Abstract
Tooth enamel, primarily composed of bioapatite, is a promising archive of endogenous organic matter for studying ancient fauna. Despite its low organic content (~1%), protein residues have been identified in teeth up to 24 million years old. This study investigates the preservation of total hydrolysable amino acids (THAAs) in fossil enamel dating back as far as 48 million years. Modern and fossil enamel from large herbivorous mammals (Equidae, Rhinocerotidae, Proboscidea) across various taphonomic settings and Cenozoic periods reveal that AAs persist at least to the Eocene. The “intra-crystalline” organic fraction stabilizes after an initial rapid decline within the first 0.10 million years. Preservation appears independent of taphonomic context, and the relative abundance of amino acids is similarly variable in both modern and fossil samples. These findings demonstrate that enamel is a diagenetically robust substrate for long-term organic preservation, with significant potential for phylogenetic and ecological reconstructions in the fossil record.
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References
Cappellini, E. et al. Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny. Nature 574, 103–107 (2019).
Welker, F. et al. Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates. Nature 522, 81–84 (2015).
Chen, F. et al. A late Middle Pleistocene Denisovan mandible from the Tibetan Plateau. Nature 569, 409–412 (2019).
Demarchi, B. Amino Acids and Proteins in Fossil Biominerals. An Introduction for Archaeologists and Palaeontologists (Wiley & Sons, 2020).
Penkman, K. E. H. et al. Dating the Paleolithic: trapped charge methods and amino acid geochronology. Proc. Natl. Acad. Sci. USA 119, 1–10 (2022).
Demarchi, B. et al. Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part I: Isolation and testing of the closed system. Quat. Geochronol. 16, 144–157 (2013).
Hendy, J. Ancient protein analysis in archaeology. Sci. Adv. 7, 1–12 (2021).
Martínez-García, A. et al. Laboratory assessment of the impact of chemical oxidation, mineral dissolution, and heating on the nitrogen isotopic composition of fossil-bound organic matter. Geochem. Geophys. Geosys. 23, e2022GC010396 (2022).
Auderset, A. et al. Enhanced ocean oxygenation during Cenozoic warm periods. Nature 609, 77–82 (2022).
Jung, J. et al. Coral photosymbiosis on Mid-Devonian reefs. Nature 636, 647–653 (2024).
Leichliter, J., Lüdecke, T., Foreman, A., Bacon, A. & Sigman, D. Nitrogen isotopic composition of tooth enamel organic matter records trophic position in modern and fossil ecosystems. Comm. Biol. 6, 373 (2023).
Scher, M. et al. Nitrogen isotopic composition of enamel-bound organic matter preserves trophic information in Mesozoic terrestrial communities. In GSA Connects 2023 Meeting in Pittsburgh, Pennsylvania 81–82 (Geological Society of America, 2023).
Comans, C. M. et al. Enameloid-bound d15N reveals large trophic separation among Late Cretaceous sharks in the northern Gulf of Mexico. Geobiology 22, e12585 (2024).
Kast, E. R. et al. Cenozoic megatooth sharks occupied extremely high trophic positions. Sci. Adv. 8, 7–17 (2022).
Lüdecke, T. et al. Australopithecus at Sterkfontein did not consume substantial mammalian meat. Science 387, 309–314 (2025).
Weber, K. et al. Diagenetic stability of non-traditional stable isotope systems (Ca, Sr, Mg, Zn) in teeth – an in-vitro alteration experiment of biogenic apatite in isotopically enriched tracer solution. Chem. Geol. 572, 120196 (2021).
Brooks, A. A. S. et al. Dating Pleistocene archeological sites by protein diagenesis in ostrich eggshell. Science 248, 60–64 (1990).
Demarchi, B. et al. Protein sequences bound to mineral surfaces persist into deep time. Elife 5, e17092 (2016).
Demarchi, B. et al. Amino acid racemization dating of marine shells: a mound of possibilities. Quat. Int. 239, 114–124 (2011).
Demarchi, B. et al. Survival of mineral-bound peptides into the Miocene. Elife 11, e82849 (2022).
Sakalauskaite, J. et al. The degradation of intracrystalline mollusc shell proteins: a proteomics study of Spondylus gaederopus. Biochim. Biophys. Acta Proteins Proteom. 1869, 140718 (2021).
Penkman, K. E. H., Kaufman, D. S., Maddy, D. & Collins, M. J. Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells. Quat. Geochronol. 3, 2–25 (2008).
Wheeler, L. J., Penkman, K. E. H. & Sejrup, H. P. Assessing the intra-crystalline approach to amino acid geochronology of Neogloboquadrina pachyderma (sinistral). Quat. Geochronol. 61, 101131 (2021).
