in scientific studies as diverse as human health, drug design, environmental responses to changing climate, and evolutionary relationships, ancient DNA is portrayed as the “holy grail” of molecular studies on fossils. Thus, DNA from dinosaurs would be the ultimate “cool science story”, the most exciting journalistic scoop, and every time a study is published on molecular analyses of dinosaur fossils, the question inevitably arises: “So do we have DNA, and how close are we to Jurassic Park?”

The first question, “Do we have DNA?’ (or any variant, such as ‘is it possible now to obtain DNA’), is simply the wrong question. The majority of bone is made up of hydroxyapatite mineral, which gives bone its hardness. This mineral has such a strong affinity for DNA and many proteins that it is used in modern labs sometimes to purify DNA. Therefore, ANY DNA, from any source, is likely to adhere to the mineral in fossil bone. This bone has been sitting in the ground for 65 million years, in a complex chemical relationship with the surrounding environment and related microbiome. The likelihood is high that if DNA were actively sought, it could be found. However, the real question (which reporters never ask), is how can we tell if DNA recovered from fossils is dinosaur DNA? And that, of course, is a lot more difficult to answer. DNA most likely WILL be found in dinosaur bone (we do not do such work here at NCSU, currently), but whether or not we could confidently say it is from the once-living dinosaur would depend upon whether or not it meets myriad criteria set in advance to rule out different potential sources of contamination. No “dinosaur-derived” DNA has yet measured up to these criteria.   Is it possible that we may someday recover authentic DNA from dinosaur bone? The scientific answer is ‘yes’; all things are possible until disproven. Have we disproven this possibility? No. Have we recovered ‘authentic’ dinosaur DNA? No.

The second question—“How close are we to Jurassic Park”? Or, “will we ever be able to clone a dinosaur”? is equally nuanced. The genetic cloning that is usually done in labs involves taking a known fragment of DNA, inserting it into a bacterial plasmid, and letting that fragment of DNA replicate over and over each time the cell divides. This results in many, many copies of identical DNA from the insert–clones. Cloning an entire vertebrate animal, on the other hand, involves taking the whole complement of DNA from cells within a tissue, and inserting it into viable cells from which the native nuclear material has been removed. This cell is then inserted into a host, and the donor DNA dictates the formation and development of the offspring, which are genetically identical to the donor—i.e., clones. Dolly the sheep is an example of this process. When people refer to cloning a dinosaur, they usually mean something along these lines. However, this is an incredibly complex process, and despite the unscientific nature of this opinion, the likelihood that we would ever be able to overcome all of the obstacles between fragments of DNA in a dinosaur bone and producing a living offspring is so incredibly small it would rank a ‘not possible’ in our book.

A third possibility exists, and has been proposed as a means to ‘clone’ a mammoth. Frozen subfossils of mammoth have been found in places like Siberia, that still retain soft parts, including reproductive organs. Scientists are analyzing this material in the hopes that some sperm will be retained in these tissues intact, and might be used to impregnate an elephant. The resulting offspring, should they be able to actually pull this off, would be half mammoth and half elephant. The idea then is to back-breed, hoping to get closer to “authentic” mammoth with each reproduction. But, that would still not result in a ‘clone’ (or identical copy) of a mammoth. (For other virtually insurmountable obstacles, see Palaeontologia Electronica, vol. 5, issue 2).

However, just because the likelihood of visiting a “real” Jurassic Park is so miniscule does not mean that it is impossible to recover original fragments of DNA or other molecules from ancient remains, nor does it mean that it is not worth the effort and expense to try to recover molecular fragments from fossils, for these ancient molecules have much to tell us. Because all evolutionary change must first occur in genes (and the proteins they encode), molecules can directly inform us of evolutionary processes. In their sequences reside information about how fast evolutionary changes occur in the genome, which bases/sequences/genes/proteins are more likely to change and which are more stable, which organisms are more closely related to others, and potentially, patterns showing co-evolution of diseases and how long pathogens have plagued certain organisms.

