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Abstract 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. 

Documenting the first appearance of modern humans in a given region is key to understanding the dispersal process and the replacement or assimilation of indigenous human populations such as the Neanderthals. The Iberian Peninsula was the last refuge of Neanderthal populations as modern humans advanced across Eurasia. Here we present evidence of an early Aurignacian occupation at Lapa do Picareiro in central Portugal. Diagnostic artifacts were found in a sealed stratigraphic layer dated 41.1 to 38.1 ka cal BP, documenting a modern human presence on the western margin of Iberia ∼5,000 years earlier than previously known. The data indicate a rapid modern human dispersal across southern Europe, reaching the westernmost edge where Neanderthals were thought to persist. The results support the notion of a mosaic process of modern human dispersal and replacement of indigenous Neanderthal populations. 

Abstract The cave site of Lapa do Picareiro, Portugal contains a deep (~10 m) sedimentary sequence representing much of the Middle and Upper Paleolithic, with abundant animal bones, lithic/bone/shell artifacts, and charcoal/ash in hearths. A deposition model based on 49 radiocarbon ages demonstrates average sedimentation rates of 0.1–0.3 mm a⁻¹between 9 and 45 ka. Extrapolation suggests an age at the base of the excavation of 65–78 ka. The cave sediments are primarily limestone éboulis derived from roof spalling, and muddy fine sediment derived from weathered soil infiltrating through bedrock joints and crevices. Bone preservation in the cave is good and disturbance by bioturbation, dissolution, or erosional processes appears to be limited. Most sedimentological parameters vary conservatively with depth, indicating stable sediment sources over time. Several parameters show good agreement with paleoclimate records over the radiocarbon‐dated section. The cold‐arid Heinrich stadials are represented by coarse‐clast beds with little fine sediment and magnetic susceptibility minima, and mild Greenland interstadials correspond with muddy beds and sharp peaks in magnetic susceptibility. This study complements ongoing archaeological excavation at the site and confirms that the sequence has the proper age, resolution, and preservation to inform on Late Pleistocene cultural and climatic transitions.