Human and cervid osseous materials used for barbed point manufacture in Mesolithic Doggerland

Barbed bone points originally deposited in Doggerland are regularly collected from the shores of the Netherlands. Their typology and direct 14C dating suggest they are of Mesolithic age. However, the species of which the barbed points were made cannot be identified based on morphological criteria. The bones used to produce the barbed points have been intensively modified during manufacture, use, and post-depositional processes. Here, we taxonomically assess ten barbed points found on the Dutch shore using mass spectrometry and collagen peptide mass fingerprinting alongside newly acquired 14C ages and δ13C and δ15N measurements. Our results demonstrate a sufficient preservation of unmodified collagen for mass spectrometry-based taxonomic identifications of bone and antler artefacts which have been preserved in marine environments since the beginning of the Holocene. We show that Homo sapiens bones as well as Cervus elaphus bones and antlers were transformed into barbed points. The 14C dating of nine barbed points yielded uncalibrated ages between 9.5 and 7.3 ka 14C BP. The δ13C and δ15N values of the seven cervid bone points fall within the range of herbivores, recovered from the North Sea, whereas the two human bone points indicate a freshwater and/or terrestrial fauna diet. The wide-scale application of ZooMS is a critical next step towards revealing the selection of species for osseous-tool manufacture in the context of Mesolithic Doggerland, but also further afield. The selection of Cervus elaphus and human bone for manufacturing barbed points in Mesolithic Doggerland is unlikely to have been opportunistic and instead seems to be strategic in nature. Further, the occurrence of Homo sapiens and Cervus elaphus bones in our random and limited dataset suggests that the selection of these species for barbed point production was non-random and subject to specific criteria. By highlighting the transformation of human bones into barbed points – possibly used as weapons – our study provides additional evidence for the complex manipulation of human remains during the Mesolithic, now also evidenced in Doggerland

Middle Pleistocene protein sequences from the rhinoceros genus Stephanorhinus and the phylogeny of extant and extinct Middle/Late Pleistocene Rhinocerotidae

Background: Ancient protein sequences are increasingly used to elucidate the phylogenetic relationships between extinct and extant mammalian taxa. Here, we apply these recent developments to Middle Pleistocene bone specimens of the rhinoceros genus Stephanorhinus. No biomolecular sequence data is currently available for this genus, leaving phylogenetic hypotheses on its evolutionary relationships to extant and extinct rhinoceroses untested. Furthermore, recent phylogenies based on Rhinocerotidae (partial or complete) mitochondrial DNA sequences differ in the placement of the Sumatran rhinoceros (Dicerorhinus sumatrensis). Therefore, studies utilising ancient protein sequences from Middle Pleistocene contexts have the potential to provide further insights into the phylogenetic relationships between extant and extinct species, including Stephanorhinus and Dicerorhinus. Methods: ZooMS screening (zooarchaeology by mass spectrometry) was performed on several Late and Middle Pleistocene specimens from the genus Stephanorhinus, subsequently followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to obtain ancient protein sequences from a Middle Pleistocene Stephanorhinus specimen.We performed parallel analysis on a Late Pleistocene woolly rhinoceros specimen and extant species of rhinoceroses, resulting in the availability of protein sequence data for five extant species and two extinct genera. Phylogenetic analysis additionally included all extant Perissodactyla genera (Equus, Tapirus), and was conducted using Bayesian (MrBayes) and maximum-likelihood (RAxML) methods. Results: Various ancient proteins were identified in both the Middle and Late Pleistocene rhinoceros samples. Protein degradation and proteome complexity are consistent with an endogenous origin of the identified proteins. Phylogenetic analysis of informative proteins resolved the Perissodactyla phylogeny in agreement with previous studies in regards to the placement of the families Equidae, Tapiridae, and Rhinocerotidae. Stephanorhinus is shown to be most closely related to the genera Coelodonta and Dicerorhinus. The protein sequence data further places the Sumatran rhino in a clade together with the genus Rhinoceros, opposed to forming a clade with the black and white rhinoceros species. Discussion: The first biomolecular dataset available for Stephanorhinus places this genus together with the extinct genus Coelodonta and the extant genus Dicerorhinus. This is in agreement with morphological studies, although we are unable to resolve the order of divergence between these genera based on the protein sequences available. Our data supports the placement of the genus Dicerorhinus in a clade together with extant Rhinoceros species. Finally, the availability of protein sequence data for both extinct European rhinoceros genera allows future investigations into their geographic distribution and extinction chronologies.The project “Hominin subsistence strategies during the Middle Pleistocene in Scho¨ningen” is a co-operation between Monrepos Archaeological Research Centre and Museum for Human Behavioural Evolution (RGZM), Johannes Gutenberg-Universita¨t Mainz, and the Niedersa¨chsisches Landesamt fu¨r Denkmalpflege, supported by the Deutsche Forschungsgemeinschaft (GA 683/9-1). This research was supported financially by the Max Planck Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The Late Middle Palaeolithic Occupation of Abri du Maras (Layer 1, Neronian, Southeast France): Integrating Lithic Analyses, ZooMS and Radiocarbon Dating to Reconstruct Neanderthal Hunting Behaviour