Bada, J. L., Shou, M. Y., Man, E. H. & Schroeder, R. A. Decomposition of hydroxy amino acids in foraminiferal tests; kinetics, mechanism and geochronological implications. Earth Planet. Sci. Lett. 41, 67–76 (1978).
Walton, D. Degradation of intracrystalline proteins and amino acids in fossil brachiopods. Org. Geochem. 28, 389–410 (1998).
Jones, J. D. & Vallentyne, J. R. Biogeochemistry of organic matter-I: Polypeptides and amino acids in fossils and sediments in relation to geothermometry. Geochim. Cosmochim. Acta 21, 1–34 (1960).
Hendy, E. J. et al. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications. Geochim. Cosmochim. Acta 86, 338–353 (2012).
Dickinson, M. R., Lister, A. M. & Penkman, K. E. H. A new method for enamel amino acid racemization dating: a closed system approach. Quat. Geochronol. 50, 29–46 (2019).
Cappellini, E. et al. Early Pleistocene enamel proteome sequences from Dmanisi resolve Stephanorhinus phylogeny performed analyses and data interpretation. Bienvenido Martínez Navarro 574, 103–107 (2020).
Abelson, P. H. Paleobiochemistry. Sci. Am. 195, 83–96 (1956).
Lacruz, R. S., Habelitz, S., Wright, J. T. & Paine, M. L. Dental enamel formation and implications for oral health and disease. Physiol. Rev. 97, 939–993 (2017).
Beniash, E. et al. The hidden structure of human enamel. Nat. Commun. 10, 1–13 (2019).
Blumenthal, S. A., Cerling, T. E., Smiley, T. M., Badgley, C. E. & Plummer, T. W. Isotopic records of climate seasonality in equid teeth. Geochim. Cosmochim. Acta 260, 329–348 (2019).
Białas, N. et al. Teeth of past and present elephants: microstructure and composition of enamel in fossilized proboscidean molars and implications for diagenesis. Geochem. Geophys. Geosys. 22, e2020GC009557 (2021).
Green, D. R. et al. Eighteen million years of diverse enamel proteomes from the East African Rift. Nature 643, 712–718 (2025).
Paterson, R. S. et al. A 20 + Ma old enamel proteome from Canada ’ s High Arctic reveals diversification of Rhinocerotidae in the middle Eocene-Oligocene. bioRxiv 6, https://www.biorxiv.org/content/10.1101/2024.06.07.597871v1 (2024).
Bada, J. L., Wang, X. S. & Hamilton, H. Preservation of key biomolecules in the fossil record: current knowledge and future challenges. Philos. Trans. R. Soc. B Biol. Sci. 354, 77–87 (1999).
Vallentyne, J. R. Biogeochemistry of organic matter-II Thermal reaction kinetics and transformation products of amino compounds. Geochim. Cosmochim. Acta 28, 157–188 (1964).
Demarchi, B. et al. An integrated approach to the taxonomic identification of prehistoric shell ornaments. PLoS One 9, e 99839 (2014).
Ortiz, J. E., Gutiérrez-Zugasti, I., Sánchez-Palencia, Y., Torres, T. & González-Morales, M. Protein diagenesis in archaeological shells of Littorina littorea (Linnaeus, 1758) and the suitability of this material for amino acid racemisation dating. Quat. Geochronol. 54, 101017 (2019).
Andrews, J. T., Miller, G. H., Davies, D. C. & Davies, K. Generic identification of fragmentary Quaternary molluscs by amino acid chromatography A tool for Quaternary and palaeontological research. Geol. J. 20, 1–20 (1985).
Kaufman, D. S., Miller, G. H. & Andrews, J. Amino acid composition as a taxonomic tool for molluscan fossils: an example from Pliocene-Pleistocene Arctic marine deposits. Geochim. Cosmochim. Acta 56, 2445–2453 (1996).
Degens, B. E. T. & Love, S. Comparative studies of amino-acids in shell structures of Gyraulus trochiformis, Stahl, from the Tertiary of Steinheim, Germany. Nature 205, 876–878 (1965).
Welker, F. et al. Enamel proteome shows that Gigantopithecus was an early diverging pongine. Nature 576, 262–265 (2020).
Collins, M. J. et al. The survival of organic matter in bone: a review. Archaeometry 44, 383–394 (2002).
Hillson, S. Teeth, 388 (Cambridge Univ. Press, 2005).
Ferretti, M. P. Structure and evolution of mammoth molar enamel. Acta Palaeontol. Pol. 48, 383–396 (2003).
