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.

Stable isotopes show Homo sapiens dispersed into cold steppes ~45,000 years ago at Ilsenhöhle in Ranis, Germany.

The spread of Homo sapiens into new habitats across Eurasia ~45,000 years ago and the concurrent disappearance of Neanderthals represents a critical evolutionary turnover in our species' history. 'Transitional' technocomplexes, such as the Lincombian-Ranisian-Jerzmanowician (LRJ), characterize the European record during this period but their makers and evolutionary significance have long remained unclear. New evidence from Ilsenhöhle in Ranis, Germany, now provides a secure connection of the LRJ to H. sapiens remains dated to ~45,000 years ago, making it one of the earliest forays of our species to central Europe. Using many stable isotope records of climate produced from 16 serially sampled equid teeth spanning ~12,500 years of LRJ and Upper Palaeolithic human occupation at Ranis, we review the ability of early humans to adapt to different climate and habitat conditions. Results show that cold climates prevailed across LRJ occupations, with a temperature decrease culminating in a pronounced cold excursion at ~45,000-43,000 cal BP. Directly dated H. sapiens remains confirm that humans used the site even during this very cold phase. Together with recent evidence from the Initial Upper Palaeolithic, this demonstrates that humans operated in severe cold conditions during many distinct early dispersals into Europe and suggests pronounced adaptability.

The ecology, subsistence and diet of ~45,000-year-old Homo sapiens at Ilsenhöhle in Ranis, Germany.

Recent excavations at Ranis (Germany) identified an early dispersal of Homo sapiens into the higher latitudes of Europe by 45,000 years ago. Here we integrate results from zooarchaeology, palaeoproteomics, sediment DNA and stable isotopes to characterize the ecology, subsistence and diet of these early H. sapiens. We assessed all bone remains (n = 1,754) from the 2016-2022 excavations through morphology (n = 1,218) or palaeoproteomics (zooarchaeology by mass spectrometry (n = 536) and species by proteome investigation (n = 212)). Dominant taxa include reindeer, cave bear, woolly rhinoceros and horse, indicating cold climatic conditions. Numerous carnivore modifications, alongside sparse cut-marked and burnt bones, illustrate a predominant use of the site by hibernating cave bears and denning hyaenas, coupled with a fluctuating human presence. Faunal diversity and high carnivore input were further supported by ancient mammalian DNA recovered from 26 sediment samples. Bulk collagen carbon and nitrogen stable isotope data from 52 animal and 10 human remains confirm a cold steppe/tundra setting and indicate a homogenous human diet based on large terrestrial mammals. This lower-density archaeological signature matches other Lincombian-Ranisian-Jerzmanowician sites and is best explained by expedient visits of short duration by small, mobile groups of pioneer H. sapiens.

Homo sapiens reached the higher latitudes of Europe by 45,000 years ago.

The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe1. Local hybridization between the two groups occurred2, but not on all occasions3. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups4. One such technocomplex for which the makers are unknown is the Lincombian-Ranisian-Jerzmanowician (LRJ), which has been described in northwestern and central Europe5-8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.

Subsistence behavior during the Initial Upper Paleolithic in Europe: Site use, dietary practice, and carnivore exploitation at Bacho Kiro Cave (Bulgaria).

The behavioral dynamics underlying the expansion of Homo sapiens into Europe remains a crucial topic in human evolution. Owing to poor bone preservation, past studies have strongly focused on the Initial Upper Paleolithic (IUP) stone tool record. Recent excavations and extensive radiocarbon dating at Bacho Kiro Cave (Bulgaria) pushed back the arrival of IUP H. sapiens into Europe to ca. 45,000 years ago. This site has exceptional bone preservation, and we present the study of 7431 faunal remains from across two IUP layers (I and J) and one Middle Paleolithic layer (K). We identified a shift in site use and occupation intensity through time, marked by increased find density and human modifications in Layer I. Alongside a decrease in carnivore presence and seasonality data demonstrating human presence in all seasons, this indicates a more frequent or prolonged occupation of the site by IUP groups. Contrarily, the dietary focus across the IUP and Middle Paleolithic layers is similar, centered on the exploitation of species from a range of habitats including Bos/Bison, Cervidae, Equidae, and Caprinae. While body parts of large herbivores were selectively transported into the site, the bear remains suggest that these animals died in the cave itself. A distinct aspect of the IUP occupation is an increase in carnivore remains with human modifications, including these cave bears but also smaller taxa (e.g., Canis lupus, Vulpes vulpes). This can be correlated with their exploitation for pendants, and potentially for skins and furs. At a broader scale, we identified similarities in subsistence behavior across IUP sites in Europe and western Asia. It appears that the first IUP occupations were less intense with find densities and human modifications increasing in succeeding IUP layers. Moreover, the exploitation of small game appears to be limited across IUP sites, while carnivore exploitation seems a recurrent strategy.

Subsistence strategies throughout the African Middle Pleistocene: Faunal evidence for behavioral change and continuity across the Earlier to Middle Stone Age transition.

