Frido Welker’s research while affiliated with IT University of Copenhagen and other places

Advances in biomolecular methods, in particular the study of ancient proteins (paleoproteomics), have revolutionized how we can taxonomically identify archaeological bone fragments. Alongside traditional zooarchaeo-logical assignments based on the visual inspection of morphological criteria, variations in collagen type I amino acid sequences can now be used to distinguish which animal a bone fragment belonged to. Using MALDI-ToF mass spectrometry, this method, known as Zooarchaeology by Mass Spectrometry (ZooMS), is now being applied regularly to archaeological faunal assemblages and, often at a large-scale, at Paleolithic sites. However, detailed explorations of how these ZooMS datasets can best be integrated with zooarchaeological and taphonomic data are only in their infancy. To further advance this field, we hosted a workshop at the University of Kent in 2023, bringing together both zooarchaeologists and ZooMS specialists, to showcase and discuss various ways of integrating ZooMS and zoo-archaeological data, especially within Paleolithic contexts. This special issue results from the papers presented at this workshop. In this introductory paper we reflect on the open discussion sessions that formed an essential part of the workshop. First, we discuss a series of methodological challenges; this includes the recording of zooar-chaeology and taphonomy on morphologically unidentifiable bone fragments, ZooMS study design and sample selection, pre-screening and sampling, pre-treatment and collagen extraction, and the acquisition, processing, and interpretation of MALDI data. Second, we delve deeper into the interpretive potential, and the wealth of future research directions, of a full contribution of ZooMS to a range of zooarchaeological research topics. In concordance with the seven research papers in this issue, this introduction illustrates how a well-designed study, integrating zooarchaeological and taphonomic observations across both the morphological and ZooMS identified fractions, cannot only increase the number of identifiable specimens at a site, but also provide novel insights into site formation histories, collection biases, carnivore behavior, environmental conditions, and past human subsistence, including site use, seasonality, carcass transport, prey preference, and butchery practices.

Abstract booklet for Zooarch/ZooMS integration workshop hosted at Kent in April 2023.

Few studies have combined the analysis of use-wear traces, traceology, and the proteomic taxonomic identification method Zooarchaeology by Mass Spectrometry (ZooMS). Traceology provides information on the usage, in this case, of bone artefacts, while ZooMS allows for taxonomic identifications where diagnostic features are otherwise gone. The approaches therefore offer complementary information on bone artefacts, allowing for insights into species selection strategies in bone tool manufacture and their subsequent use. Here we present a case study of 20 bone artefacts, mainly bone points, from the Early Neolithic cave site of Coro Trasito located on the southern slope of the Central Pyrenees. Hitherto, studies on Early Neolithic bone artefacts from the Iberian Peninsula have suggested based on morphological assessments that Ovis aries/Capra hircus constituted the majority of the bone material selected for bone tool production. However, the taxonomic identification in this study suggests that, at this site, Cervidae was selected equally to that of O. aries/C. hircus. Furthermore, bone artefacts made from Cervidae specimens seem to be utilised in a wider range of artefact types compared to O. aries/C. hircus. Coro Trasito’s bone artefact species composition is probably site-specific to some degree, however, morphological assessments of bone artefacts might not be representative and could be biased towards certain species. Therefore, research on bone artefacts’ usage could possibly gain new insights by implementing ZooMS in combination with traceology.

Genetic and fragmented palaeoanthropological data suggest that Denisovans were once widely distributed across eastern Eurasia1–3. Despite limited archaeological evidence, this indicates that Denisovans were capable of adapting to a highly diverse range of environments. Here we integrate zooarchaeological and proteomic analyses of the late Middle to Late Pleistocene faunal assemblage from Baishiya Karst Cave on the Tibetan Plateau, where a Denisovan mandible and Denisovan sedimentary mitochondrial DNA were found3,4. Using zooarchaeology by mass spectrometry, we identify a new hominin rib specimen that dates to approximately 48–32 thousand years ago (layer 3). Shotgun proteomic analysis taxonomically assigns this specimen to the Denisovan lineage, extending their presence at Baishiya Karst Cave well into the Late Pleistocene. Throughout the stratigraphic sequence, the faunal assemblage is dominated by Caprinae, together with megaherbivores, carnivores, small mammals and birds. The high proportion of anthropogenic modifications on the bone surfaces suggests that Denisovans were the primary agent of faunal accumulation. The chaîne opératoire of carcass processing indicates that animal taxa were exploited for their meat, marrow and hides, while bone was also used as raw material for the production of tools. Our results shed light on the behaviour of Denisovans and their adaptations to the diverse and fluctuating environments of the late Middle and Late Pleistocene of eastern Eurasia.

Circular structures made from woolly mammoth bones are found across Ukraine and west Russia, yet the origin of the bones remains uncertain. We present ten new mammoth radiocarbon dates from the largest circular structure at Kostenki 11-Ia, identifying two mammoth mandibles ∼200-1,200 years older than the other dated materials from the site, suggesting skeletal material from long-dead individuals was scavenged and used in the site construction. Biomolecular sexing of 30 individuals showed a predominance of females, suggesting the Kostenki mammoths are primarily from herds. We identify six mitochondrial lineages across 16 samples, showing they are not all from the same matriline. Integrating biomolecular sexing with stable δ ¹³ C and δ ¹⁵ N isotope analysis, we find no isotopically-differentiated resource use by females and males, providing the first analysis of foraging differences between sexes in any Late Pleistocene megafauna. Our study highlights the significance of integrating ancient biomolecular approaches in archaeological inference. Teaser Integrating ¹⁴ C dating, ancient DNA, palaeoproteomics, and stable isotopes improves our understanding of Kostenki 11-Ia

The study of ancient proteins preserved in a range of archaeological, cultural heritage, and palaeontological materials is increasingly contributing to our understanding of human evolution and archaeological research questions. Many of the specimens studied have been excavated and stored for a significant duration prior to their proteomic analysis. Human handling and storage environments therefore provide ample opportunities for protein contamination onto and into specimens of interest to palaeoproteomic studies. As such, modern protein contamination limits access to endogenous proteomes. Here, we compare five approaches of bone protein decontamination applied to a Pleistocene Equus sp. bone fragment contaminated with a modern dog salivary proteome. We find that all tested methods reduce the protein contamination, but with different efficiencies. We find that a brief bleach wash is the most effective approach in removing modern protein contamination, and that no additional damage is caused to the endogenous proteome by this treatment. Next, we apply this approach to a hominin tooth found at Khudji, a Late Pleistocene archaeological site in Tajikistan. We demonstrate that a brief bleach wash removes almost all human skin protein contamination while retaining the endogenous hominin dentine proteome. Subsequent phylogenetic analysis of the Khudji dentine proteome allowed determination that the specimen likely represents a Neanderthal, extending the fossil evidence for Neanderthals in Central Asia.

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3-4 working days, while subsequent data analysis usually takes 2-5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.