1 Looking for constraints: exploring proteins characterisation in arid environments to unravel past human adaptations to climate change

3 Louise Le Meillour¹, Pernille Bangsgaard², Lisa Yeomans³, ⁴, Gaudry Troché¹, Jesper V. Olsen⁵,

4 Tobias Richter⁶, Matthew J. Collins³, ⁷, Frido Welker¹

5

6 ¹ Section for Molecular Ecology and Evolution, Globe Institute, University of Copenhagen, Øster Farimagsgade 5, 1353 København, Denmark

7 ¹ Section for Molecular Ecology and Evolution, Globe Institute, University of Copenhagen, Øster Farimagsgade 5, 1353 København, Denmark

8 louise@palaeome.org

9 ² Section for Geogenetics, Globe Institute, University of Copenhagen, Øster Voldgade 5-7, 1353 København, Denmark

10 ² Section for Geogenetics, Globe Institute, University of Copenhagen, Øster Voldgade 5-7, 1353 København, Denmark

11 ³ Section for Geobiology, Globe Institute, University of Copenhagen, Øster Voldgade 5-7, 1353 København, Denmark

12 ³ Section for Geobiology, Globe Institute, University of Copenhagen, Øster Voldgade 5-7, 1353 København, Denmark

13 ⁴ University College London, Institute of Archaeology, 31-34 Gordon Square, London, WC1H 0PY, United Kingdom

14 ⁵ Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 København, Denmark

15 ⁵ Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 København, Denmark

16 ⁶ Department of Cross-Cultural and Regional Studies, University of Copenhagen, Karen Blixens Plads 8, 2300 København, Denmark

17 ⁶ Department of Cross-Cultural and Regional Studies, University of Copenhagen, Karen Blixens Plads 8, 2300 København, Denmark

18 ⁷ McDonald Institute for Archaeological Research, University of Cambridge, Henry Wellcome Building, Fitzwilliam Street, Cambridge CB2 1QH, United Kingdom

19 ⁷ McDonald Institute for Archaeological Research, University of Cambridge, Henry Wellcome Building, Fitzwilliam Street, Cambridge CB2 1QH, United Kingdom

20 Building, Fitzwilliam Street, Cambridge CB2 1QH, United Kingdom

21

22 Keywords

23 Prehistoric archaeology; climate change; subsistence strategies; ancient proteins; arid environments
24 Résumé

La pression environnementale est l'un des facteurs ayant probablement affecté les populations humaines passées et leur développement cognitif. L'adoption progressive d'animaux et de plantes domestiques représente un tournant dans la Préhistoire humaine. Or, retracer l'adoption de ces pratiques et leur potentiel lien avec des événements climatiques majeurs peut s'avérer un véritable défi pour l'archéologie préhistorique. En effet, les dernières décennies ont montré que les périodes de refroidissement ou d'assèchement environnemental ont pu jouer un rôle plus important qu'on ne le pensait dans le développement de la cognition humaine et, par conséquent, dans l'adoption de certaines pratiques de subsistance. L'avènement des méthodes biomoléculaires, qui permettent de caractériser le matériel génétique conservé dans les écofacts et les artefacts archéologiques, a permis d'apporter de nouvelles données concernant notre compréhension des populations passées.

Avec de meilleures chances de survie dans les contextes archéologiques, les protéines anciennes ont d'ores et déjà prouvé leur potentiel pour aider à la compréhension de nombreuses problématiques archéologiques. Afin d'apporter de nouvelles données concernant les réponses humaines aux pressions climatiques dans l'adoption de pratiques d'élevage, cet article présente une étude paléoprotéomique préliminaire axée sur les restes de Bovidae provenant de sites archéologiques de contextes secs à arides en Jordanie, au Soudan et en Namibie. Les spécimens ont été sélectionnés pour couvrir une période couvrant la fin du *Younger Dryas* en Jordanie au *Little Ice Age* en Namibie.

