We present here evidence for an early Holocene case of decapitation in the New World (Burial 26), found in the rock shelter of Lapa do Santo in 2007. Lapa do Santo is an archaeological site located in the Lagoa Santa karst in east-central Brazil with evidence of human occupation dating as far back as 11.7–12.7 cal kyBP (95.4% interval). An ultra-filtered AMS age determination on a fragment of the sphenoid provided an age range of 9.1–9.4 cal kyBP (95.4% interval) for Burial 26. The interment was composed of an articulated cranium, mandible and first six cervical vertebrae. Cut marks with a v-shaped profile were observed in the mandible and sixth cervical vertebra. The right hand was amputated and laid over the left side of the face with distal phalanges pointing to the chin and the left hand was amputated and laid over the right side of the face with distal phalanges pointing to the forehead. Strontium analysis comparing Burial 26’s isotopic signature to other specimens from Lapa do Santo suggests this was a local member of the group. Therefore, we suggest a ritualized decapitation instead of trophy-taking, testifying for the sophistication of mortuary rituals among hunter-gatherers in the Americas during the early Archaic period. In the apparent absence of wealth goods or elaborated architecture, Lapa do Santo’s inhabitants seemed to use the human body to express their cosmological principles regarding death.

Competing interests: The authors of this manuscript have the following competing interests: Author Renato Kipnis is employed by Scientia Consultoria Científica Ltda. There are no patents, products in development or marketed products to declare. This does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

Funding: The excavation of Lapa do Santo was significantly funded by the São Paulo Research Foundation (FAPESP) through two research grants obtained by WN (99/0670-7 and 04/01321-6). FAPESP also provided a post-doctoral fellowship to RK (01/06881-1), PhD scholarship (08/58729-8 to DVB), and a MSc scholarship (08/51747-0 to AS). The Brazilian National Council for Scientific and Technological Development (CNPq) WAN with a productivity grant (300818/2007-6). The Max Planck Society provided a PhD scholarship for AS. DCSG acknowledges the Generalitat Valenciana (VALi+d APOSTD/2014/123) and the European Union (FP7/2007-2013 - MSCA-COFUND, n°245743) through a Braudel-IFER-FMSH in collaboration with the LAMPEA lab at the Université d'Aix-Marseille. RK is currently employed in a commercial company that provided support only in the form of salaries, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section”.

Copyright: © 2015 Strauss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Introduction

Few Amerindian habits impressed the European colonizers more than the taking and displaying of human body parts, especially when decapitation was involved [1]. Although disputed by some authors [2], it has become widely accepted that decapitation was common among Native Americans across the entire continent and the archaeological evidence confirms that the practice has deep chronological roots [3]. In South America, the oldest decapitation is reported for the Andean region and dates to ca. 3000 BP at the site of Asia 1, Peru. Since all other South American archaeological cases occur in the Andes (e.g., Nazca, Moche, Wari, Tiwanaco) it was assumed that decapitation was an Andean phenomenon in both its origins and in its most unambiguous expression. In the present contribution we review the available evidence on decapitation in South America and report the discovery in east-central Brazil of a case of human decapitation directly dated to 9127–9438 cal BP (all chronological ranges reported here are based on a 95.4% interval). Excavated at the Lapa do Santo rock shelter in Lagoa Santa, Central Brazil, this is the oldest case of decapitation found in the New World, leading to a re-evaluation of the previous interpretations of this practice, particularly with regards to its origins and geographic dispersion.

