Heritage Cases

Examining the spread of herding into southern Africa using proteomics and beads Overview The transition from foraging to food producing economies marks a major transformation for African Holocene populations. The domestication of plants and animals fundamentally changed human subsistence, allowing for resource scheduling and food surpluses, which facilitated population growth and social stratification. Therefore, documenting the early stages of this transition provides a crucial framework for our understanding of present lifeways, economies, and social interactions. Herd animals entered Africa via southwest Asia, and spread down the continent from north to south over a span of 5-6,000 years. Archaeological evidence for pastoralism includes domesticated fauna, ceramics, and informal lithic typologies. Beginning ~7-8 thousand years ago (ka), sheep and goats entered North Africa (di Lernia, 2013). There are also signs of cattle at this time, though it is debated whether they were brought from southwest Asia or were domesticated from wild North African aurochs (Gifford-Gonzalez & Hanotte, 2011; Marshall & Hildebrand, 2002). Lipid evidence has shown the use of milk in North Africa from 7200 BP (Dunne et al., 2012, 2018), suggesting that secondary animal products were already being exploited shortly after the introduction of domesticated ruminants. A few thousand years later, signs of herding lifeways emerge in eastern Africa. Monumental cemeteries and elaborate grave goods are found at Lothagam North in Turkana by 5ka (Hildebrand et al., 2018). This intense land use was facilitated by food production, which provided regular access to resources, thereby supporting larger population sizes. However, the transition to food production is particularly patchy in eastern Africa, perhaps owing to an uneven distribution of resources (Marshall & Hildebrand, 2002). Lipid signals for milk use in eastern Africa are evident by ca 3000 years ago (Grillo et al., 2020). This apparent time lag between the arrival of herding animals and the use of secondary products may be the result of poor preservation, the slow uptake of herding, or both. Finally, pastoralism enters southern Africa, with signs of herding by 2ka. Spoegrivier on the western cape of South Africa has sporadic sheep remains from layers dated to 2400-2100 BP (Vogel et al., 1997), including one directly dated bone at 2105±65 BP (Sealy & Yates, 1994). By 1300 BP, a dozen sites throughout Namibia, Botswana and South Africa had yielded evidence for domesticated caprines (Pleurdeau et al., 2012). South Africa in particular has been the focus of research for the spread of herding, and it has an abundance of well-researched Holocene sites. Thus, South Africa is an ideal location to study early contact between foragers and herders. The timing of pastoral animals entering southern Africa is well-established, however the mechanisms of their spread are debated. The two dominant hypotheses are a) the first livestock were brought by herders via waves of migration into southern Africa, or b) existing foragers obtained sheep or goats through trade, and practiced low-intensity pastoralism while maintaining their traditional lifeways (Sadr, 2013a, 2013b; Smith, 2021). In order to test these hypotheses, it is imperative to study forager sites that show early signs of contact with herding populations. Previous research has drawn upon the presence of domesticated faunal remains as evidence of contact with herding populations; however, it can be difficult to distinguish wild from domesticated fauna by morphology alone. A decade ago, two faunal teeth from Leopard Cave in Namibia were identified as sheep based on morphology. They were directly radiocarbon dated (2190±40 BP and 2270±60 BP), and reported as the earliest evidence for domesticated fauna in southern Africa (Pleurdeau et al., 2012). A later paper assessed the teeth using ZooMS (zooarchaeology by Mass Spectrometry), which uses molecular protein markers to make taxonomic identifications. The re-evaluation of these samples revealed them to belong to wild antelope (Le Meillour et al., 2020), illustrating that morphological identifications can be unreliable. In addition, sites left by foragers practicing small-scale pastoralism would not have abundant (domesticated) faunal remains (Sadr, 2013a). This presents a challenge: how can we identify early herder contact, without the typical traces of domesticated fauna? One solution is to leverage the power of proteomics to identify molecular traces of domesticates. Proteomics provides a method to identify tissue-specific proteins, which can often be taxonomically classified to the family, order, genus, or species level. It has proven to be an effective method to identify species tissues in human dental calculus (Bleasdale et al., 2021; Hendy, Warinner, et al., 2018; Warinner et al., 2015; Wilkin et al., 2020, 2021) and food preparation materials (Hendy, Colonese, et al., 2018). Furthermore, proteomic analysis requires very small samples (only a few milligrams), and when applied to residues the method should cause no damage