During times of global change, many organisms go extinct. Other groups are threatened because they have been greatly compromised by “bottleneck” events, where living representatives contain a mere fraction of the genetic diversity the species once held. By studying molecules from fossils of groups before and after such events, we may learn about the degree of diversity that once existed in natural populations, and learn ways to increase diversity in compromised descendants.

We can also learn about the durability of molecules under naturally occurring conditions more directly than using lab proxies, such as heat, to estimate rates of molecular degradation. Knowing which bases of DNA, or which residues of proteins are more likely to persist for long periods may aid in directed drug design. We might not want to design drugs that target a molecular region that lasts for tens of millions of years! And, tangentially, understanding the molecular make-up of materials that can last for millions of years and still retain their original flexibility, transparency, or other components might help us in designing biomaterials used for organo-electronics.

Finally, recovering molecules from fossils, including dinosaurs, yields important information on the origin and distribution of evolutionary novelties. For example, take feathers. These are the most complex epidermal appendage (i.e., structures that arise from the skin, including hair, nails, claws, etc) to evolve. Their function in flight is obvious, and is what we most associate with the rise of feathers, since they were observed in the first “true” bird, the Jurassic Archaeopteryx. But, because we have found feathers in many dinosaurs that obviously did not, and could not fly, they clearly did not evolve “for” flight. Obtaining molecular sequence data from fossil feathers would let us ask questions like: what structures were the likely precursors to feathers? Did dinosaur feathers differ, at the molecular level, from those of birds? When in the dinosaur-bird lineage did the particular modifications to keratin proteins that comprise feathers first occur?

But, even if we can’t get sequence data from fossils except rarely, there might be just enough molecular fragments remaining to allow us to ask deeper questions anyway, and that was the point of our recent paper. Although we have obtained and published protein sequence data from this particular dinosaur, now we show that by understanding the molecular make up of tissues in modern animals, especially those with unique and novel structures, we can apply these data (and methods) to fossils. We reported the presence of this reproductive bone tissue, medullary bone, ten years ago (Schweitzer et al., 2005)! At the time, the identification of this tissue as reproductive medullary bone was based on morphological similarities. But, in the intervening years, we have learned a lot about what technology can tell us, and how to best use that technology to get at important questions in extinct groups. We also learned that there are a few other things that, superficially, might resemble some aspects of medullary bone—the random and disorganized “woven” nature of medullary bone, for example, might also resemble fracture callus or some diseases. So to rule out these alternatives, we capitalized on the molecules that were originally produced by the dinosaur and which still remain in her bones. By using chemistry, microscopic structure, location and CT imaging, we can show that, not only is this reproductive medullary bone, it is composed of the same unique chemical signatures as medullary bone found in birds. And from that, we can say that this chemistry was acquired in the ancestor of T.rex and all living birds, and that the physiology of reproduction, and how they shelled and laid their eggs, was the same as modern birds, but very different from crocodiles, their other closest living relatives. Multiple tests ruled out the possibility that what we were seeing was either disease or a bony response to fracture. In light of all these data, it is pretty hard to argue that this dinosaur is NOT a reproducing female.

Although medullary bone is very short lived, and it might be a long time before we can find another T.rex (or other dinosaur) that retains it, we can now go back to the rest of this dinosaur skeleton, and see if there are features present that we might find in other T.rex specimens. Then, maybe we can tell if there are specific traits that are linked to “female-ness” in dinosaurs. And from that, we can, perhaps, get a better idea of questions like population structure, individual variation, and other questions about the evolution and function of this dinosaur group.

But…what about the DNA question? How long can DNA last in the fossil record, and how can we tell for certain that it is dinosaurian, and not a modern lab contaminant, or DNA that has leached in from the environment?