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44 Abstract

Environmental pressure is one of the factors that probably affected past human populations and their cognitive behaviour. One spinning point in human prehistory is the progressive adoption of domestic animals and plants. However, tracing the adoption of these practices and their relation to major climatic events could be a real challenge in prehistoric archaeology. In fact, these past
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decades have proven that the cooling or dryer environmental periods might have played a greater
role than assumed in the development of human cognition, and consequently, for the adoption of
certain subsistence practices. The advent of biomolecular methods, allowing for the
characterisation of genetic material preserved in archaeological ecofacts and artefacts have
increasingly extended our knowledge of past populations. With higher chances of survival within
archaeological contexts, ancient proteins have revealed their potential in helping to shed light on
numerous archaeological questions. For the purpose of trying to infer human responses to climatic
pressure in the adoption of herding practices, this paper presents a preliminary palaeoproteomics
study focusing on remains of Bovidae from dry to arid archaeological sites from Jordan and
Namibia. Specimens were selected so that they cover a period from hunting and gathering practices
to established pastoralism and covering environmental conditions from the end of the Younger
Dryas in Jordan to the Little Ice Age in Namibia.
62 **Introduction**

63 *Cognition and influence of climate on adoption of various subsistence strategies*

64 The relationship existing between human cognition and culture (in a very broad acceptance) has
65 already been discussed extensively, especially in the frame of thinking and material culture
66 production. This entire discipline called cognitive archaeology, has greatly improved our
67 understanding of the links between cognitive development and production (whether it be material,
68 spiritual or social)¹. However, assessing these links is a real archaeological challenge when
69 concerning changes in subsistence strategies. There is, in fact, no *production* per se when focusing
70 on subsistence in Prehistory. One way of characterising subsistence in archaeology is through the
71 study of faunal remains. Multiple lines of evidence can be used to do so, from comparative
72 anatomy to traceology or molecular identifications.

73 *The Levant and Africa as case studies: arid environments and the adoption of herding practices*

74 One of the major shifts human populations had to experienced lies in the advent of
75 domestication, also referred to as the “Neolithic revolution”. Not only did it lead to major
76 biological and societal changes (both for animals/plants and humans), but it also re-shaped the
77 environment. Rather seen as a complex process than an abrupt shift, the domestication of animals
78 happened in the Zagros mountain in current Middle East²,³. Domestication is a relatively long
79 process occurring over generations that implies the replacement of one wild population by a
80 domestic one. Nowadays, sheep, *Ovis aries* and goats, *Capra hircus*, lie at the centre of many
81 economies across the globe and are thus of interest as to where and how they were domesticated
82 in the first place. The two species thus result from the management of their wild ancestors, the
83 Asiatic mouflon, *Ovis orientalis* and the wild bezoar, *Capra aegagrus*, initiated after the end of the
84 Younger Dryas, around 9,600 before common era (BCE)⁴. The initial management of these species
85 lead to their domestic forms by 7000 BCE⁵. Introduced in Africa later in comparison, around the
86 seventh millenium BCE in the northeastern part of the continent, they are documented around
87 the turn of the era in the southernmost tip⁶.
State of the art on studies of ancient proteins in the Levant and Africa

Molecular methods employed in archaeology are numerous and one could think about
stable isotopes, dating methods or ancient DNA (aDNA) analyses. These methods can be
employed to recover information about past diet, environments, chronologies or past populations
gene pools. One molecule of choice is thus aDNA, allowing to infer

<figure><img src="image_1.png" /><figcaption>Figure 1: General description of the palaeoproteomics workflow when looking for species identification of archaeological remains.</figcaption></figure>