to the original artifact. One type of artifact with appropriate residue, that was used by foragers and early herders alike, is ostrich eggshell (OES) beads. Beads of this type were first produced at least 42,000 years ago in southern Africa (d’Errico et al., 2012), and are still worn in some areas today, making them one of the most enduring craft traditions in the world. Many ethnographic OES beaded garments have visible residues, and the work by Miller (2019) documented that archaeological beads can yield similar residues. In addition to residues, OES beads also contain stylistic information that documents the introduction of herding into southern Africa. This stylistic shift was first documented by Jacobson (Jacobson, 1987b, 1987a), who noted that forager and herder assemblages in Namibia could be distinguished in part by their OES bead diameters. Forager assemblages had small consistently sized OES beads, while herding assemblages had larger and more variably sized beads (Jacobson, 1987b). Over the next decades, research across southern Africa confirmed this general pattern to be true (e.g. (Miller & Sawchuk, 2019; Sadr et al., 2003; Smith et al., 1991, 1995). Therefore, OES beads with residue can yield symbolic information, as well as proteomic data about pastoralism. In advance of this proposal, we conducted a small pilot study on the residue from an OES bead from a modern context. From this single sample, we were able to recover milk proteins from four different types of animals (sheep, goat, cow, and camel), muscle and blood proteins, and plant proteins from beans and corn. In addition to these traces, we also recovered many human keratins (skin and hair) and collagen (skin and bone) proteins, as well as those deriving from the ostrich eggshell itself. While previous proteomics research has focused primarily on dietary sources (from dental calculus or vessel residues), our evidence may not be directly related to consumption. The presence of animal proteins on OES beads seems unusual, however several ethnographic examples document the application of secondary animal products to the skin or hair. Women of the Ovahimba, forager-pastoralists in Namibia, are known for covering their hair and skin in otjise (a mix of clarified butter and red ochre) (Rifkin, 2015). Mao women and children, in Ethiopia, likewise cover their hair with a mixture of ochre and butter (Cerulli, 1956), as do Tutsi women from Burundi (Albert, 1963). These are often for cosmetic purposes, though informal interviews suggest the mixture may also protect against sunburns and insects (Rifkin et al., 2015). Through application to the body, caesins from dairy products can transfer directly onto clothing or other adornments, building up over time. These ethnographic examples suggest one way that milk casein could transfer onto an OES beads, but there may be a plethora of activities that leave the same result. In this study, we will test ethnographic and archaeological residues for animal proteins, as a new avenue for studying the spread of herding. This project will advance in two stages. First, we will sample residues from ethnographic OES bead collections stored at the Iziko Museum. These analyses will reveal whether the appropriate proteins preserve on the historic scale. If this is successful, we will sample residues from archaeological OES beads from foraging and herding sites, also stored at the Iziko Museum. Our specific questions for these stages are: Which animal species specifically were exploited for secondary resources? Did secondary resources from pastoral animals also transform other practices, not related to consumption? The ultimate goal of this study is to better understand the spread of herding practices in southern Africa, however these more targeted questions can also reveal new insights into behaviors of herders and foragers. The potential proteomic applications to archaeological questions are still being explored, and this study is a novel approach to examine the spread of herding in southern Africa. By examining residues for molecular traces, analysis can proceed even in the absence of identifiable faunal bones or teeth. The resolution available with protein sequences effectively removes any uncertainty about morphological identification. Further, proteomics is sensitive enough to detect trace amounts of secondary animal products. This evidence will be crucial to test whether foragers adopted low-intensity pastoralism in conjunction with traditional lifeways. Combining this evidence with established information for OES bead diameters, we can also test whether new cultural styles arrived alongside early domesticates. Our study unlocks new avenues to explore the long-standing debates about the spread of herding in South Africa. Methods Heat alteration (natural variations in shell colour; these are not residues): The natural colour of an ostrich eggshell is a creamy white. From personal observation, most modern and ethnographic OES beads retain this natural colour. Archaeological OES shells however may take on a variety of colours as the result of heat. This may even