The exact strategies and technologies underlying Neanderthal hunting events remain open for debate with lithic points being sparse across the European Middle Palaeolithic. An exception is the Neronian entity in southeast France, defined by ventrally retouched Soyons points. This study contextualises one of the largest Neronian assemblages, layer 1 at Abri du Maras. Our lithic analyses focussed on attributes described as indicative of projectile use or hafting to contextualise the morphometric and technological characteristics of the pointed implements at an assemblage level. We found that retouched points were made on a variety of blank types (including Levallois, laminar and discoidal flaking techniques) and ventral retouch is present across different artefact types (including points, scrapers and denticulates). Next, these lithic data were compared to similar typo-technological data recorded on a sample from the recently excavated and well-contextualised point-rich layer 4.1 of Abri du Maras (MIS-3). Zooarchaeology by Mass Spectrometry (ZooMS) was applied to 280 faunal remains from layer 1 and indicated significant proportions of equids, bovids, cervids and reindeer. Carnivore remains and carnivore modifications are absent, while human bone surface modifications are present across a variety of species. Five bones had sufficient collagen for radiocarbon dating but returned dates younger than expected (ca. 41–31 ka cal BP). Finally, we place Abri du Maras layer 1 in its broader regional context and discuss its relation to other Neronian assemblages and more general problematics inherent to studying material from old excavations

SPIN enables high throughput species identification of archaeological bone by proteomics

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.info:eu-repo/semantics/publishedVersio

Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points.

Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology

Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

Palaeoproteomic analysis of skeletal proteomes is used to provide taxonomic identifications for an increasing number of archaeological specimens. The success rate depends on a range of taphonomic factors and differences in the extraction protocols employed. By analyzing 12 archaeological bone specimens from two archaeological sites, we demonstrate that reducing digestion duration from 18 to 3 hours has no measurable impact on the obtained taxonomic identifications. Peptide marker recovery, COL1 sequence coverage, or proteome complexity are also not significantly impacted. Although we observe minor differences in sequence coverage and glutamine deamidation, these are not consistent across our dataset. A 6-fold reduction in digestion time reduces electricity consumption, and therefore CO2 emission intensities. We furthermore demonstrate that working in 96-well plates further reduces electricity consumption by 60%, in comparison to individual microtubes. Reducing digestion time therefore has no impact on the taxonomic identifications, while reducing the environmental impact of palaeoproteomic projects

The Northern Route for Human dispersal in Central and Northeast Asia: New evidence from the site of Tolbor-16, Mongolia.

The fossil record suggests that at least two major human dispersals occurred across the Eurasian steppe during the Late Pleistocene. Neanderthals and Modern Humans moved eastward into Central Asia, a region intermittently occupied by the enigmatic Denisovans. Genetic data indicates that the Denisovans interbred with Neanderthals near the Altai Mountains (South Siberia) but where and when they met H. sapiens is yet to be determined. Here we present archaeological evidence that document the timing and environmental context of a third long-distance population movement in Central Asia, during a temperate climatic event around 45,000 years ago. The early occurrence of the Initial Upper Palaeolithic, a techno-complex whose sudden appearance coincides with the first occurrence of H. sapiens in the Eurasian steppes, establishes an essential archaeological link between the Siberian Altai and Northwestern China . Such connection between regions provides empirical ground to discuss contacts between local and exogenous populations in Central and Northeast Asia during the Late Pleistocene

Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identification

High-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research

Author Correction : The dental proteome of Homo antecessor (Nature, (2020), 580, 7802, (235-238), 10.1038/s41586-020-2153-8)

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