Disembodied heads and decapitation in South America In South America, the practice of decapitation is reported in both the ethnographic and archaeological literature. Tupinamba groups from coastal Brazil, famous for their rituals, including exo-cannibalism [4], used to collect body parts, including heads, as war trophies [5]. The Arara Indians, in the Brazilian Amazon, performed the Ieipari ceremony in which the cranium of the defeated enemy, also used as a musical instrument, was displayed on the top of a pole [6]. Among the Uru-Uru Chipayas, in Bolivia, skulls were used as part of a syncretic Christian liturgy [7]. Among the Inca, decapitation was a common means of establishing and reinforcing positions of status and power. The head of important enemies were turned into trophies and the skulls into drinking jars in a clear message of military supremacy [8]. However, among the ethnographic examples in which decapitation was prominent, the trophy heads made by the Munduruku and Jivaros are the most famous. The Munduruku Indians from the Tapajós River in northern Brazil used to behead the defeated enemy immediately after death [9–16]. The spine was sectioned near the foramen magnum and the head removed. The internal muscles, brain, eyes and tongue were then removed [16] and the head mummified through immersion in hot oil and subsequent smoking [15]. The trophy would be brought to the village and designated as the focus of a series of ceremonies over several years. At first, the ritual involved the cultural appropriation of the trophy by adding ornaments and tattoos to it. Subsequently, as the power of the head faded away, the skin and the ornaments were removed. Finally, the dentition was extracted from the skull and attached to a cotton belt that would remain with the owner of the head indefinitely, while the skull itself would be left in some corner of his habitation to be forgotten [13]. For the Munduruku, the head of the defeated enemies clearly served the role of a war trophy and symbol of belligerent superiority [14,16]. The head was sometimes positioned on the end of a long pole [11] or carried by strings attached to the cranium, clearly characterizing the importance of public display [13]. At the same time, the head was an empowering object capable of increasing success in hunting and incorporating a female semiology of fertility. Although the Munduruku would remove other body parts of their own dead, they only produced trophy heads with enemies. The enemy’s children were commonly captured and incorporated into the community but never used to generate trophy heads [15]. In Ecuador, the Jivaros produced shrunken heads (tsantsa) from dead enemies. The head was quickly removed from the body with a “v-shaped” incision made above the clavicles. Later, in a safer location, the skin of the head was removed from the skull. This scalp was then washed with boiling water for 15–30 minutes resulting in a 50% reduction of the head’s dimensions. The shrunken head was equipped with cords to facilitate transport and handling [17]. Jivaro’s tsantsa had the power to imprison the soul of the dead enemy precluding it from perpetrating any vengeance [18–21] (but see Fausto and Rodgers (1999) [22] for a broader perspective on the meaning of tsantsa). Some authors suggested that the practices of head-hunting were not a truly indigenous phenomena but a result of the western commercial demands for trophy heads [2]. However, although the European market certainly catalyzed the practice of head-hunting in South America, leading to a transformation of the reasoning behind it, archaeological evidence confirms that similar practices were common long before the arrival of the European colonizers [23,24]. The Chimus (900AD-1470AD) in Peru incorporated decapitation as a standard procedure in human sacrifices. In the Huaca 1 Complex of Pacatnamu, the mutilated skeletons of 14 individuals were found within a defensive trench of three meters deep. The ubiquitous presence of young males, many of which were tied and left exposed after death, suggests that these were sacrificed defeated warriors. Among the diverse types of mutilation to which they were subjected, decapitation was one of them [25]. Chimu human sacrifices also took place in the Temple of the Sacred Stone in Tucume [26]. Osteological analysis suggests a ritual sequence starting with throat cutting followed by heart extraction and ending with decapitation (a total of 72 individuals presented explicit osteological evidence of decapitation). The severed heads were buried in the same pit with the correspondent headless body. The presence of children among the sacrificed individuals makes it unlikely that these were defeated warriors, pointing to a different sort of sacrificial ritual compared to Pacatnamu. Disembodied skulls of both adults and children were also used as dedicatory offerings and were included in tombs as individualized objects wrapped in textile accompanying the remains of sacrificed individuals [27]. Among the Chachapoyas from the Peruvian Amazon, disembodied skulls are found on top of elaborated anthropomorphic sarcophagi used