Hypothesis and question(s) driving the study

This project revokes around the question of the influence of climatic events on the adoption
of herding practices in challenging environments. The archaeological material was selected for the
triple purpose of: covering a large chronology covering the advent of herding practices as long as
various climatic events that might have influenced the adoption of such practices; securely
identifying the species of the specimens (i.e. either from domestic sheep or goat, wild gazelles or
wild caprines in the case of the Jordanian sites); and finally, confirming the effectiveness of ancient
proteins characterisation in the dry and arid environments of the Near East and Africa from
various time-scales. In order to gain insights at these questions, we selected three archaeological
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sites located in Jordan and covering a large chronology from the Natufian to the end of the PPNA: Shubayqa 1 and 6 and Shkarāt Msaied (see extensive description in the Material and Methods section following). We also included two African sites corresponding to different environmental conditions: SJE 270 in Sudan and Leopard Cave in Namibia, representing the current climatic conditions and the Little Ice Age, respectively. We selected samples along the stratigraphy to identify potential shifts in subsistence strategies, although our sample size remains sparse due to unknown protein preservation. We compared two ancient proteins extraction protocols to identify a potential ideal extraction strategy for the sampled remains and analysed them using liquid chromatography coupled to tandem mass spectrometry. Finally, we used a database partially developed during the study, to identify the archaeological specimens as specifically as possible.

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## Material and method

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### *About having the proper references – establishment of bovids COL1 sequences*

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Since (palaeo)proteomics rely on the comparison of the obtained data to available protein references, many bioarchaeologists already faced the issue of the lack of reference in previous studies<sup>7,8</sup>. A substantial amount of time of this work was thus dedicated to the re-analysis of COL1 sequences data to allow for the species identification of arid environments Bovidae (for a detailed description, see ref. <sup>9</sup>). The establishment of this accurate referential to work with is however critical to go beyond “classical” comparative anatomy and morphology-based methods and thus, enhance our comprehension of species distribution, ecology and subsequent human exploitation.

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Then, we recently published a new COL1 reference database accessible to anyone willing to use it<sup>9</sup> and including nineteen species of wild Bovidae falling in the “size 2/3 bovids” category (classically employed by zooarchaeologists when preservation of the archaeological material does not allow to identify the remains more specifically). Mostly composed of wild gazelles that can be confused with sheep and goat, the database also includes two species of wild caprines, the Barbary sheep, *Ammotragus lervia*, and the Nubian ibex, *Capra nubiana*. Even though the database used to
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assign the studied archaeological specimens is lacking endemic species from Jordan, it presents
African species of the same genera. Indeed, given the consistency of COL1 sequences within the
genera of family Bovidae (we did not observe any amino acid substitution from genus *Gazella*, and
very few in the different Antilopinae species for instance), we did not expect any mis assignation
that might have been due to a database bias.

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*Archaeological context*

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Shkarāt Msaied and Shubayqa 1 & 6

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Located in current Jordan, the archaeological sites of Shkarāt Msaied and Shubayqa 1 and 6 are covering a large chronology from early Natufian to the pre-pottery neolithic B (PPNB), with remains of domesticated sheep and goats morphologically identified at both sites.

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Shubayqa 1 and 6 are located less than a kilometre apart, on the edge of the Qa' Shubayqa basin in the Harrat ash-Sham desert in north-eastern Jordan (Fig. 2,¹⁰,¹¹). The stratigraphy indicates sporadic occupations from 12400 BCE to 9650 BCE at Shubayqa 1¹⁰, when Shubayqa 6 was occupied continuously from 10420 to 8640 BCE¹². Both sites thus cover a time-range spanning from the Natufian to the PPNA, covering environmental conditions including the Bølling-Allerød, the Younger Dryas and the return to milder conditions during the Pre-Boreal oscillation⁵.

<figure><img src="image_2.png" /></figure>

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<caption>Figure 2: Locations of the Levantine archaeological sites included in the project.</caption>
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Shkarāt Msaied is almost at the opposite side of the country, north of Petra in south-western Jordan, close to the Israeli border (Fig. 2)¹³. The stratigraphy indicates human occupation starting during the middle PPNB (MPPNB) around 8000 BCE¹⁴,¹⁵. Circular stone buildings and material culture related to daily life (cooking areas) have been excavated, associated with what was described as a “small village”¹⁶,¹⁷. Thus, occupations at Shkarāt Msaied correspond to the Holocene climatic optimum, environmental conditions resembling the ones currently observed in the region.

