DOI: 10.5281/zenodo.15050021
title: Archaeozoological insights into avian exploitation in the Magdalenian of Belgium at the caves of Trou du Frontal and Trou des Nutons

description: The karstic area of the Lesse valley, located in the south of Belgium, has been at the heart of the development of archaeological research in the country during the 19th century. Numerous cavities have revealed remains of human activities documenting their use during the Palaeolithic period.

We present the results of the archaeozoological analysis of bird bones from the Trou des Nutons and Trou du Frontal recovered during the 19th century excavations in connection with Magdalenian artefacts. These two caves are located in the Lesse valley, close to each other, and near the Trou de Chaleux cave which is the most important Magdalenian site yet uncovered in Belgium. These new data allow us to compare the variability of human behaviours in contemporary Magdalenian occupations within the same microregion.

Our analysis documents the first evidence of human exploitation of birds at the Trou des Nutons and Trou du Frontal. Bird species exploited include geese, merganser, ptarmigan, black grouse and snowy owl. The use of large birds for crafts is only attested at Trou du Frontal and is consistent with what has been observed at the Trou de Chaleux. Tool marks linked to the removal of meat are observed at both the Trou des Nutons and Trou du Frontal. Given uncertainty regarding the chronocultural attribution of certain bones bearing anthropogenic marks, new radiocarbon dates were specifically obtained on these specimens. To maximize the chances of successful and reliable results, given the small size and light weight of bird bones, a dedicated protocol was designed. This involved near-infrared screening, proteomic analyses to detect potential contaminants, and dating using the MICADAS accelerator with a gas ion source, enabling the dating of very small carbon samples.  Experimentation were conducted to obtain a referential of scraping marks produced with metal-tools to compare with some marks observed on the archaeological material.
description: Protocole for proteomic analyses.

The digestion of the bones was described in Bray et al. (2023). In brief, 1–5 mg of bone samples were placed in 96-well MultiScreenHTS-IP plates (0.45 μm, MSIPS4510, Millipore, Billerica, MA, USA). The bone powder was demineralized, washed, gelatinized, and digested in these plates. The demineralization solution was digested in classical 96-well plates. Peptides from the bones and demineralized solution were purified on C18 96-well plates (Affinisep, Petit-Couronne, France). Purification of peptides involved washing the plates once with 500 μL of acetonitrile (ACN), followed by washing with 80% ACN, 0.5% acetic acid in water (repeated three times), and a second washing with water alone (0.5% acetic acid, repeated three times). Tryptic peptides from the bone powder were resuspended in 200 μL of water, 0.5% acetic acid solution. Tryptic peptides from both solutions were transferred to C18 96-well plates and eluted using a vacuum manifold. The plate was washed three times with 200 μL of water, 0.5% acetic acid. Peptides were recovered in a V-bottom well collection plate using 100 μL of 80% ACN, 0.1% acetic acid solution, followed by 100 μL of ACN. The plate was evaporated using the TurboVap 96 Evaporator (Caliper LifeScience, Hopkinton, USA). For mass spectrometry analysis, the sample was re-dissolved in 10 μL of water, 0.1% formic acid. Mass spectrometry analysis was performed by depositing desalted peptides (1 μL) onto 384-massive MALDI plates (Bruker Daltonics, Bremen, Germany), followed by the addition of 1 μL of HCCA matrix at 10 mg/mL in ACN/H2O (70:30 v/v), 0.1% TFA for each sample. The spots were allowed to dry at room temperature. MALDI FTICR experiments were conducted on a Bruker 9.4 Tesla SolariX XR FTICR mass spectrometer (Bruker Daltonics, Bremen, Germany). The laser frequency was set to 1,000 Hz using the “Minimum” predefined shot pattern. MALDI FTICR spectra were generated in the m/z range from 693.01 to 5,000 by accumulating 10 scans. Two M data points were used per spectrum, corresponding to a transient duration of 5.0332 s. The transfer time to the ICR cell was set to 1.2 ms, and the quadrupole mass filter was set at m/z 600, operated in RF-only mode. MS raw data from MALDI FTICR were processed using DataAnalysis 5.0.