Scientists have proposed that DNA has a pretty short shelf-life, most saying that it is unlikely to persist as long as a million years, and surely not more than 5 or 6 million years at the most. That sort of leaves out the possibility that we will ever obtain it from dinosaurs that last walked the earth over 65 million years ago! How do they arrive at this number though? By carefully trying to recover it from progressively older fossils? Well, not really. Some have studied DNA degradation by placing molecules of known length and composition into hot acid, and monitoring how long it takes to fall apart. They use heat and acidity as proxies for time, and declare that DNA falls apart quite rapidly. One study set out to recover DNA from progressively older fossils—from a few hundred years old, to about 8000 years. They found that the amount of recoverable DNA declined with age, and they used this to model a ‘rate of decay’. They also showed that DNA degradation only loosely correlated to age of the fossil! They then predicted, but did not test, that DNA as small as 175 base pairs (about the minimum length that could possibly yield phylogenetic information) was extremely unlikely to persist in Cretaceous bone.

On the other hand, in our labs, we have used 4 different lines of evidence to suggest that a molecule chemically similar to DNA that is localized to the center of what remains of bone-forming cells in a duck-billed dinosaur called Brachylophosaurus, which is consistent with what we might expect to find, if it were dinosaurian DNA. So, how do we tell if DNA recovered from bone is truly dinosaurian, and not contaminant?

The idea that DNA can persist that long is a long shot at best, so anyone claiming to find or recover dinosaur DNA has to meet the most stringent of criteria. We suggest the following:

  1. DNA sequences recovered from bone should match what we would expect from other data. For example, there are over 300 characters that link dinosaurs to birds, and strongly suggest that the origin of birds lies within theropod (meat eating) dinosaurs. So, DNA sequences obtained from dinosaurs should also follow that pattern, being more similar to bird DNA than to crocodile DNA, but clearly different from both. Because we did not attempt to sequence the material that gave a positive response, we cannot claim it is dinosaurian, and we will not make that statement.
  2. If DNA is original, it is likely to be highly fragmented, and difficult to analyze by our current methods, which were developed to sequence happy healthy DNA. If “rex DNA” comes in long pieces that are relatively easy to sequence, it is likely to be a contaminant.
  3. DNA is proposed to be fragile, relative to other molecules. So, if DNA is present, other, more durable molecules should also be present. DNA sequence from dinosaur bone should always be accompanied by the sequences of other molecules that are known to be more decay resistant while in bone. Collagen I protein, for example, is a major constituent of bone that has been shown to be more resistant to degradation than DNA . Therefore, if one can show DNA that is similar in sequence to avian and crocodilian DNA, and can also produce, from the same bone, collagen sequences showing a similar evolutionary relationship, the case for “real” dinosaur DNA goes up. Similarly, one should also be able to demonstrate the persistence of lipids. Lipids are more resistant overall than either protein or DNA, and make up the membranes of cells that would have been originally present.
  4. IF DNA, proteins, and lipids are shown to persist, other methods in addition to sequencing should also support this conclusion. For example, binding of proteins to specific antibodies can be used to show that protein signal is localized to the tissues, and not present in surrounding sediments. In previous studies, we were able to localize a substance similar to DNA inside the bone cells recovered from this rex, using both DNA specific stains, and antibodies to proteins associated with vertebrate DNA.
  5. Finally, and probably most importantly, for all steps of any test, adequate controls should be employed. Samples that yield DNA should be co-extracted with the sediments that surrounded the fossil, and also, all buffers and chemicals used in the lab should also be treated to the exact same conditions as the fossil bone. If these non-fossil samples also contain the same sequences found in the fossil, then they are most likely from a contaminant.

We still have a lot to learn in the molecular analysis of fossils, and we should proceed with the utmost caution, never overstating the data we obtain. But there is so much we can learn from molecules preserved in fossils that we believe it is worth the effort. For example, by many standards we are undergoing potentially severe and long-term climate change. But so far, this is minimal compared to the global change the dinosaurs experienced from their origins in the Triassic to their demise at the end of the Cretaceous. If we could recover DNA and/or proteins across this time period and compare the molecules in animals before and after these events, perhaps we could gain insight into how they responded, at the molecular level, to these changes. And this may help us deal with our own environmental changes.

-Mary Schweitzer

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