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SJE 170, Sudan

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Between 1960-1964, extensive rescue excavation campaigns were conducted by a team of Nordic prehistorian in Nubian Sudan. The sites discovered and documented by the Scandinavian Joint Expedition in Sudanese Nubia (SJE) are curated in different places of Scandinavia, including Denmark, at the Zoological Museum (Univ. of Copenhagen)¹⁸. We accessed domesticated caprines material from site number 270, a funerary context in which the caprines were originally associated with human burials¹⁹ (Fig. 3). The 270 site has previously been dated from 1950 to 1750 BCE, period associated with C-group culture, with large buildings and wealthy burials. Resembling Bronze Age cultures in other parts of the world²⁰, and contemporary of the Middle Kerma phase for site 270, C-group Nubian populations were close to the river Nile and experienced environmental conditions resembling the ones occurring nowadays in south Egypt and north Sudan, consecutive to the mid-third millennium BCE aridification²¹.
<figure><img src="image_3.png" /><figcaption>Figure 3: Location of the African sites included in the project.</figcaption></figure>

## Leopard Cave, Namibia

Leopard Cave is located in the Erongo mountains, north of the capital of Namibia and has been discovered in 2007²² (Fig. 3). The rock shelter presents a chronologically long sequence, from the end of the Middle Stone Age (MSA) to the Later Stone Age (LSA)²³. Recently, our team has revised the morphological assignation to sheep for some of the faunal remains of the sites, pushing back the arrival of the domesticates at the site to ca 1000 CE⁷. We indeed confirmed the presence of sheep at the site during the Little Ice Age, but not earlier.