Cring, F. D. Enamel prism patterns in proboscidean molar teeth. Elephant 2, 72–79 (1986).
Tabuce, R., Delmer, C. & Gheerbrant, E. Evolution of the tooth enamel microstructure in the earliest proboscideans (Mammalia). Zool. J. Linn. Soc. 149, 611–628 (2007).
Dirks, W., Bromage, T. G. & Agenbroad, L. D. The duration and rate of molar plate formation in Palaeoloxodon cypriotes and Mammuthus columbi from dental histology. Quat. Int. 255, 79–85 (2012).
Berkovitz, B. & Shellis, P. The Teeth of Mammalian Vertebrates (Academic Press, 2018).
Kaboth-Bahr, S. et al. Improved chronostratigraphy for the Messel Formation (Hesse, Germany) provides insight into early to middle Eocene climate variability. Newslett. Stratigr. 57, 153–170 (2024).
Smith, K. T., Schaal, S. F. K. & Habersetzer, J. Messel: an ancient greenhouse ecosystem. Geoconserv. Res. 4, 1–355 (2018).
Tütken, T. Isotope compositions (C, O, Sr, Nd) of vertebrate fossils from the Middle Eocene oil shale of Messel, Germany: implications for their taphonomy and palaeoenvironment. Palaeogeogr. Palaeoclimatol. Palaeoecol. 416, 92–109 (2014).
Hoppe, K. A., Stover, S. M., Pascoe, J. R. & Amundson, R. Tooth enamel biomineralization in extant horses: implications for isotopic microsampling. Palaeogeogr. Palaeoclimatol. Palaeoecol. 206, 355–365 (2004).
Uno, K. T. et al. Forward and inverse methods for extracting climate and diet information from stable isotope profiles in proboscidean molars. Quat. Int. 557, 92–109 (2020).
Bada, J. L. Amino acid cosmogeochemistry. Philos. Trans. R. Soc. Lond. B 333, 349–358 (1991).
Saitta, E. T. et al. Non-avian dinosaur eggshell calcite can contain ancient, endogenous amino acids. Geochim. Cosmochim. Acta 365, 1–20 (2023).
Sato, N., Quitain, A. T., Kang, K., Daimon, H. & Fujie, K. Reaction kinetics of amino acid decomposition in high-temperature and high-pressure water. Ind. Eng. Chem. Res. 43, 3217–3222 (2004).
Gehler, A., Tütken, T. & Pack, A. Oxygen and carbon isotope variations in a modern rodent community - implications for palaeoenvironmental reconstructions. PLoS One 7, 16–27 (2012).
Acknowledgements
This study was funded by the Max Planck Society. We thank the project PE5 CHANGES: Cultural Heritage Active Innovation for Sustainable Society. PE 0000020 CHANGES, - CUP B53C22003780006, PNRR Missione 4 Componente 2 Investimento 1.3, NextGenerationEU for contribution. We thank the following museums for providing access to modern or fossil tooth enamel samples: Staatliches Museum für Naturkunde Stuttgart Museum am Löwentor, Naturhistorisches Museum Basel, Bayerische Staatssammlung für Paläontologie und Geologie München, Geowissenschaftliches Museum Universität Göttingen, Naturhistorisches Museum Mainz, Naturalis Leiden, Paläontologisches Institut und Museum Universität Zürich, JURASSICA Museum Porrentruy, Naturhistoriska riksmuseet Stockholm, Archäozoologische Sammlung Universität Tübingen, Zoologische Schausammlung Universität Tübingen, Naturhistorisches Museum of Bern, Staatliches Museum für Naturkunde of Karlsruhe, Hessisches Landesmuseum of Darmstadt, Senckenberg Naturmuseum of Frankfurt, Archäologisches Forschungszentrum und Museum für menschliche Verhaltensevolution Monrepos. TT acknowledges funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program grant agreement no. 681450. The authors thank Mareike Schmitt, and Barbara Hinnenberg for their technical support during the sample analysis. The authors thank Prof. Matthew Collins for his precious suggestions and contribution to this work.
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L.G.: performed research, data evaluation, original draft preparation; F.L.: data evaluation, writing – review and editing; F.R.: performed research, writing – review and editing; J.L.: performed research, writing – review and editing; G.S.: writing – review and editing; S.P.: resources, supervision, writing – review and editing, T.T.: designed research, sampled teeth, resources, writing – review and editing; A.M.G.: designed research, supervision, writing – review and editing.
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Gatti, L., Lugli, F., Rubach, F. et al. Deep-time preservation of amino acids in mammalian fossil tooth enamel. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09716-6
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DOI: https://doi.org/10.1038/s42003-026-09716-6
