Alkylresorcinol detection and identification in archaeological pottery using ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry.

RATIONALE: Alkylresorcinols (AR) are cereal-specific biomarkers and have recently been found in archaeological pots. However, their low concentrations and high susceptibility to degradation make them difficult to detect using conventional gas chromatography mass spectrometry (GC/MS). Here we describe the development of a more sensitive liquid chromatography mass spectrometry (LC/MS) method to detect these compounds. METHOD: A method based on the use of ultra-high-performance liquid chromatography (UHPLC) coupled to an Orbitrap mass analyser was established and validated for the detection of low-concentration ARs in pottery. During the preliminary experiments, UHPLC-Q/Orbitrap MS (ultra-high-performance liquid chromatography-quadrupole/Orbitrap mass spectrometry) was demonstrated to be more sensitive, and a wide range of AR homologues in cereal extracts were detected, unlike UHPLC-QTOFMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry) and GC/MS. The developed method was utilised to profile AR homologue distribution in modern cereal samples and reanalyse AR-containing pots from the archaeological site of Must Farm. RESULTS: A highly sensitive LC/MS method with a limit of detection (LOD) of 0.02 µg/g and a limit of quantification (LOQ) of 0.06 µg/g was used to profile ARs in five modern cereal grains. The obtained LOD is 250 times lower than that obtained using the conventional GC/MS approach. AR 21:0 was the most abundant homologue in all four Triticum spp.-einkorn, emmer, Khorasan wheat and common wheat. Meanwhile, AR 25:0 was the predominant homologue in barley, potentially enabling differentiation between wheat and barley. The developed LC/MS-based method was successfully used to analyse ARs extracted from Must Farm potsherds and identified the cereal species most likely processed in the pots-emmer wheat. CONCLUSION: The described method offers an alternative and more sensitive approach for detecting and identifying ARs in ancient pottery. It has been successfully utilised to detect AR homologues in archaeological samples and discriminate which cereal species-wheat and barley-were processed in the pots.

Dating the Paleolithic: Trapped charge methods and amino acid geochronology.

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.

Estimating the dwarfing rate of an extinct Sicilian elephant.

Evolution on islands, together with the often extreme phenotypic changes associated with it, has attracted much interest from evolutionary biologists. However, measuring the rate of change of phenotypic traits of extinct animals can be challenging, in part due to the incompleteness of the fossil record. Here, we use combined molecular and fossil evidence to define the minimum and maximum rate of dwarfing in an extinct Mediterranean dwarf elephant from Puntali Cave (Sicily).1 Despite the challenges associated with recovering ancient DNA from warm climates,2 we successfully retrieved a mitogenome from a sample with an estimated age between 175,500 and 50,000 years. Our results suggest that this specific Sicilian elephant lineage evolved from one of the largest terrestrial mammals that ever lived3 to an island species weighing less than 20% of its original mass with an estimated mass reduction between 0.74 and 200.95 kg and height reduction between 0.15 and 41.49 mm per generation. We show that combining ancient DNA with paleontological and geochronological evidence can constrain the timing of phenotypic changes with greater accuracy than could be achieved using any source of evidence in isolation.

Author Correction: The dental proteome of Homo antecessor.

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

The dental proteome of Homo antecessor.

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 debated1-5. 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 challenge6-8. 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.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 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 I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. 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 epoch7-9, 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 vertebrates11, 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.

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

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.