## Proteins extraction and characterisation

A total of 47 archaeological remains morphologically identified as sheep/goat, antelopes/gazelles or 'unidentified Bovidae' were selected. An additional set of eight samples from Shkarāt Msaied from which shotgun proteomics data were already available were included to our analyses. All processed sampled are described in Table 1.
<table><caption>Table 1: Archaeological specimens and corresponding dating, culture and morphological identification selected for the study. Specimens marked with * stand for the data re-analysis performed by the first author for the Jensen et al. 2023 paper. All other samples were prepared and analysed following the arid environment protocol.</caption><thead><tr><th>Archaeological site</th><th>Sample code</th><th>Dating (years BCE)</th><th>Culture</th><th>Skeletal element</th><th>Morphological ID</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="6">Shubayqa 1</td><td>SH_1</td><td>12300-12000</td><td>Natufian</td><td>L. humerus</td><td>Gazella sp.</td><td>Richter et al. 2017</td></tr><tr><td>SH_2</td><td>12300</td><td>Natufian</td><td>R. tibia</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td>SH_3</td><td>12300</td><td>Natufian</td><td>L. tibia</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td>SH_4</td><td>12300-12000</td><td>Natufian</td><td>L. tibia</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td>SH_5</td><td>11200-10000</td><td>Natufian</td><td>L. humerus</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td>SH_6</td><td>10000-9800</td><td>Natufian</td><td>Tibia</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td rowspan="3">Shubayqa 6</td><td>SH_7</td><td>10400-10000</td><td>Natufian</td><td>L. humerus</td><td>Gazella sp.</td><td>Richter et al. 2016</td></tr><tr><td>SH_8</td><td>9000</td><td>PPNA</td><td>Humerus</td><td>Capra/Ovis</td><td></td></tr><tr><td>SH_9</td><td>test pit</td><td>N/A</td><td>Tibia</td><td>Capra/Ovis</td><td></td></tr><tr><td rowspan="20">Shkarāt Msaied</td><td>SM_20</td><td>7000</td><td>LPPNB</td><td>Femur</td><td>Capra/Ovis</td><td>Bangsgaard 2008</td></tr><tr><td>SM_21</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra/Ovis</td><td>Bangsgaard 2009</td></tr><tr><td>SM_22</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra sp.</td><td>Bangsgaard 2010</td></tr><tr><td>SM_23</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra/Ovis</td><td>Bangsgaard 2011</td></tr><tr><td>SM_24</td><td>7000</td><td>LPPNB</td><td>Tibia</td><td>Capra/Ovis</td><td>Bangsgaard 2012</td></tr><tr><td>SM_25</td><td>7000</td><td>LPPNB</td><td>Mandible</td><td>Capra/Ovis</td><td>Bangsgaard 2013</td></tr><tr><td>SM_26</td><td>7000</td><td>LPPNB</td><td>Tibia</td><td>Capra/Ovis</td><td>Bangsgaard 2014</td></tr><tr><td>SM_27</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra/Ovis</td><td>Bangsgaard 2015</td></tr><tr><td>SM_28</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra/Ovis</td><td>Bangsgaard 2016</td></tr><tr><td>SM_29</td><td>7000</td><td>LPPNB</td><td>Mandible</td><td>Capra/Ovis</td><td>Bangsgaard 2017</td></tr><tr><td>SM_30</td><td>7000</td><td>LPPNB</td><td>Mandible</td><td>Capra/Ovis</td><td>Bangsgaard 2018</td></tr><tr><td>SM_31</td><td>7000</td><td>LPPNB</td><td>Tibia</td><td>Capra sp.</td><td>Bangsgaard 2019</td></tr><tr><td>SM_32</td><td>7000</td><td>LPPNB</td><td>Tibia</td><td>Capra/Ovis</td><td>Bangsgaard 2020</td></tr><tr><td>SM_33</td><td>7000</td><td>LPPNB</td><td>Mandible</td><td>Capra/Ovis</td><td>Bangsgaard 2021</td></tr><tr><td>SM_34</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra sp.</td><td>Bangsgaard 2022</td></tr><tr><td>SM_35</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra sp.</td><td>Bangsgaard 2023</td></tr><tr><td>SM_36</td><td>7000</td><td>LPPNB</td><td>Tibia</td><td>Capra/Ovis</td><td>Bangsgaard 2024</td></tr><tr><td>SM_37</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra sp.</td><td>Bangsgaard 2025</td></tr><tr><td>SM_38</td><td>7000</td><td>LPPNB</td><td>Humerus</td><td>Capra sp.</td><td>Bangsgaard 2026</td></tr><tr><td>SM4*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM5*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM8*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM10*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM12*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM18*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM23*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td>SM26*</td><td>8000</td><td>MPPNB</td><td>Petrous</td><td>Caprinae</td><td>Jensen et al. 2023</td></tr><tr><td rowspan="2">SJE 270</td><td>SJE_10</td><td>2500-2050</td><td>C-group</td><td>Tibia</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr><tr><td>SJE_11</td><td>2500-2050</td><td>C-group</td><td>Tibia</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr></tbody></table>
<table><tbody><tr><td></td><td>SJE_12</td><td>2500-2050</td><td>C-group</td><td>Cranium</td><td>Ovis aries</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_13</td><td>2500-2050</td><td>C-group</td><td>Tibia</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_14</td><td>2500-2050</td><td>C-group</td><td>Mandible</td><td>Ovis aries</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_15</td><td>2500-2050</td><td>C-group</td><td>Mandible</td><td>Ovis aries</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_16</td><td>2500-2050</td><td>C-group</td><td>Mandible</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_17</td><td>2500-2050</td><td>C-group</td><td>Vertebra</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_18</td><td>2500-2050</td><td>C-group</td><td>Mandible</td><td>Ovis aries</td><td>Johansson 2014</td></tr><tr><td></td><td>SJE_19</td><td>2500-2050</td><td>C-group</td><td>Tibia</td><td>Capra/Ovis</td><td>Johansson 2014</td></tr><tr><td rowspan="10">Leopard Cave</td><td>LC_166</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_167</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_168</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_169</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_170</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_171</td><td>1st c.</td><td>LSA</td><td>Metatarsal</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_172</td><td>1st c.</td><td>LSA</td><td>Vertebra</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_173</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr><tr><td>LC_174</td><td>1st c.</td><td>LSA</td><td>Unidentified</td><td>Size 2/3 bovids</td><td>Pleurdeau et al. 2012</td></tr></tbody></table>

Approximately 20 mg of bone powder was sampled for each of the 47 archaeological specimens using a diamond drill or disc, cleaned thoroughly in between samples with 96% ethanol to avoid cross-contamination. Proteins extractions were carried out as follows: the 47 specimens and one experimental blank (hereafter, first batch) were treated using the 'arid environment protocol' that was optimised<sup>24,25</sup>; while the other eight (hereafter, second batch) were treated using the new 'Tryps-IN' simplified protocol that our team published recently<sup>15</sup>. All samples underwent enzymatic hydrolysis using trypsin, allowing to break down the proteins into smaller pieces, or peptides.

