Can ZooMS help assess species abundance in highly fragmented bone assemblages? Integrating morphological and proteomic identifications for the calculation of an adjusted ZooMS-eNISP

Zooarchaeology by Mass Spectrometry (ZooMS) is a rapid, low-cost, collagen-based method for the taxonomic identification of animal tissues. It is now increasingly applied to bone fragments from archeological contexts, creating large taxonomic datasets. How to integrate these ZooMS identifications within general zooarchaeological theoretical frameworks, such as estimates of species abundance and taxonomic richness, remains problematic. Past large-scale ZooMS analyses of Eurasian Paleolithic faunal assemblages have shown a general trend towards an increased representation of large ungulates (mainly Bos/Bison) in the ZooMS fraction, often coupled with a decrease in medium-sized taxa (e.g., reindeer). Here we propose several hypotheses to explain these identification discrepancies, involving identification biases and differential fragmentation patterns across various taxa, and test them using the case study of the Paleolithic site of Cassenade. At the Châtelperronian site of Cassenade (France), nearly all bone fragments larger than 20mm (n=1,119) have been identified to taxa, either through comparative morphology (n=364) or ZooMS (n=755). Each of these fragments was weighed and measured, creating a unique database to explore the relation between fragmentation and identification. Analysis shows that fragment size and mass distributions are distinct across taxa if only bones identified by morphology are considered, but, somehow counter-intuitively, extremely similar across taxa of various body sizes when all their bones are integrated. In particular, the bones of larger ungulates tend to be broken into a higher number of fragments, inducing an over-representation of larger taxa in ZooMS-NISP. Our dataset also shows that sorting long-bone shaft fragments by body size classes should be seen by zooarchaeologists as a process that, in addition to being prone to risks of misidentification, provides highly biased information of little use for estimating species abundance. To overcome this issue, we propose the calculation of an adjusted equivalent ZooMS NISP (ZooMS-eNISP) by dividing, for each taxon, the total ZooMS mass of identified bones (g) by the mean mass of morphologically identified bones for that taxon (g/NISP). The advantage of this method is that it considers site-specific characteristics of the faunal assemblage, notably bone preservation, which is especially important in Paleolithic contexts. Finally, we propose that ZooMS-eNISP, despite its limits, can facilitate the integration of both identification methods to produce a more refined picture of patterns of species representation, site formation, and human behavior at an archaeological site

Integrating ZooMS and Zooarchaeology to assess the Châtelperronian and carnivore occupations at Cassenade (Dordogne, France)

Archaeological animal bone assemblages are often highly fragmented, meaning that for over 70% of the recovered bone fragments we do not know what animal (or human) species they belonged to. This is especially problematic in Paleolithic contexts, when both humans and carnivores repeatedly occupied the same caves and rock shelters. Identifying bone fragments from these dual occupation contexts through Zooarchaeology by Mass Spectrometry (ZooMS) can provide additional insights into both carnivore and human behavior. In this paper, we apply ZooMS to the majority of morphologically unidentifiable bone fragments larger than 20mm (n=840) recovered from the 2012–2013 excavated Châtelperronian layer of Cassenade (France). Collagen was extracted using an ammoniumbicarbonate (AmBic) buffer and over 99% of the sampled bone fragments could be identified taxonomically. While the proportion of Equidae is similar in both ZooMS and zooarchaeological components, Bos/Bison is represented by a threefold increase in the ZooMS fraction (50.5% vs. 16.6%). Conversely, Ursidae, the dominant taxa in the morphologically identifiable remains (36.4%), only formed 7.3% of the ZooMS fragments. Carnivores are also present, but in low numbers (0–2%), and include Hyaenidae, Panthera and Canidae. In the ZooMS fraction, only few bones show traces of human activity (ca. 2%), which is most likely related to low bone surface readability. These show that human groups at Cassenade were processing Bovinae and Equidae, as well as Ursidae and rhinoceros. Conversely, traces of carnivore activity are abundant, and we were able to taxonomically identify 334 bone fragments that were digested by carnivores (as indicated by acid etched surfaces). While large proportions of the Rhinocerotidae (63.79%), Elephantidae (52%), Equidae (48%), and Bos/Bison (45%) remains were digested by carnivores, this is only the case for 1.7% of the Ursidae ZooMS fragments. Threedimensional data are available for all the ZooMS-identified fragments and confirm the near-exclusive presence of cave bear in the lower part of the sequence. Further, the ZooMS spatial data identified a restricted presence of mammoth in the middle part of the sequence and a diverging presence of reindeer and Cervid/saiga remains at the bottom and top. Overall, this study illustrates the added value of integrating zooarchaeological and ZooMS datasets to obtain additional insights into past ecologies, changing site use, carnivore diets, and human subsistence practices

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, defned 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 faking techniques) and ventral retouch is present across diferent 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 signifcant proportions of equids, bovids, cervids and reindeer. Carnivore remains and carnivore modifcations are absent, while human bone surface modifcations are present across a variety of species. Five bones had sufcient 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

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

Investigating species composition in the Early Aurignacian of le Piage (France) through collagen fingerprinting (ZooMS) of screen-recovered small bone fragments

In the field of paleoproteomics, ZooMS (Zooarcheology by Mass Spectrometry) has been developed to identify morphologically non-diagnostic animal remains to taxon, offering insights into human subsistence practices. Here, we report new ZooMS analyses of 1,050 Early Aurignacian (ca. 37,000–34,000 cal BP) bone fragments from the site of Le Piage (Lot, France). The studied sample is heavily fragmented and was retrieved through water sieving. In our analysis, we compare the taxonomic identifications of bone remains using traditional morphological attributes with remains identified using ZooMS and discuss the implications of the taxonomic patterns that we uncovered. Our results indicate that, despite small effect sizes, the faunal spectrum identified through ZooMS differs from that obtained through morphological analyses. While reindeer remains the dominant species, bovids and other cervids are more abundantly represented in the ZooMS fraction. Two rare taxa, a hare (Lepus sp.) and a previously unidentified carnivore (Pantherinae/Hyaenidae/Mustelidae), were also identified using ZooMS. In addition, we note an increase of Bos/Bison remains in the sample of spongy fragments that is possibly explained by the use of grease-rich bones and bone portions as fuel. Our work adds new data on patterns of reindeer dominance during the Early Aurignacian and illustrates how ZooMS identifications of screen-recovered fragments can enhance our understanding of Paleolithic subsistence strategies and patterns of site occupation

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 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. [Abstract copyright: © 2024. The Author(s).

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. [Abstract copyright: © 2024. The Author(s).

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 Europe5,6,7,8. 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