The African Middle Pleistocene (781-126 ka) is a key period for human evolution, witnessing both the origin of the modern human lineage and the lithic turnover from Earlier Stone Age (ESA) Acheulean bifacial tools to Middle Stone Age (MSA) prepared core and point technologies. This ESA/MSA transition is interpreted as representing changing landscape use with greater foraging distances and more active hunting strategies. So far, these behavioral inferences are mainly based on the extensive stone tool record, with only a minor role for site-based and regional faunal studies. To provide additional insights into these behavioral changes, this paper details a pan-African metastudy of 63 Middle Pleistocene faunal assemblages from 40 sites. A hierarchical classification system identified 26 well-contextualized assemblages with quantitative paleontological and/or zooarcheological data available for detailed comparative analyses and generalized linear mixed modeling. Modeling of ungulate body size classes structured around three dimensions (context, antiquity and technology) illustrates no one-to-one correlation between changes in lithic technology (Acheulean vs. MSA) and changes in prey representation. All assessed faunal assemblages are dominated by medium-sized bovids, and variations between smaller and larger body size classes are linked to site context (cave vs. open-air), with an increase in cave sites during the Middle Pleistocene. Current data do not signal a broadening of the hominin dietary niche during the Middle Pleistocene; no meaningful variation was visible in the exploitation of smaller-sized bovids or dangerous game, with coastal resources exploited when available. Proportions of anthropogenic bone surface modifications, and hence carcass processing intensity, do increase over time although more zooarcheological data is crucial before making behavioral inferences. Overall, this paper illustrates the potential of broad scale comparative faunal analyses to provide additional insights into processes of human behavioral evolution and the mechanisms underlying patterns of technological, chronological and contextual change.

On the local Mousterian origin of the Châtelperronian: Integrating typo-technological, chronostratigraphic and contextual data.

Across Europe the period 45-40 ka (thousands of years ago) is associated with several technological changes, including the emergence of the Châtelperronian technocomplex in France and northern Spain. The Châtelperronian, stratigraphically located between the Mousterian and Aurignacian, is characterized by Upper Palaeolithic features, such as volumetric blade reduction, curved backed blades, end-scrapers, bladelets, bone tools and ornaments. Concurrently, repeated, though debated, associations with Neanderthal remains and Mousterian elements suggest a local technological development. Following recent critiques and cumulating technological studies, this paper provides data-driven contextualisations of the Châtelperronian and late Mousterian archaeological records and a primary comparative assessment of a major linking element, backed knives, to re-assess the origin of the Châtelperronian. The results demonstrate the challenging nature of the 50-35 ka record, with many interpretive problems caused by poorly recorded excavations, resulting in only 25 well-contextualised assemblages from the claimed 143 Châtelperronian find spots. These 25 assemblages facilitate more detailed chronostratigraphic and typo-technological assessments and show that the Châtelperronian has a homogenous set of technologies and tools. A similar evaluation of the late Mousterian indicates a wide-ranging late Neanderthal skill set, commonly including laminar blank production and backing. Further, conceptual similarities were noted both in blank selection and edge modification between Mousterian and Châtelperronian backed knives, alongside their near-absence in other, contemporaneous technocomplexes. A Europe-wide contextualisation shows that while the current coarse-grained record still allows for several potential scenarios, the data throughout this paper point towards a most parsimonious model of a Châtelperronian made by Neanderthals, with roots in the late Middle Palaeolithic technological skill set. However, this change seems triggered by early arrivals of modern humans either indirectly, through stimulus diffusion, or directly, after ca. 42 ka. Fully testing this model requires an ongoing focus on site formation and assemblage integrity, alongside in-depth analyses of recently excavated assemblages and existing collections.

Regional behaviour among late Neanderthal groups in Western Europe: a comparative assessment of late Middle Palaeolithic bifacial tool variability.

Population dynamics between and within Pleistocene groups are vital to understanding wider behavioural processes like social transmission and cultural variation. The late Middle Palaeolithic (MIS 5d-3, ca. 115,000-35,000 BP [years before present]) permits a novel, data-driven assessment of these concepts through a unique record: bifacial tools made by classic Neanderthals. Previously, studies of late Middle Palaeolithic bifacial tools were hampered by a convoluted plethora of competing terms, types and regional entities. This paper presents a large-scale intercomparison of this tool type, and bridges typo-technological and spatio-temporal data from across Western Europe (Britain, Belgium, the Netherlands, France and Germany). Results indicate a high level of variation among individual bifacial tools and assemblages. Each bifacial tool concept is correlated with various methods of production, resulting in large degrees of morphological variation. Despite such variation, a distinct three-fold, macro-regional pattern was identified: the Mousterian of Acheulean Tradition (MTA) in the southwest dominated by handaxes, the Keilmessergruppen (KMG) in the northeast typified by backed and leaf-shaped bifacial tools, and, finally a new unit, the Mousterian with Bifacial Tools (MBT), geographically situated between these two major entities, and characterised by a wider variety of bifacial tools. Differing local conditions, such as raw material or function, are not sufficient to explain this observed macro-regional tripartite. Instead, the MTA and KMG can be viewed as two distinct cultural traditions, where the production of a specific bifacial tool concept was passed on over generations. Conversely, the MBT is interpreted as a border zone where highly mobile groups of Neanderthals from both the east (KMG) and west (MTA) interacted. Principally, this study presents an archaeological contribution to behavioural concepts such as regionality, culture, social transmission and population dynamics. It illustrates the interpretive potential of large-scale lithic studies, and more specifically the presence of regionalised cultural behaviour amongst late Neanderthal groups in Western Europe.