All 56 samples were analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the Center for Protein Research (CPR, Novo Nordisk Foundation, Univ. of Copenhagen). First, the peptides contained in each sample were separated regarding their affinity with the liquid chromatography column internal phase. Consequently, molecules were ionised (addition of a positive charge), separated based on their mass over charge ratio (m/z) in the mass spectrometer, and detected. The resulting data for each sample consist in a set of spectra,
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each corresponding to a specific peptide, allowing the bioinformatic reconstruction of the initial
protein.
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All raw data were search against the reference Bovidae COL1 database described in a
previous section and available in ref.⁹ using the MaxQuant software (v. 2.3.0, ²⁶). Parameters for
the different searches were set as follow: we allowed 2 tryptic missed-cleavages, post-translational
modifications included oxidation (M), hydroxyproline, deamidation (N, Q) and false discovery rate
(FDR) was set to 1%. All other parameters were set as default. Identifications were considered
confident when at least 2 peptide spectral matches (PSMs) for one species were observed. A more
descriptive methodology is available in refs. ²⁵, ⁹ and ¹⁵.

## Results and discussion

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*Molecular identifications of remains from arid environments*

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Overall, data quality was uneven in between the different archaeological sites. Only three
samples from Shubayqa (1&6) allowed for a very limited number of characterised peptides,
preventing more specific identification than to family level. Over the initial 18 samples from
Shkarāt Msaied, seven allowed for identification from family to species level. However, it is worth
noticing that the samples prepared using the “Tryps-IN” protocol yielded at least genus level
identifications, even though they were almost two centuries older than the previous set. Samples
from site 270 of the SJE were all assigned to sheep, when six out of nine samples from Leopard
Cave present preserved peptides allowing assignation from family to species level (Fig. 4).
<figure><img src="image_4.png" /><figcaption>Figure 4: Molecular taxonomic identifications for all the studied sites of the project.</figcaption></figure>

## Towards an ideal protocol for ancient protein extraction from Holocene arid environments?

Two preparation protocols were used here. Although our initial intention was not to compare their efficiency, the analysed specimens originated from the same site, allowing for comparison. We observe enhanced identification rates for the specimens analysed using the Tryps-IN protocol compared to the ones using the “arid environment” protocol. Not only the number of “identifiable” samples was higher with Tryps-IN (100% against 39% for the other protocol), but identification were more specific. Given these conclusions, we have confidence that if the Tryps-IN protocol was going to be used again on all other samples analysed in this paper, it will result in enhance number of identified specimens.