as funerary monuments (e.g., Karajia) [28]. Disembodied skulls were also found in the walled city of Kuelap. In either case, detailed osteological analyses are not available, and the interpretations about the disembodied skulls range from them being considered simply delayed burials to being war trophies [29,30]. In the Wari Empire (600AD-1100AD), in southern Peru, disembodied heads were transformed into trophies and played a central role in ritualistic traditions [31]. In the site of Conchopata at least 31 trophy heads were recovered from ritual structures (EA143 and EA72) [32]. The skulls show drill holes near the bregma and, sometimes, at the occipital bone [7]. The demographic profile of Wari’s trophy heads shows a predominance of male individuals of all ages, including children [32]. Isotopic analyses suggest a non-local origin for some of the decapitated individuals and osteological evidence points to high levels of inter-personal violence [33]. Altogether, and including the practice of child abduction, decapitation in Wari is understood as a strategy adopted by military and ritual elites to legitimate their authority in the eyes of their enemies. However, not all disembodied skulls found in Wari contexts were trophy heads. In the site of Wari, a non-modified skull wrapped in cloth and pinned with four copper tupus was found under the floor of an architectural construction and was probably a dedicatory offering [34]. During the Tiwanaku period (300AD-1000AD), in the Titicaca basin in Bolivia, scenes involving decapitation or disembodied human heads were a common theme in the etchings of their rock sculptures and panels [35]. The osteoarchaeological record for the corresponding period confirms that these were indeed a real practice. In the high-status residential complex of Putuni (west to the Kalasasaya) a total of fifteen articulated and disarticulated individuals were buried as a dedicatory offering to the building, including a disembodied human skull [36,37]. In the pyramid of Akapana, a site of communal ritual in the core of the Tiwanaku complex, isolated human bones or partially articulated skeletons were recovered from the base of some of the excavated pyramid’s walls. Several skulls were found isolated (in one case, three skulls were grouped together), and eighteen skeletons lacked their skulls [35]. In the absence of cut marks, the skulls must have been removed from the skeleton in secondary contexts, which has been suggested to be a part of an “esoteric cult of the head” [36]. In the site of Wata Wata, human heads were presented as dedicatory offerings [38]. Three disembodied skulls were found displaying different signs of perimortem violence, including beheading, cranial and facial fracturing, defleshing, jaw removal, and possible eye extraction. The extreme violence characterizing these findings suggests this was done to remove power from those individuals and legitimize the authority of the expanding influence of Tiwanaku into the region [38]. Head removal is a common theme in Moche (100AD-700AD) iconography, in northern Peru [39–42], and archaeological and osteological evidence abound to confirm this was not merely figurative but a real practice. In Plaza 3A and Plaza 3C of Huaca de la Luna [34,43–50], articulated severed heads and decapitated bodies were found in a context of generalized sacrifice of defeated warriors [49,51,52]. In Plaza 3C, in addition to the ritual of sacrifice, the severed skulls were also subject to both peri and postmortem intentional manipulation which could imply some sort of ritual cannibalism [49]. Nearby, at the complex named ZUM 8, two disembodied skulls altered to function as jars show the diversity of purposes head removal had among the Moche, going beyond the immediate needs of sacrificing defeated warriors [45,48]. In Huaca Dos Cabezas, a cache of 18 severed skulls with cut marks on the anterior portion of the cervical vertebrae was found [41]. Nearby, the complete skeleton of a tall man was found with a tumi (ceremonial axe characterized by a semi-circular blade) in his left hand and a pottery human head in his right hand, suggesting he was an actual decapitator. In San José del Moro (tomb M-U1221), seven individuals were buried together and eight disembodied skulls were placed on top of the burial [53]. The presence of several pottery artifacts related to shamanistic activities [54] suggests that the skulls are grave offerings, possibly holding some supernatural power. During the Moche period, human bones from reopened tombs were used as dedicatory offerings. Skulls were the most commonly selected anatomical part and therefore not all disembodied heads or headless bodies are a product of decapitation (i.e., perimortem removal of the head) [55,56]. In addition to humans, llamas’ decapitated heads were also included in tombs and graves (e.g., Huaca Rajada Sipán [57] and Dos Cabezas Tomb 2 [58,59]). During the earlier Gallinazo period, in Huacas de Moche, a single case of skull removal is known for burial G2. The skull was removed and replaced by a pottery jar with the figure of a human head stamped on it. It is not