The publication of “Fibres and cellular structures preserved in 75-million–year-old dinosaur specimens”, by Bertazzo et al. (2015), seems a fitting way to inaugurate the NCSU Molecular Paleontology website and blog. In this paper, the authors using FIB (focused ion beam), ToF-SIMS (time of flight secondary ion mass spectrometry), EDS (energy dispersive x-ray) and TEM (transmission electron microscopy) to support the idea that soft tissues and original organic material persist in a number of Cretaceous dinosaur bones. According to the authors (at least, as they are quoted in the press), the really important thing about this paper was that the bones were 1. Not well preserved, and 2. Drawn from museum collections where they had resided for many, many years. Their conclusion is that the preservation of ‘fibers’ and ‘blood cells’ may not be unusual, rather the norm, and may not require otherwise “exceptional” preservation; thus they suggest that a host of fossils may be available for molecular studies.
Some things I like about the paper: they were relatively cautious in their interpretation, hedging their bets with phrases like “putative erythrocyte remains”, and “fibrous structures similar to calcified collagen fibres”. They note “peaks consistent with fragments of amino acids present in collagen”. This is all suggestive, and they treat it as such; the data they present are suggestive, but are not conclusive.
Some problems I have with the paper:
1. The amino acids they identified are indeed present in collagen, but they are also present in a lot of other proteins, including proteins produced by soil microbes, graduate students, museum curators and just about everything else. They do not show di- or tri-peptides, which would give an indication that maybe unfragmented proteins might be present, and they do not show the ‘fingerprint’ amino acids indicative of collagen, hydroxylated proline and/or hydroxylated lysine. These unique post translational modifications are only found in collagen proteins, and the addition of hydroxyl groups is critical to the formation of intramolecular cross links that give collagen its stability. This can be identified in fossil bone, as we showed in our 2007 paper, so I was surprised that these were not identified in the paper.
2. The ‘putative’ red blood cells averaged 2 um, and were described as biconcave. This is significantly smaller than even the smallest mammalian red blood cells (at ~8-10 um) and much smaller than those of living birds, which average around 20-25 um. And, the biconcave nature of the structures they present is not consistent with RBCs in birds. Both birds and archosaurs (indeed, all vertebrates except mammals), retain a fully functioning nucleus in circulating red blood cells; thus it is reasonable to predict that dinosaurs would also have nucleated RBCs. And retention of a nucleus results in a biconvex structure with a ‘nuclear bulge’, which is very distinctive. We demonstrated this morphology in small intravascular structures within a well preserved T.rex, structures that were high in iron. We mapped iron to these intravascular structures, using an x-ray map in our 1999 paper, and a line scan in our 1997 paper. And, we showed that these structures were within the same size range, and retained a nuclear bulge, similar to all living archosaurs. However, despite the suggestive nature of these structures, we have not claimed these structures were RBCs, because the data were not conclusive (see below).
3. The authors refer frequently to ‘cement’ surrounding the putative erythrocytes, (e.g. “Erythrocyte-like structures composed of carbon surrounded by cement”, figure 1 caption), but they do not describe this cement nor posit its origin. This is particularly confusing because it is in opposition to their statement “Specimens from the Dinosaur Park Formation and Lance Formation were selected because pore spaces in the trabecular bone tissue were not infilled with matrix or cement” p.6, methods,emphasis mine). Is the cement diagenetic? Related to the purported erythrocytes? Part of the organism? It is referred to repeatedly, but not discussed.
4. The ‘fibres’ imaged in SEM in the Bertazzo paper were claimed to be collagen. This is reasonable because collagen in the dominant protein in bone, and because bone collagen has high preservation potential when associated with the mineral phase of bone. In particular, the TEM images they produced (fig 3a) for collagen show the 67nm banding pattern consistent with collagen, and indeed are almost indistinguishable from images produced by Weiner et al (1998, figure 1, ‘level 2’). As mentioned above, their MS data show amino acids found in collagen proteins, but these are not conclusive for collagen, and their paper would have been greatly strengthened by in situ localization of collagen antibodies binding to the tissues, identification of collagen specific amino acids, or MS sequence data.
I was also confused by the statement in their paper that “Before this finding, the oldest undegraded collagen recorded (based on mass spectrometry sequencing and peptide fingerprinting) was about 4 million years old” (p. 3). We produced collagen sequence data from two different dinosaurs, one about 67 MYO, and the other at ~80 MYO. These were degraded, but I don’t think there is any evidence presented in the Bertazzo paper to indicate there was no degradation in their ‘collagen’ either. So this statement is not accurate as it stands.
Identification of red blood cells/hemoglobin in fossils
So, what, in my opinion, would it take to demonstrate conclusively the presence of heme, hemoglobin, or red blood cells in dinosaur or other skeletal remains? Certainly more than morphology! Size (within the range of RBCs in extant animals), morphology (biconvex if non mammalian, biconcave if mammalian), and location (within vessels or vascular channels) are important first steps. Erythrocytes that retain their nuclei (all vertebrates except mammals) are generally speaking larger than anucleate cells. Birds and crocodiles have cells that are around 20-25 um, but some amphibian cells can be much larger, up to 60 um. It is plausible to propose that labile cells, such as erythrocytes, may undergo shrinkage as a result of diagenensis, but how much shrinkage? Too much, and will they retain their hallmark features? How can they shrink an order of magnitude and still retain basic morphology? This can be addressed by taphonomic experimentation, to be sure, but this was not done by the authors of this study.
However, as we showed in our 1997 paper, heme and heme-containing compounds have very unique fingerprint characteristics, because of the association of iron to organics. Heme and those proteins containing it are paramagnetic, making them responsive to a host of different methods, such as spectroscopy, resonance Raman, high-resolution solution phase proton nuclear magnetic resonance, and electron spin resonance. The iron-organic association pulls signals out of the normal ‘protein window’, causing specific responses by these methods. Additionally, the sensitivity of biological iron to oxygen can be monitored by absorbance in the oxidized and reduced states. Heme-containing compounds are red-brown when oxidized, and dark purple when reduced. Our T.rex bone extracts showed this repeatable absorbance switch, a reaction that is not common to geological iron compounds. And yet, with all of this data, we did not claim that those rounded biconvex intravascular structures were cell remnants, because we were unable to localize those signals to the structures; rather they came from chemical extracts of whole bone. In 1997, we did not have the highly sensitive methods, such as micro-Raman, that could be conducted on the microstructures directly, and thus could be diagnostic.
I think it is a great thing that more and more high resolution technologies are being applied to fossils to retrieve biological and evolutionary information, as in the Bertazzo et al paper. However, it is equally important not to overstate the data, and to apply multiple methods to these samples rather than relying on just one or two methods. All methods have limitations. Similarly, methods cannot be applied to fossils that have not been applied to extant analogues. And above all, adequate and appropriate controls should be applied at every step. Caution in interpretation, multiple methods and a conservative approach will make the entire field of molecular paleontology more robust