happen after deposition, if heat penetrates sediments and reaches buried OES. Light heat application creates yellow or orange tones. Higher temperatures create brown, and even black colours. Very high temperatures damage the shell structure, and may appear as iridescent blue. These heat-induced colours are not what we are seeking for this project. Identifying residues: For the present study, we are targeting visible residues on OES beads. Ethnographic documentation shows that OES beads were worn in a variety of ways: sewn flat onto garments, hung dangling as embellishments, woven into brick-like patterns, or worn in single strands as necklaces or bracelets. When worn for long periods, beads will naturally accumulate trace residues from daily life. These residues could include mixtures of butter and ochre that have been traditionally applied to the skin (for decoration, sunscreen, insect repellent). These residues may be adhering to the shell surface, caked within the bead aperture, or even trapped between two beads binding them together. Residues are not present on all OES beads. Of the more than 1300 completed archaeological beads JM has assessed in person, 546 had the presence of ochre, but only 178 had an additional residue. These residues may be yellow, orange, red, brown, black. For archaeological specimens, prioritise sampling beads that are bound together by residue. This binding will have protected the residues from degradation, thus better preserving the ancient proteins, and will be more likely to yield a positive result. If two beads are stuck together, photograph them together, then use a bamboo skewer to try and loosen them apart, while observing under the microscope. Do not apply pressure within the bead apertures, only from the outer rim. If they are able to be separated, photograph them again before residue sampling. If they do not come apart, do not force it, but note this in the word/excel file. Name the picture files with clear reference to the site, provenience, museum accession, and/or ethnographic information. At the end of name, use bracketed numbers to indicate photos of the same bead (e.g. HxJf2.lvl4.NW (1), HxJf2.lvl4.NW (2), HxJf2.lvl4.NW (3)). Enter the picture file name into a word or excel document, and document in words any museum information about the site, provenience, museum accession, and/or ethnographic information. If possible, also photograph the beads after residue sampling. Obtaining residue samples for proteomic analysis Equipment: Nitrile gloves Eppendorf tubes, tube rack Laboratory scale (preferably with sensitivity in milligrams) Computer/laptop Scalpel Aluminum foil Alcohol wipes Sampling protocol: Put on nitrile gloves before sampling any beads Sample one bead at a time (or two if they are stuck together) as to reduce sample to sample contamination Label the tube, making sure all of the bead information (as provided on photograph naming system) is included If you have a sensitive scale, weigh the tube and then TARE the scale (bring to 0) Enter the bead information into an excel table, name the entry using the same information as used in the photograph naming system Lay out a piece of clean aluminum foil under where the sampling will take place Place a folder square of foil on to the tube rack (See example below) Wrap the tube rack in foil (in case some residue goes flying, it won’t fall into another tube hole). Place a tube rack onto the foil and place the tube into one of the holes. Using a scalpel, carefully scrape the visible residue into the tube. Once the residue is in, check the foil to see if there is any other residue that landed outside of the tube. Close the tube and weigh it and note the final weight. Should be only the weight of the residue as the scale was zeroed out with the weight of the tube. For modern/ethnographic specimens, >5 milligrams of sample weight is sufficient. Ancient specimens will have poorer protein preservation, so obtain as much residue as possible. Wipe down the scalpel/dental pick and surfaces and then replace the foil for the next sample. Change your gloves or wipe them with an alcohol wipe between samples. Protein extraction (will be overseen by SW in Zurich) Bead residues will be scraped and collected from beads in the Iziko Museum in 2 mL clean Eppendorf tubes and shipped to the Institute of Evolutionary Medicine at the University of Zurich for protein extractions. Upon arrival, proteins from each residue will be extracted. Ancient samples will be processed in a dedicated ancient protein laboratory, while modern samples will be processed in a modern laboratory setting to avoid contamination. Samples will be treated with chemicals and reagents to denatured, reduced and alkylated, and then enzymatically digested with trypsin. Once digested, proteins will be purified and taken to the Functional Genomics Center Zurich for measurement via liquid chromatography, tandem mass spectrometry (LC-MS/MS). Following measurement, the data will be compared against existing and curated databases of annotated and translated proteins. 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