possible, however, to determine if this was a peri or postmortem removal [60]. The Nazca (100BC-800AD), in southern coastal Peru, produced elaborate trophy heads that were characterized by a drill hole in the front of the head and an enlargement of the foramen magnum [61–67]. The lips and eyes were usually sealed with spines and the head was equipped with a carrying string [34]. The available iconography and the predominance of adult males among trophy heads [34] indicates that decapitation took place in the battlefield, and that the severed head functioned as a trophy of war. Isotopic analysis indicates that these were intra-valley battles involving local Nazca warriors [68,69]. The heads were commonly interred in caches in numbers ranging from three to groups of 40 or more [66,70]. Therefore, their significance went far beyond signaling military supremacy, and it is assumed they were a central element in rituals aiming to control the forces of nature, particularly concerning crop fertility [64,71–73]. In the site of Chavín de Huantar (1200BC-500BC), in the northern Peruvian highlands, four disembodied skulls were found on a platform (Urabarriu phase, 900BC-500BC). Since the skulls were from an old adult male, a young adult male, an adolescent female and an infant, they are sometimes thought to represent an extended family [74]. The skulls show no signs of modification. Another isolated skull in Chavin de Huantar was recovered from the Galeria de Ofrendas and, although a precise date is not available, this could represent the earliest modified trophy head in the Andes [31,75]. During the Formative period, five disembodied skulls were found in the site of Wichquana, in Peru. Buried in individual pits within a ceremonial structure these skulls still had the cervical vertebrae articulated to them supporting the interpretation that they were decapitated when soft tissue was still present, which suggests that they were sacrificed [76]. The site of Asia 1 [77], in central coastal Peru, is usually considered the oldest possible case of decapitation in South America (ca. 3000 BP) [31,45]. However, in the absence of a detailed osteological description accounting for the presence of cut marks in the cranium and associated cervical vertebrae, it is not possible to determine if this in indeed a case of decapitation. The findings consisted of three wrapped bundles containing a total of eight disembodied heads that were found in separate graves. In addition, two headless bodies were also present. One skull had cut marks on the frontal bone that were interpreted as resulting from the scalping of the face [77]. The funerary context included several textiles, a necklace of bone disks, shell pendants, a bone pin, feathers, red pigment and an “engraved tray holding a mirror” [77]. Such an elaborate treatment indicates that the practice of removing skulls in Asia 1 could have been reserved to individuals of special status. Altogether, and considering the lack of any further modification to the skulls, it seems they were less likely trophy heads, but instead venerated members of this society. Accordingly, it has been suggested that the flayed skull might represent a local individual who was mutilated somewhere else and later brought back to Asia 1 [34]. The site of Asia 1 is commonly mentioned as the first appearance of disembodied heads in the South American archaeological record. However, Aguazuque (5025–2725 BP) might be a better candidate. Located in Sabana de Bogotá, Colombia, at least two cases of disembodied skulls and one headless body were identified among a total of 59 burials. The site presents one of the most elaborate funerary records of the Archaic period and the disembodiment of the skulls were part of a broader mortuary context that was focused on the manipulation of bones and body parts [78–80]. Long bones, for example, were sectioned into diaphyses and epiphyses and further painted with geometric motifs. Once again, in the absence of a detailed osteological description accounting for the presence or absence of cut marks, it is not possible to determine if these were true cases of decapitations. Notwithstanding, the fact that one of the disembodied skulls was articulated with the cervical vertebrae is highly suggestive that the removal occurred while soft tissue was still present and therefore characterizes a case of decapitation. In Brazil, as far as we could determine, there is only one single case of a possible decapitation reported for the entire pre-history of the country. This finding comes from the shellmound of Forte Marechal Luz [81], but no detailed chronology or osteological descriptions are available. Therefore, it is clear that almost all reported archaeological cases of decapitation and disembodied heads in South America are concentrated in the Andean region [82]. For this reason it is commonly assumed that this was an Andean phenomenon in both its origins and in its most unambiguous expression [2,24,40,72]. The purpose of the present publication is to contribute to the field by reporting an early Holocene case of decapitation found in Lagoa Santa, east-central Brazil.

