Much of my current work involves understanding how soft tissues preserve in exceptional fossils and what biomolecules persist deep into the geologic record. Doing so will lead to better interpretations of fossil data and a better understanding of the biology of extinct organisms. My work involves experimental taphonomy utilizing decay and maturation experiments as well as modern analytical techniques on fossils themselves. These projects are highly collaborative in nature given their span of paleontology, geology, biology, and chemistry.
My first foray into this subject was during my masters thesis. This thesis is available through my Researchgate page. The thesis was also presented at the 2016 Society of Vertebrate Paleontology annual meeting. The slides from the talk are also available through Researchgate. I also presented the thesis in a poster at the 2015 annual meeting of the Palaeontological Association (again available through Researchgate). The first paper to come from my masters thesis, showing through decay and maturation experiments common to experimental taphonomy that keratin is not likely to preserve in fossils of appreciable age or burial depth, was published in Palaeontology:
Saitta, E. T., Rogers, C., Brooker, R. A., Abbott, G. D., Kumar, S., O’Reilly, S. S., Donohoe, P., Dutta, S., Summons, R.E., & Vinther, J. (2017). Low fossilization potential of keratin protein revealed by experimental taphonomy. Palaeontology, 60(4), 547-556.
Recent studies have suggested the presence of keratin in fossils dating back to the Mesozoic. However, ultrastructural studies revealing exposed melanosomes in many fossil keratinous tissues suggest that keratin should rarely, if ever, be preserved. In this study, keratin’s stability through diagenesis was tested using microbial decay and maturation experiments on various keratinous structures. The residues were analysed using pyrolysis-gas chromatography-mass spectrometry and compared to unpublished feather and hair fossils and published fresh and fossil melanin from squid ink. Results show that highly matured feathers (200–250°C/250 bars/24 h) become a volatile-rich, thick fluid with semi-distinct pyrolysis compounds from those observed in less degraded keratins (i.e. fresh, decayed, moderately matured, and decayed and moderately matured) suggesting hydrolysis of peptide bonds and potential degradation of free amino acids. Neither melanization nor keratin (secondary) structure (e.g. ⍺- vs β-keratin) produced different pyrograms; melanin pyrolysates are largely a subset of those from proteins, and proteins have characteristic pyrolysates. Analyses of fossil fur and feather found a lack of amides, succinimide and piperazines (present even in highly matured keratin) and showed pyrolysis compounds more similar to fossil and fresh melanin than to non-matured or matured keratin. Although the highly matured fluid was not water soluble at room temperature, it readily dissolved at elevated temperatures easily attained during diagenesis, meaning it could leach away from the fossil. Future interpretations of fossils must consider that calcium phosphate and pigments are the only components of keratinous structures known to survive fossilization in mature sediments.
A second paper deriving from my masters thesis discusses the early taphonomic changes that might occur in keratinous structures and whether the resulting textures would ever be expected in the fossil record. It also challenges structures identified as dinosaur erythrocytes, suggesting that they are taphonomic artifacts of degraded organic material. This paper was published in Palaios and reported by the University of Bristol. It also formed the basis of an article I wrote for The Conversation.
Saitta, E. T., Rogers, C. S., Brooker, R. A., & Vinther, J. (2017). Experimental taphonomy of keratin: a structural analysis of early taphonomic changes. Palaios, 32(10), 647-657.
The evolution of integumentary structures, particularly in relation to feathers in dinosaurs, has become an area of intense research. Our understanding of the molecular evolution of keratin protein is greatly restricted by the fact that such information is lost during diagenesis and cannot be derived from fossils. In this study, decay and maturation experiments are used to determine if different keratin types or integumentary structures show different patterns of degradation early in their taphonomic histories and if such simulations might advance our understanding of different fossilization pathways. Although different distortion patterns were observed in different filamentous structures during moderate maturation and ultrastructural textures unique to certain types of scales persisted in moderate maturation, neither of these have been observed in fossils. It remains uncertain whether these degradation patterns would ever occur in natural sediment matrix, where microbial and chemical decay happens well before significant diagenesis. It takes some time for remains to be buried, meaning that keratin may not be left for moderate maturation to produce such patterns. Higher, more realistic maturation conditions produce a thick, and water soluble fluid that lacks all morphological and ultrastructural details of the original keratin, suggesting that such textural or distortion patterns are unlikely to be preserved in fossils. Although different degradation patterns among keratinous structures are intriguing, it is unlikely that such information could be recorded in the fossil record. Calcium phosphates and pigments are the surviving components of integumentary structures. Thus, the results here are likely of more relation to the archaeological record than fossil record. Other noteworthy results of these experiments are that melanin may not be the leading factor in determining the rate of microbial decay in feathers but may reduce the rate of degradation from maturation, that the existence of rachis filamentous subunits similar to plumulaceous barbules is supported, and that previously reported dinosaur ‘erythrocytes’ may be taphonomic artifacts of degraded organic material.
