Explore the vast range of titles available.

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. The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria. To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source. Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world.

The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor , and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens , are highly debated 1 – 5 . For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge 6 – 8 . Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain) 9 , 10 and Homo erectus from Dmanisi (Georgia) 1 , two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor —that is, similar to that of modern humans—may have a considerably deep ancestry in the genus Homo , and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo . Analyses of the proteomes of dental enamel from Homo antecessor and Homo erectus demonstrate that the Early Pleistocene H. antecessor is a close sister lineage of later Homo sapiens , Neanderthal and Denisovan populations in Eurasia.

Despite the vast array of different geochronological tools available, dating the Paleolithic remains one of the discipline’s greatest challenges. This review focuses on two different dating approaches: trapped charge and amino acid geochronology. While differing in their fundamental principles, both exploit time-dependent changes in signals found within crystals to generate a chronology for the material dated and hence, the associated deposits. Within each method, there is a diverse range of signals that can be analyzed, each covering different time ranges, applicable to different materials and suitable for different paleoenvironmental and archaeological contexts. This multiplicity of signals can at first sight appear confusing, but it is a fundamental strength of the techniques, allowing internal checks for consistency and providing more information than simply a chronology. For each technique, we present an overview of the basis for the time-dependent signals and the types of material that can be analyzed, with examples of their archaeological application, as well as their future potential.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa 1 . However, the irreversible post-mortem degradation 2 of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr) 3 . By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I 4 , and suggested the presence of protein residues in fossils of the Cretaceous period 5 —although with limited phylogenetic use 6 . In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch 7 – 9 , using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia) 10 . Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros ( Coelodonta antiquitatis ) and Merck’s rhinoceros ( Stephanorhinus kirchbergensis ). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates 11 , and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation. Palaeoproteomic analysis of dental enamel from an Early Pleistocene Stephanorhinus resolves the phylogeny of Eurasian Rhinocerotidae, by enabling the reconstruction of molecular evolution beyond the limits of ancient DNA preservation.

Ancient DNA (aDNA) sequencing has enabled reconstruction of speciation, migration, and admixture events for extinct taxa1. Outside the permafrost, however, irreversible aDNA post-mortem degradation2 has so far limited aDNA recovery to the past ~0.5 million years (Ma)3. Contrarily, tandem mass spectrometry (MS) allowed sequencing ~1.5 million year (Ma) old collagen type I (COL1)4 and suggested the presence of protein residues in Cretaceous fossil remains5, although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae7–9 using a ~1.77 Ma old dental enamel proteome of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus)10. Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly (Coelodonta antiquitatis) and Merck’s rhinoceros (S. kirchbergensis) clade. We show that Coelodonta evolved from an early Stephanorhinus lineage and that the latter includes at least two distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel proteome sequencing overcomes the limits of ancient collagen- and aDNA-based phylogenetic inference. It also provides additional information about the sex and taxonomic assignment of the specimens analysed. Dental enamel, the hardest tissue in vertebrates11, is highly abundant in the fossil record. Our findings reveal that palaeoproteomic investigation of this material can push biomolecular investigation further back into the Early Pleistocene.

Analysis of the predictable breakdown of proteins and amino acids in ancient biominerals enables age estimation over the Quaternary. We postulate that enamel is a suitable biomineral for the long-term survival of endogenous amino acids. Analysis of multiple amino acids for geochronological studies is typically achieved using a RP-HPLC method. However, the low concentrations of amino acids coupled with high concentrations of inorganic species make accurate determination of amino concentrations challenging. We have developed a method for the routine preparation of multiple enamel samples using biphasic separation. Furthermore, we have shown that amino acids that exhibit effectively closed system behaviour can be isolated from enamel through an exposure time of 72 h to bleach. Elevated temperature experiments investigating the processes of intra-crystalline protein degradation (IcPD) do not appear to match the patterns from fossil samples, reinforcing the need for a comprehensive understanding of the underlying mechanisms of protein degradation. This novel preparative method isolates intra-crystalline amino acids suitable for the development of mammalian geochronologies based on enamel protein degradation. The lower rates of racemisation in enamel (cf. Bithynia opercula) suggest that the enamel AAR may be able to be used as a relative dating technique over time scales > 2.8 Ma. Enamel AAR has the potential to estimate the age of mammalian remains past the limit of all other current direct dating methods, providing an invaluable tool for geochronological studies.

Ancient DNA (aDNA) sequencing has enabled unprecedented reconstruction of speciation, migration, and admixture events for extinct taxa. Outside the permafrost, however, irreversible aDNA post-mortem degradation has so far limited aDNA recovery within the ~0.5 million years (Ma) time range. Tandem mass spectrometry (MS)-based collagen type I (COL1) sequencing provides direct access to older genetic information, though with limited phylogenetic use. In the absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae using ~1.77 million years (Ma) old dental enamel proteome sequences of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus). Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly (Coelodonta antiquitatis) and Merck's rhinoceros (S. kirchbergensis) clade. We show that Coelodonta evolved from an early Stephanorhinus lineage and that this genus includes at least two distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel proteome sequencing overcomes the limits of ancient collagen- and aDNA-based phylogenetic inference, and also provides additional information about the sex and the taxonomic assignment of the specimens analysed. Dental enamel, the hardest tissue in vertebrates, is highly abundant in the fossil record. Our findings reveal that palaeoproteomic investigation of this material can push biomolecular investigation further back into the Early Pleistocene.

In the past decade, ancient protein sequences have emerged as a valuable source of data for deep-time phylogenetic inference. Still, the recovery of protein sequences providing novel phylogenetic insights does not exceed 3.7 Ma (Pliocene). Here, we push this boundary back to 21-24 Ma (early Miocene), by retrieving enamel protein sequences of an early-diverging rhinocerotid (Epiaceratherium sp. - CMNF-59632) from the Canadian High Arctic. We recover partial sequences of seven enamel proteins (AHSG, ALB, AMBN, AMELX, AMTN, ENAM, MMP20) and over 1000 peptide-spectrum matches, spanning over at least 251 amino acids. Authentic endogeneity of these sequences is supported by indicators of protein damage, including several spontaneous and irreversible post-translational modifications accumulated during prolonged diagenesis and reaching near-complete occupancy at many sites. Bayesian tip-dating, across 15 extant and extinct perissodactyl taxa, places the divergence time of CMNF-59632 in the middle Eocene-Oligocene, and identifies a later divergence time for Elasmotheriinae in the Oligocene. The finding weakens alternative models suggesting a deep basal split between Elasmotheriinae and Rhinocerotinae. This divergence time of CMNF-59632 coincides with a phase of high diversification of rhinocerotids, and supports a Eurasian origin of this clade in the late Eocene or Oligocene. The findings are consistent with previous hypotheses on the origin of the enigmatic fauna of the Haughton crater, which, in spite of their considerable degree of endemism, also display similarity to distant Eurasian faunas. Our findings demonstrate the potential of palaeoproteomics in obtaining phylogenetic information from a specimen that is ten times older than any sample from which endogenous DNA has been obtained.