## Morphology vs molecular identifications

The analyses presented here allowed to refine the morphological identifications in some cases. Proteins did not seem to be preserved enough at Shubayqa 1 and 6 to allow any identification, except for one sample for which the preserved peptides did not allow enough sequence coverage to confidently identify below family level.
<table><caption>Table 2: Comparison of morphological and molecular identifications obtained for all the analysed samples.</caption><thead><tr><th>Archaeological site</th><th>Sample code</th><th>Morphological ID</th><th>Molecular ID</th></tr></thead><tbody><tr><td rowspan="6">Shubayqa 1</td><td>SH_1</td><td>Gazella sp.</td><td></td></tr><tr><td>SH_2</td><td>Gazella sp.</td><td></td></tr><tr><td>SH_3</td><td>Gazella sp.</td><td></td></tr><tr><td>SH_4</td><td>Gazella sp.</td><td></td></tr><tr><td>SH_5</td><td>Gazella sp.</td><td>Bovidae?</td></tr><tr><td>SH_6</td><td>Gazella sp.</td><td></td></tr><tr><td rowspan="3">Shubayqa 6</td><td>SH_7</td><td>Gazella sp.</td><td></td></tr><tr><td>SH_8</td><td>Capra/Ovis</td><td>Bovidae?</td></tr><tr><td>SH_9</td><td>Capra/Ovis</td><td>Bovidae?</td></tr><tr><td rowspan="20">Shkarāt Msaied</td><td>SM_20</td><td>Capra/Ovis</td><td>Capra hircus</td></tr><tr><td>SM_21</td><td>Capra/Ovis</td><td>Caprinae</td></tr><tr><td>SM_22</td><td>Capra sp.</td><td></td></tr><tr><td>SM_23</td><td>Capra/Ovis</td><td>Gazella sp.</td></tr><tr><td>SM_24</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_25</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_26</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_27</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_28</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_29</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_30</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_31</td><td>Capra sp.</td><td>Bovidae?</td></tr><tr><td>SM_32</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_33</td><td>Capra/Ovis</td><td></td></tr><tr><td>SM_34</td><td>Capra sp.</td><td>Capra nubiana</td></tr><tr><td>SM_35</td><td>Capra sp.</td><td>Caprinae</td></tr><tr><td>SM_36</td><td>Capra/Ovis</td><td>Bovidae?</td></tr><tr><td>SM_37</td><td>Capra sp.</td><td></td></tr><tr><td>SM_38</td><td>Capra sp.</td><td></td></tr><tr><td>SM4</td><td>Caprinae</td><td>Capra sp.</td></tr><tr><td>SM5</td><td>Caprinae</td><td>Capra sp.</td></tr><tr><td>SM8</td><td>Caprinae</td><td>Panthera sp.</td></tr><tr><td>SM10</td><td>Caprinae</td><td>Capra sp.</td></tr><tr><td>SM12</td><td>Caprinae</td><td>Gazella sp.</td></tr><tr><td>SM18</td><td>Caprinae</td><td>Capra sp./Antilopinae</td></tr></tbody></table>
<table><tr><td></td><td>SM23</td><td>Caprinae</td><td>Capra sp.</td></tr><tr><td></td><td>SM26</td><td>Caprinae</td><td>Capra sp.</td></tr><tr><td rowspan="9">SJE 270</td><td>SJE_10</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td>SJE_11</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td>SJE_12</td><td>Ovis aries</td><td>Ovis aries</td></tr><tr><td>SJE_13</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td>SJE_14</td><td>Ovis aries</td><td>Ovis aries</td></tr><tr><td>SJE_15</td><td>Ovis aries</td><td>Ovis aries</td></tr><tr><td>SJE_16</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td>SJE_17</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td>SJE_18</td><td>Ovis aries</td><td>Ovis aries</td></tr><tr><td>SJE_19</td><td>Capra/Ovis</td><td>Ovis aries</td></tr><tr><td rowspan="8">Leopard Cave</td><td>LC_166</td><td>Size 2/3 bovids</td><td>Raphicerus campestris</td></tr><tr><td>LC_167</td><td>Size 2/3 bovids</td><td></td></tr><tr><td>LC_168</td><td>Size 2/3 bovids</td><td>Antidorcas marsupialis</td></tr><tr><td>LC_169</td><td>Size 2/3 bovids</td><td>Bovidae?</td></tr><tr><td>LC_170</td><td>Size 2/3 bovids</td><td>Bovidae?</td></tr><tr><td>LC_171</td><td>Size 2/3 bovids</td><td>Bovidae?</td></tr><tr><td>LC_172</td><td>Size 2/3 bovids</td><td></td></tr><tr><td>LC_173</td><td>Size 2/3 bovids</td><td>Oreotragus oreotragus</td></tr><tr><td>LC_174</td><td>Size 2/3 bovids</td><td></td></tr></table>

One domestic goat, one gazelle (genus *Gazella*) and a wild goat were identified, along with two Caprinae and two Bovidae. The domestic and wild goat were discriminated using the single peptide variant described in ref. ⁹, located on the alpha 1 chain of COL1. Since our database contains only two species of genus *Capra*, the domestic goat *Capra hircus* and the wild Nubian ibex, *Capra nubiana*, we can confidently argue that the sample identified as *C. hircus* indeed belong to the latter species. However, the case of the wild *Capra* is different. Since *C. nubiana* is the only reference in our database, and even though the species is indeed ecologically distributed in the vicinity of Shkarāt Msaied, we cannot rule out the possibility of the specimen belonging to another species of ibex, such as *C. aegagrus*. We therefore need to expand our reference sequences of the genus in order to assign the archaeological specimen to one species or the other.
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All specimens sampled from the 270 site of the SJE were identified as domestic sheep.