I was coauthor of a paper providing an overview of the various processes involved in fossilization, highlighting how decay is far from the only factor to consider when studying fossil taphonomy or designing experiments. The paper was published in BioEssays.
Parry, L. A., Smithwick, F., Norden, K., Saitta, E. T., Lozano-Fernandez, J., Tanner, A., Bernard Caron, J., Edgecombe, G. D., Briggs, D. E. G., & Vinther, J. (2017). Soft-bodied fossils are not simply rotten carcasses—towards a holistic understanding of exceptional fossil preservation. BioEssays. doi: 10.1002/bies.201700167
Exceptionally preserved fossils are the product of complex interplays of biological and geological processes including burial, autolysis and microbial decay, authigenic mineralization, diagenesis, metamorphism, and finally weathering and exhumation. Determining which tissues are preserved and how biases affect their preservation pathways is important for interpreting fossils in phylogenetic, ecological, and evolutionary frameworks. Although laboratory decay experiments reveal important aspects of fossilization, applying the results directly to the interpretation of exceptionally preserved fossils may overlook the impact of other key processes that remove or preserve morphological information. Investigations of fossils preserving non-biomineralized tissues suggest that certain structures that are decay resistant (e.g., the notochord) are rarely preserved (even where carbonaceous components survive), and decay-prone structures (e.g., nervous systems) can fossilize, albeit rarely. As we review here, decay resistance is an imperfect indicator of fossilization potential, and a suite of biological and geological processes account for the features preserved in exceptional fossils.
One project involves improved equipment and methods for maturation experiments using a large-scale, sediment-based approach with the goal of modeling fossilization processes to create ‘synthetic fossils’ in the lab, in an analogous manner to the production of synthetic diamonds. Such a technique holds great potential for better understanding fossilization and testing a wide range of hypotheses for many years to come. The initial results were presented in a pre-conference, informal series of talks prior to the 2016 annual meeting of the Palaeontological Association. I also presented this work during the Romer session of the 2017 annual meeting of the Society of Vertebrate Paleontology. The method was mentioned by my collaborator, Tom Kaye, in the early part of a podcast interview with Palaeocast. The paper itself was published in Palaeontology. The work was covered in a variety of media outlets including Science.
Saitta, E. T., Kaye, T. G., & Vinther, J. (2018). Sediment-encased maturation: a novel method for simulating diagenesis in organic fossil preservation. Palaeontology. doi: 10.1111/pala.12386
Exceptional fossils can preserve diagenetically‐altered biomolecules. Understanding the pathways that lead to such preservation is vital to utilizing fossil information in evolutionary and palaeoecological studies. Experimental taphonomy explores the stability of tissues during microbial/autolytic decay or their molecular stability through maturation under high pressure and temperature. Maturation experiments often take place inside sealed containers, preventing the loss of labile, mobile or volatile molecules. However, wrapping tissues inside aluminium foil, for example, can create too open a system, leading to loss of both labile and recalcitrant materials. We present a novel experimental procedure for maturing tissues under elevated pressure/temperature inside compacted sediment. In this procedure, porous sediment allows maturation breakdown products to escape into the sediment and maturation chamber, while recalcitrant, immobile components are contained, more closely mimicking the natural conditions of fossilization. To test the efficacy of this procedure in simulating fossil diagenesis, we investigate the differential survival of melanosomes relative to proteinaceous tissues through maturation of fresh lizard body parts and feathers. Macro‐ and ultrastructures are then compared to fossils. Similar to many carbonaceous exceptional fossils, the resulting organic components are thin, dark films composed mainly of exposed melanosomes resting on the sediment in association with darkened bones. Keratinous, muscle, collagenous and adipose tissues appear to be lost. Such results are consistent with predictions derived from non‐sediment‐encased maturation experiments and our understanding of biomolecular stability. These experiments also suggest that organic preservation is largely driven by the original molecular composition of the tissue and the diagenetic stability of those molecules, rather than the tissue’s decay resistance alone; this should be experimentally explored in the future.
