Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C).DOI: http://dx.doi.org/10.7554/eLife.17092.001

The living tree sloths Choloepus and Bradypus are the only remaining members of Folivora, a major xenarthran radiation that occupied a wide range of habitats in many parts of the western hemisphere during the Cenozoic, including both continents and the West Indies. Ancient DNA evidence has played only a minor role in folivoran systematics, as most sloths lived in places not conducive to genomic preservation. Here we utilise collagen sequence information, both separately and in combination with published mtDNA evidence, to assess the relationships of tree sloths and their extinct relatives. Results from phylogenetic analysis of these datasets differ substantially from morphology-based concepts: Choloepus groups with Mylodontidae, not 64 Megalonychidae; Bradypus and Megalonyx pair together as megatherioids, while monophyletic Antillean sloths may be sister to all other folivorans. Divergence estimates are consistent with fossil evidence for mid-Cenozoic presence of sloths in the West Indies and an early Miocene radiation in South America. The sloths (Xenarthra, Folivora), nowadays a taxonomically narrow (6 species in 2 genera) component of the fauna of South and Central America 1,2 were once a highly successful clade of placental mammals as measured by higher-level diversity (Fig. 1). Diverging sometime in the Palaeogene from their closest relatives, the anteaters (Vermilingua), folivorans greatly expanded their diversity and range, eventually reaching North America as well as the West Indies 3-8. During the late Cenozoic sloth lineage diversity may have expanded and contracted several times 9. Final collapse occurred in the late Quaternary (end-Pleistocene on the continents, mid

Fourier transform infrared (FTIR) spectroscopy using attenuated total reflection (ATR) is commonly used for the examination of bone. During sample preparation bone is commonly ground, changing the particle size distribution. Although previous studies have examined changes in crystallinity caused by the intensity of grinding using FTIR, the effect of sample preparation (i.e. particle size and bone tissue type) on the FTIR data is still unknown. This study reports on the bone powder particle size effects on mid-IR spectra and within sample variation (i.e. periosteal, mesosteal, trabecular) using FTIR-ATR. Twenty-four archaeological human and faunal bone samples (5 heated and 19 unheated) of different chronological age (Neolithic to post-Medieval) and origin (Belgium, Britain, Denmark, Greece) were ground using either (1) a ball-mill grinder, or (2) an agate pestle and mortar, and split into grain fractions (>500 m, 250-500 m, 125-250 m, 63-125 m, and 20-63 m). Bone powder particle size has a strong but predictable effect on the infrared splitting factor (IRSF), carbonate/phosphate (C/P) ratio, and amide/phosphate (Am/P) values. The absorbance and positions of the main peaks, the 2nd derivative components of the phosphate and carbonate bands, as well as the full width at half maximum (FWHM) of the 1010 cm-1 phosphate peak are particle size dependent. This is likely to be because of the impact of the particle size on the short-and long-range crystal order, as well as the contact between the sample and the prism, and hence the penetration depth of the IR light. Variations can be also observed between periosteal, cortical and trabecular areas of bone. We therefore propose a standard preparation method for bone powder for FTIR-ATR analysis that significantly improves accuracy, consistency, reliability, replicability and comparability of the data, enabling systematic evaluation of bone in archaeological, anthropological, paleontological, forensic and biomedical studies.

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Avian eggshell survives well in alkaline and neutral soils, but its potential as an archaeological resource remains largely unexplored, mainly due to difficulties in its identification. Here we exploit the release of novel bird genomes and, for the first time on eggshell, use MALDI-ToF (matrix-assisted laser desorption ionisation-time of flight) mass spectrometry in combination with peptide sequencing by LC-MS/MS. The eggshell proteome is revealed as unexpectedly complex, with 5755 proteins identified for a reference collection comprising 23 bird species. We determined 782 m/z markers useful for eggshell identification, 583 of which could be assigned to known eggshell peptide sequences. These were used to identify eggshell fragments recovered from a medieval site at Freeschool Lane, Leicester. We discuss the specificity of the peptide markers and highlight the importance of assessing the level of taxonomic identification achievable for archaeological interpretation.

Ancient protein analysis is a rapidly developing field of research. Proteins ranging in age from the Quaternary to Jurassic are being used to answer questions about phylogeny, evolution, and extinction. However, these analyses are sometimes contentious, and focus primarily on large vertebrates in sedimentary fossilisation environments; there are few studies of protein preservation in fossils in amber. Here we show exceptionally slow racemisation rates during thermal degradation experiments of resin enclosed feathers, relative to previous thermal degradation experiments of ostrich eggshell, coral skeleton, and limpet shell. We also recover amino acids from two specimens of fossil feathers in amber. The amino acid compositions are broadly similar to those of degraded feathers, but concentrations are very low, suggesting that much of the original protein has been degraded and lost. High levels of racemisation in more apolar, slowly racemising amino acids suggest that some of the amino acids were ancient and therefore original. Our findings indicate that the unique fossilisation environment inside amber shows potential for the recovery of ancient amino acids and proteins.

13The exceptional eggshell assemblage from Çatalhöyük was studied using an integrated approach 14 combining morphology (by optical and scanning electron microscopy) and palaeoproteomics (by 15 mass spectrometry). We provide taxonomic classification for 90 fragments, of which only 11 16 remain undetermined. The striking predominance of Anseriformes (probably including greylag 17 geese, as well as ducks and swans) in all types of deposits examined, including middens and 18 burial fills, suggests that these eggs were exploited as food and, at the same time, had a special 19 significance for the inhabitants of the site. We detected the presence of crane eggshell in contexts 20 associated with both the world of the living and the world of the dead (consistent with the well-21 known importance of this bird at Çatalhöyük), as well as the possible but infrequent occurrence 22 of bustards and herons. Overall, we suggest that eggshell analysis, together with osteological 23 data and the archaeological context, can provide the basis for a nuanced understanding of the 24 relationship between humans and birds in the past. 25 Keywords 26

Eggshells are unusual finds in the Iron Age of eastern Europe (500 BC-1200 AD) deserving extra attention in terms of analysis as well as interpretation. This paper discusses two rare eggshell finds, discovered in female burials at the conversion period (12 th-13 th century AD) cemetery at Kukruse, NE Estonia. Our multianalytical study combining FT-IR, SEM(-EDS), microscopy and ZooMS provides an overview of methods applicable for identifying egg species, their predepositional history and curation. Based on the analytical results and the comparative analysis of the content and context of these two burials, we argue that different aims and connotations lay behind depositing eggs as burial goods, allowing well-supported interpretations of both pagan and Christian religious worldviews simultaneously.