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Samples all presented the alpha 1 chain of COL1 peptide that differs from all other species of either Caprinae or Antilopinae observed in both Barbary (*Ammotragus lervia*) and domestic sheep.

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Considering the provenance and dating of the samples, we can confidently rule out the possibility of them belonging to another species (or wild sheep for instance). Indeed, domestic sheep arrived in Sudan around the Vth millennium BCE and thus, the eventuality of the studied samples to belong to the wild species *A. lervia*, can be excluded.

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Out of nine samples from Leopard Cave, six presented preserved peptides. For three of them, the number of peptides was too low to go further than Family level identification. The other three were assigned to species level, all three to different wild antelopes species. One present the protein signature of a steenbok, *Raphicerus campestris*; another of a springbok *Antidorcas marsupialis* and finally, the last sample present peptides of a klipspringer, *Oreotragus oreotragus*. Springbok and klipspringer have specific peptides that allow to distinguish them from all other Antilopinae, when steenbok present a combination of COL1 peptides that makes the species rather easily distinguishable from the other antelopes. The identified samples present either the corresponding species-specific peptides or combination, allowing to be confident in the subsequent species assignation. Moreover, these three species are easily observable nowadays in southern Africa, and especially in Namibia where the site is located²⁷. Considering that the current environmental conditions were established after the end of the AHP, the probability for finding these species in a first century BCE archaeological site is more than probable.

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*Climatic influence over subsistence strategies in past Levantine and African populations*

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**Conclusion**

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Palaeoproteomics is rather new field in the landscape of archaeological sciences²⁸ and it might be tempting to see it as a more “efficient” way than comparative anatomy or morphometrics for identifying osteological remains. However, it might be necessary to remind several critical points

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when working with the molecular method: first, even if the method falls into the “-omics”, it
should be seen as part of archaeometry, and therefore as a methodology in the service of
archaeology and its -many- questions; second, without the initial morphological identification, we
would be completely blind in our analyses and even though it is technically feasible, it is also time
and resources consuming.

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Data availability

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Data will be made available upon publication.

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Acknowledgements

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I would firstly like to express my gratitude to the Fyssen Foundation for funding this post-doctoral project for two years. This has been an incredible chance and I am very grateful. I would also like to warmly thank L. Wastin for always being so prompt and kind at answering my many questions! I would also take the opportunity to thank my mentor from these two years of fellowship, Prof. M. Soressi and in particular her availability, support and encouragements all along the time of my Fyssen grant. Even though this paper is authored by my name only, this work would not have been possible without the multiple collaborations it has involved. I first thank my wonderful supervisors, Prof. Matthew J. Collins and Dr. Frido Welker for their invaluable help, support, patience and enthusiasm before, during and after the conduction of this Fyssen fellowship. The LC-MS/MS analysis of the here-discussed specimens, was made possible with funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement no. 948365 (PROSPER), awarded to F.W. My second set of warm gratitude goes to Dr. P. Bangsgaard and Dr L. Yeomans, who have been so kind at introducing me into the world of Levantine prehistory and for having spent numerous hours in the collections of Shkarāt Msaied and Shubayqa with me. D. Pleurdeau and J. Lesur are also acknowledged here, who respectively are director of the Franco-Namibian archaeological mission and zooarchaeologist of the latter. I thank the MAEDI for funding fieldwork in Namibia
297 and J. L. for granting me access to zooarchaeological material from Leopard Cave. Last but not

298 least, I would also like to thank our very pleasant intern, Pauline Poujois, for all the hard work she

299 has provided during her internship on the palaeoproteomics identification of specimens from all

300 the sites presented here.

301
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