Similarly, my colleagues and I have used a variety of chemical analyses to understand the organics present in fossil dinosaur (e.g., Mesozoic) bone. This work involves samples that I aseptically excavated from Dinosaur Provincial Park in the summer of 2016. I wish to understand whether or not these organics are ancient, endogenous, and/or well-preserved. I presented a poster on this topic entitled “Life inside a dinosaur bone: a thriving microbiome” at the 2017 annual meeting of the Palaeontological Society. We have published the final paper in eLife where we discovered a unique and unusual microbiome living inside buried dinosaur bones. The preprint was covered in a piece by The Atlantic. The main paper was covered in The Scientist and Chemical & Engineering News and the podcasts Skeptics Guide to the Universe and Palaeocast.
Saitta, E.T., Liang, R., Lau, M.C., Brown, C.M., Longrich, N.R., Kaye, T.G., Novak, B.J., Salzberg, S.L., Norell, M.A., Abbott, G.D. and Dickinson, M.R., 2019. Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities. eLife, 8, p.e46205.
Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These ‘soft tissues’ have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil ‘soft tissues’ differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.
In a follow up study, we ran further metagenomic analyses of these Centrosaurus bones to find 46 unique strains of bacteria and archaea living within them, the majority of which could be new species, and several potentially represent a new class. Some have the ability to breakdown collagen as well as kerogen or humic-like organic geopolymers, making it further unlikely that dinosaur proteins can preserve. This paper was published in Environmental Microbiome.
Liang, R., Lau, M.C., Saitta, E.T., Garvin, Z.K. and Onstott, T.C., 2020. Genome-centric resolution of novel microbial lineages in an excavated Centrosaurus dinosaur fossil bone from the Late Cretaceous of North America. Environmental Microbiome, 15(1), pp.1-18.
Exceptional preservation of endogenous organics such as collagens and blood vessels has been frequently reported in Mesozoic dinosaur fossils. The persistence of these soft tissues in Mesozoic fossil bones has been challenged because of the susceptibility of proteins to degradation and because bone porosity allows microorganisms to colonize the inner microenvironments through geological time. Although protein lability has been studied extensively, the genomic diversity of microbiomes in dinosaur fossil bones and their potential roles in bone taphonomy remain underexplored. Genome-resolved metagenomics was performed, therefore, on the microbiomes recovered from a Late Cretaceous Centrosaurus bone and its encompassing mudstone in order to provide insight into the genomic potential for microbial alteration of fossil bone. Co-assembly and binning of metagenomic reads resulted in a total of 46 high-quality metagenome-assembled genomes (MAGs) affiliated to six bacterial phyla (Actinobacteria, Proteobacteria, Nitrospira, Acidobacteria, Gemmatimonadetes and Chloroflexi) and 1 archaeal phylum (Thaumarchaeota). The majority of the MAGs represented uncultivated, novel microbial lineages from class to species levels based on phylogenetics, phylogenomics and average amino acid identity. Several MAGs from the classes Nitriliruptoria, Deltaproteobacteria and Betaproteobacteria were highly enriched in the bone relative to the adjacent mudstone. Annotation of the MAGs revealed that the distinct putative metabolic functions of different taxonomic groups were linked to carbon, nitrogen, sulfur and iron metabolism. Metaproteomics revealed gene expression from many of the MAGs, but no endogenous collagen peptides were identified in the bone that could have been derived from the dinosaur. Estimated in situ replication rates among the bacterial MAGs suggested that most of the microbial populations in the bone might have been actively growing but at a slow rate. Our results indicate that excavated dinosaur bones are habitats for microorganisms including novel microbial lineages. The distinctive microhabitats and geochemistry of fossil bone interiors compared to that of the external sediment enrich a microbial biomass comprised of various novel taxa that harbor multiple gene sets related to interconnected biogeochemical processes. Therefore, the presence of these microbiomes in Mesozoic dinosaur fossils urges extra caution to be taken in the science of paleontology when hunting for endogenous biomolecules preserved from deep time.
