DOI: 10.5061/dryad.xd2547dnw
title: The name of the game: Palaeoproteomics and radiocarbon dates further refine the presence and dispersal of caprines in Eastern and Southern Africa

description: We report the first large-scale palaeoproteomics research on eastern and
 southern African zooarchaeological samples, thereby refining our
 understanding of early caprine (sheep and goat) pastoralism in Africa.
 Assessing caprine introductions is a complicated task because of their
 skeletal similarity to endemic wild bovid species and the sparse and
 fragmentary state of relevant archaeological remains. Palaeoproteomics has
 previously proved effective in clarifying species attributions in African
 zooarchaeological materials, but few comparative protein sequences of wild
 bovid species have been available. Using newly generated collagen type I
 sequences for wild species, as well as previously published sequences, we
 assess species attributions for elements originally identified as caprine
 or “unidentifiable bovid” from seventeen eastern and southern African
 sites that span seven millennia. We identified over 70% of the
 archaeological remains and the direct radiocarbon dating of domesticate
 specimens allows refinement of the chronology of caprine presence in both
 African regions. These results thus confirm earlier occurrences in eastern
 Africa and the systematic association of domesticated caprines with wild
 bovids at all archaeological sites. The combined biomolecular approach
 highlights repeatability and accuracy of the methods for conclusive
 contribution in species attribution of archaeological remains in dry
 African environments.
description: <em>“Modern” and
 archaeological samples protein
 extraction</em> Both reference specimen samples and
 archaeological samples underwent the same preparation protocol for protein
 extraction. Briefly, ten to twenty milligrams of bone or tooth powder were
 sampled using an ethanol clean diamond drill (Supplementary Table 2). The
 archaeological specimens from Wakarida were mandibles with their teeth and
 were thus sampled twice to assess the best tissue for sampling. For these,
 we took one bone sample and another dentine sample, for which organic
 preservation was assessed using the method described in
 Lebon et al.
 (2016). Only the
 best-preserved sample according to the threshold discussed in
 Le Meillour et al.
 (2018) is
 presented here (Supplementary Table 2). 
 After sampling, bone powders
 were placed in protein LoBind 2mL tubes (Eppendorf, Germany). We followed
 the protocol for African remains, which is appropriate for the extraction
 and characterisation of proteins from remains recovered from arid
 environments (Le Meillour et
 al., 2020). Bone
 or tooth powders were decalcified in a neutral pH buffer
 (tris(hydroxymethyl)aminomethane (Tris) 0.05 M and
 ethylenediaminetetraacetic acid (EDTA) 0.5 M, final pH 7.4, Sigma Aldrich,
 Germany) for 1-8 days, depending on the sample. Solutions were first
 replaced every ½ day, then every 24 hours. When completely decalcified
 (i.e., until only a collagen “phantom” remained), the pellets were rinsed
 5 times with milliQ water. The extracted proteins were solubilized in 50
 mM ammonium bicarbonate (ABC, pH 8, Sigma Aldrich) for 3 hours at 65 °C.
 Solutions were then centrifuged for 10 min at 3000 g and collected into
 new microtubes. Extracted proteins were finally reduced with
 dithiothreitol (DTT, final concentration 10 mM, 20 min, 56 °C, 350 rpm),
 alkylated with iodoacetamide (final concentration 10 mM, 30 min, in the
 dark, at room temperature) and hydrolysed using trypsin (0.01 μg/μL,
 Trypsin Gold, Promega). Finally, samples were transferred into clean vials
 and kept at -20°C until mass spectrometry analyses.
 <em>Mass spectrometry -
 UHPLC-MS/MS</em> Bovid species digested extracts and
 archaeological samples were analysed independently by UHPLC-MS/MS using a
 workflow described previously (Le Meillour et
 al., 2020).
 Separation was performed on an Ultimate 3000-RSLC system (Thermo Fisher
 Scientific) with a RSLC Polar Advantage II Acclaim column (2.1 × 100 mm,
 120 Å, 2.2 μm) using a flow rate of 300 μL/min and mobile phase gradient
 of A: H<sub>2</sub>O + formic acid (FA) 0.1% and B:
 acetonitrile + FA 0.08%. We used a high-resolution ESI-Q-TOF mass
 spectrometer (Maxis II ETD, Bruker Daltonics) in positive mode and
 data-dependent auto-MS/MS mode on the <em>m/z</em> range
 200-2200. MS/MS spectra were generated using collision induced
 dissociation by selection of ions with charge states between
 2<sup>+</sup> and 5<sup>+</sup> and on
 <em>m/z </em>range 300-2200. Calibration was carried out for
 each run with sodium formate clusters.