I have also started to investigate the organics of fossil eggshells. Preliminary results were presented at the 2017 International Workshop on Konservat-Lagerstätten in Cork, Ireland. This work has now been posted as a preprint to bioRxiv.
Rates of peptide bond hydrolysis and other diagenetic reactions are not favourable for Mesozoic protein survival. Proteins hydrolyse into peptide fragments and free amino acids that, in open systems such as bone, can leach from the specimen and be further degraded. However, closed systems are more likely to retain degradation products derived from endogenous proteins. Amino acid racemisation data in experimental and subfossil material suggests that mollusc shell and avian eggshell calcite crystals can demonstrate closed system behaviour, retaining endogenous amino acids. Here, high-performance liquid chromatography reveals that the intra-crystalline fraction of Late Cretaceous (estimated ~80 Ma) titanosaur sauropod eggshell is enriched in some of the most stable amino acids (Glx, Gly, Ala, and possibly Val) and those that racemise are fully racemic, despite being some of the slowest racemising amino acids. These results are consistent with degradation trends deduced from modern, thermally matured, sub-fossil, and ~3.8 Ma avian eggshell, as well as ~30 Ma calcitic mollusc opercula. Selective preservation of certain fully racemic amino acids, which do not racemise in-chain, along with similar concentrations of free versus total hydrolysable amino acids, likely suggests complete hydrolysis of original peptides. Liquid chromatography-tandem mass spectrometry supports this hypothesis by failing to detect any non-contamination peptide sequences from the Mesozoic eggshell. Pyrolysis-gas chromatography-mass spectrometry reveals pyrolysates consistent with amino acids as well as aliphatic hydrocarbon homologues that are not present in modern eggshell, suggestive of kerogen formation deriving from eggshell lipids. Raman spectroscopy yields bands consistent with various organic molecules, possibly including N-bearing molecules or geopolymers. These closed-system amino acids are possibly the most thoroughly supported non-avian dinosaur endogenous protein-derived constituents, at least those that have not undergone oxidative condensation with other classes of biomolecules. Biocrystal matrices can help preserve mobile organic molecules by trapping them (perhaps with the assistance of resistant organic polymers), but trapped organics are nevertheless prone to diagenetic degradation even if such reactions might be slowed in exceptional circumstances. The evidence for complete hydrolysis and degradation of most amino acids in the eggshell raises concern about the validity of reported polypeptide sequences from open-system non-avian dinosaur bone and other Mesozoic fossils.
I have published a commentary on the fossilization of keratinous structures, criticizing the reliance upon antibody methods to study ancient putative proteins that might not actually be present. I also discuss scientific epistemology and replication versus validation. This commentary was published in Palaeontologia Electronica.
Saitta, E.T. and Vinther, J., 2019. A perspective on the evidence for keratin protein preservation in fossils: An issue of replication versus validation.
The preservation potential of biomolecules within vertebrate integument through deep time has recently been subject to much research and controversy. In particular, the preservation potential of proteins, such as collagen and keratin, is currently debated. Here, we examine claims from a recent study (Schweitzer et al., 2018, PLoS One), which concludes that feather keratin has a high preservation potential. We argue that this work provides insufficient evidence for protein preservation due to issues of methodology and data interpretation. Additionally, we contrast their approach and claims to those of other recently published studies in relation to the question of keratin protein preservation in fossils. We worry that most of the perceived evidence for Mesozoic polypeptide survival stems from repeated replication of methods prone to false detection, rather than triangulation by validating these claims with alternative methods that provide independent lines of evidence. When alternative explanations exist for the evidence cited as support for dinosaur proteins far exceeding their predicted preservation limits, it is most parsimonious to reject the more extreme taphonomic hypotheses. The evidence is instead more consistent with a mode of preservation in which keratinous structures do not fossilize organically as polypeptides, but rather as largely pigment and/or calcium phosphate remnants, which were originally held within the keratin matrix that is now lost. Unsupported taphonomic models (e.g., keratin polypeptide preservation) have the potential to influence our interpretation of fossil data, potentially resulting in erroneous paleobiological or evolutionary conclusions, as illustrated in another recent paper (Pan et al., 2019, PNAS) that we also discuss.
As part of my post-doc, I am examining trace metal accumulation in exceptionally preserved fossils from various parts of the world using synchrotron x-ray fluorescence.