 <em>Type 1 collagen
 sequences de novo
 reconstruction</em> The raw data were converted to .mgf using the
 mass spectrometer manufacturer software, DataAnalysis (version 4.4, Bruker
 Daltonics). The sequences were reconstructed using a database assisted
 tool using the Byonic workflow C57 (Byologic, Protein Metrics). The
 restricted database used for the search contained COL1A1 and COL1A2
 sequences of the following bovid species: <em>Bos taurus</em>
 (P02453 and P02465),<em> B. mutus</em> (L8IV51 and L8HQF7),
 <em>B. indicus</em> (A0A4W2FAL4 and A0A4W2FTM9), <em>C.
 hircus </em>(A0A452FHU9 and A0A452G3V6), <em>O. aries
 </em>(W5P481 and W5NTT7) and <em>Pantholops
 hogsonii</em> (XM_005964647.1 and XM_005985683.1). The referenced
 sequences can be found on NCBI and UniProt repositories and correspond to
 entire translated protein sequences, including signal peptide and
 tropocollagen. Then, in order to reconstruct only the secreted protein
 sequences, we excluded these parts of the sequence from further analyses.
 When referring to amino acid positions, we use the sheep reference protein
 sequences. The alpha 1 chain then starts (further first position in
 Supplementary Information 2) at position 170 (Q) and stops at position
 1229 (K) in the UniProt reference (W5P481). Similarly, the alpha 2 chain
 then starts at position 80 (Q) and stops at position 1117 (A) in the
 UniProt reference (W5NTT7). The software parameters were set to 1% FDR,
 and post-translational modifications as follows: we allowed 3 tryptic
 missed cleavages; carbamidomethylation of cysteines was set as fixed
 modification; deamidation of N and Q, Gln to pyro-Glu (N term Q),
 phosphorylation of S and T and oxidation of M and P were set as variables
 modifications, with a maximum of 5 modifications allowed for one peptide.
 In the Byonics workflow, proline oxidations (HyP) were considered as
 “common modification” and every amino acid substitution was included in
 the “rare modification” list. Once results were obtained, peptides with
 potential single amino acid substitution (SAPs) were manually assessed by
 verifying MS/MS spectra with at least 2 peptide spectral matches (PSMs) in
 each of the two samples’ specimens for one species. For each
 species-specific peptide detected in a sample, we verified that the
 alternate sequence was absent by assessing PSMs manually (Supplementary
 Data 2). We confirmed every sequence by aligning them using the Geneious
 Prime® software (v. 2023.0.4) before building the final fasta file
 (Supplementary Information 2).
 <em>Archaeological
 samples identification using type I collagen
 sequences</em> Since Janzen et al. (2021) published COL1
 sequences of some of the species we included in our initial dataset, the
 “overlapping” species sequences were excluded from this paper. Here we
 present data from only 9 species (compared to the 19 initially sampled for
 reference purposes): <em>Ammordorcas clarkei, Eudorcas rufifrons,
 Gazella dorcas, Nanger dama, N. soemmeringi, Ammotragus lervia, Capra
 nubiana, Addax nasomaculatus and Pelea capreolus</em>. All other
 species COL1 sequences were taken from Janzen et al. (2021). All
 archaeological samples were searched using the MaxQuant software
 (v.2.1.3.0, Cox and Mann,
 2008) against
 the updated database of bovid species (Supplementary Information 2), and
 using the following parameters: trypsin allowed missed cleavages was set
 on 3; carbamidomethylation of cysteines was set as fixed modification, and
 deamidation (N,Q), Gln to pyro-Glu (N term Q), phosphorylation (S, T) and
 oxidation (M, P) as variable modifications; mass tolerances were set to 10
 ppm for precursor and 0.02 Da for fragment ions; all other parameters were
 left as default. We considered species identification confident if at
 least 2 razor and unique peptides from non-overlapping parts of the
 sequence were covered by inspecting the evidence file provided after the
 MaxQuant search (Supplementary Data 3 and 4). In addition, we performed
 manual assessment of the species-specific peptides
 spectra.
description: MaxQuant 2.3.0 (open source)