The Lagoa Santa region Lagoa Santa is an environmentally protected area comprising 360 km2 located in east-central Brazil (Fig 1). The vegetation is dominated by cerrado (a savannah-like vegetation) and semi-deciduous forest. The rivers Mocambo, Samambaia, Jaguara and Gordura make up a tributary net that flows west to east towards the Velhas River, the main river in the area. Geomorphologically, Lagoa Santa is a karstic terrain that can be divided into four distinct domains [83]: 1) below 660 meters above sea level (masl), the terrain is characterized by a fluvial plain connected with the regional base level (Velhas River); 2) between 660 and 750 masl, there is a karstic plain with dolines and lakes 3) between 750 and 850 masl, there are karstic plateaus characterized by the presence of limestone outcrops (reaching up to 75 meters in height); 4) above 850 masl, residual peaks composed of the non-soluble meta-sedimentary rocks from the Serra da Santa Helena Formation. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 1. Map of South America. The location of Lagoa Santa is indicated by the dot. https://doi.org/10.1371/journal.pone.0137456.g001 The region’s geology comprises the Sete Lagoas Formation and the Serra da Santa Helena Formation, both part of the Upper Proterozoic meta-sediments of the Bambuí Group [84] of the São Francisco craton. This cratonic cover metamorphosed during the Brazilian Cycle (700–450 million years ago) in a process that resulted in planar structures, such as lineation and foliation, and sub-vertical structures, such as normal and revert faults. The combination of these structures provides the path for the geomorphologic evolution that leads to the rock shelter configurations found in the region. The regional rock shelters and outcrops are developed in the limestone of the Sete Lagoas Formation. More specifically, Lapa do Santo rock shelter developed in the Member Pedro Leopoldo that is composed of very pure limestone with more than 90% calcite [84]. The annual mean temperature is 23°C, with lower temperatures (11°C) occurring between June and July and higher temperatures (35°C) occurring between October and November. The mean humidity is around 65% in the dry season, from May to September, and around 85% in the rainy season, from November to April, with a pluviometric mean of 1,400 mm/year. The major climatic characteristic of this region is the high concentration of rain during the rainy season (93% of total volume). When evaporation is analyzed, the region presents an annual deficit of 176 mm [85]. Despite these particular variations, the regional climate is classified as tropical, with a rainy summer and a dry winter [86]. During the dry period, the above ground water sources can become very scarce, although underground drainages are capable of preserving the discharge in the Velhas River. The first prehistoric human bones in Lagoa Santa were found by the Danish naturalist Peter Lund between 1835 and 1844 [87–91]. Due to the putative coexistence of humans and megafauna, Lagoa Santa became a well-known region for 19th century scholars [92–95]. During the 20th century different teams went to the region to find evidence that could confirm the coexistence hypothesis [96–100]. As a result of more than 170 years of excavations, a large collection of early Holocene skeletons was gathered [101–103]. However, all those excavations were done without proper documentation and therefore they lack detailed contextual information. Coordinated by WAN and funded by the São Paulo State Grant Foundation (FAPESP), the project “Origins and Microevolution of Man in America: a Paleoanthropological Approach” aimed to overcome this problem by identifying and excavating new sites in the Lagoa Santa region. Lapa do Santo was excavated within the midst of these efforts.

Lapa do Santo archaeological record Lapa do Santo (“Saint’s rock shelter”) is an archaeological site located in the northern part of the Lagoa Santa karst (city of Matozinhos, state of Minas Gerais, Brazil, coordinates of the site 19°28'37.86"S and 44°2'17.00"W) (Fig 2) [104]. The site has an associated sheltered area of ca. 1300 m2 (Fig 3a) developed under the negative slope of a 30-meter high limestone massif (Fig 4). The southern region of the sheltered area has a relatively flat, high and dry area located immediately in front of the cave’s entrance. The floor of the shelter has a strong descending inclination towards the north, which becomes flat again near a natural sinkhole located in the northern extreme of the sheltered area. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 2. Map of the Lagoa Santa region. The dots indicate all early Holocene sites where human skeletal remains were found. https://doi.org/10.1371/journal.pone.0137456.g002 PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 3. Plan of Lapa do Santo. a) The grid corresponds to 1 square meter units. Purple and orange areas indicate excavated surfaces. Pink area indicates the main excavation area (MEA). The bedrock is depicted in gray, and secondary deposits such as breccia and stalagmites in beige. The topographic lines are 10 cm equidistant and the associated values correspond to the z-value of the site coordinate system. b) Detail of the MEA area. Black disk and the black arrow indicate the position of Burial 26. Numbers in the lower and right margin indicate the x and y values, respectively, from the coordinate system of site. https://doi.org/10.1371/journal.pone.0137456.g003 PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 4. Lapa do Santo massif. a) Aerial view of the massif in which the rock shelter is located; b) ground view of the massif, the site is located just behind the vegetation. The individual in panel 4b has given written informed consent (as outlined in PLOS consent form) to publish this image. https://doi.org/10.1371/journal.pone.0137456.g004 Excavations took place between 2001 and 2009 under the coordination of RK, AGMA and DVB. Starting in 2001 several units were opened in distinct areas of the shelter, which showed that the richest archaeological deposits were located in its southern part, immediately in front of the cave`s entrance. An ample excavation surface was established in this region, becoming the Main Excavation Area (MEA, the highlighted area in Fig 3b). Excavations ended in 2009 when, in accordance to Brazilian laws, the excavated area was filled with sediments to reconstitute the original topography of the shelter`s floor. In 2011 a new excavation area was opened as part of a new research project (“The Mortuary Rituals of the First Americans”), coordinated by AS, and a joint venture between the Department of Human Evolution of the Max Planck Institute for Evolutionary Anthropology (Germany) and the Laboratório de Estudos Evolutivos Humanos da Universidade de São Paulo (Brazil). The chronology of the site is based on OSL and radiocarbon dates and points to the human presence starting at 12.7–11.7 cal kyBP (95.4% interval). Three distinct periods of occupation were determined based on the radiocarbon dates. Lapa do Santo’s Period 1 (LSP-1) starts at 12.7 cal kyBP and ends at 7.9 cal kyBP; Lapa do Santo’s Period 2 (LSP-2) starts at 5.4 cal kyBP and ends at 3.9 cal kyBP; Lapa do Santo’s Period 3 (LSP-3) starts at 2.1 cal kyBP and ends at 0.0 cal kyBP (see [105] for a detailed account on the site chronology). Lithic technology [106,107], zooarchaeology [108], and multi-isotopic analyses [109] indicate typical early Archaic groups of hunter-gathers with low mobility and a subsistence strategy focused on gathering plant foods and hunting small and mid-sized mammals [104]. Together with reported frequencies of dental caries comparable to those observed among agricultural populations [103,110,111], the emerging picture for Lagoa Santa during early Holocene is an economy structured around staple carbohydrates complemented by hunting of small and mid-sized animals. Formation process analysis characterizes the Lapa do Santo’s deposits as mainly anthropogenic and composed of repeated combustion activities, indicating an intense occupation of the same locality. The oldest evidence of rock art in South America, including a pictorial tradition that depicts phallic imagery, was also found engraved on the bedrock of Lapa do Santo, under four meters of excavated sediments [112]. A total of 26 human burials dating to early Holocene (LSP-1) were exhumed from Lapa do Santo between 2001 and 2009 (see [105] for a comprehensive depiction of the mortuary practices in Lapa do Santo and the Lagoa Santa region). The use of Lapa do Santo as an interment ground started between 10.3–10.6 cal kyBP. Lapa do Santo Mortuary Pattern 1 (LSMP-1) was characterized by articulated skeletons in flexed position buried in shallow graves and covered by limestone blocks and occurred between 9.7–10.6 cal kyBP. Lapa do Santo Mortuary Pattern 2 (LSMP-2) took place between 9.4–9.6 cal kyBP and was characterized by an emphasis on the reduction of the body by means of mutilation, defleshing, tooth removal and exposure to fire followed by the secondary burial of the remains according to specific rules. The case of decapitation reported here is part of LSMP-2. In the absence of monumental architecture or grave goods, during this period the local groups elaborated their funerary rituals through the use of the human body as a symbol [113]. Lapa do Santo Mortuary Pattern 3 (LSMP-3) took place between 8.2–8.6 cal kyBP when another change occurred whereby pits were instead filled with disarticulated bones of a single individual without signs of body manipulation. In some cases the long bones were highly comminuted in order to fit the small pit.

Dating A fragment of the sphenoid from Burial 26 was pretreated at the Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany, using the method described by Talamo and Richards [115]. The outer surface of the bone sample was first cleaned by a shot blaster and then 500mg of bone powder was removed. The sample was then decalcified in 0.5M aq. HCl at room temperature for about 4 hours, until no CO 2 effervescence was observed. 0.1M aq. NaOH was added for 30 minutes to remove humic acids. The NaOH step was followed by a final 0.5M HCl step for 15 minutes. The resulting solid was gelatinized in a pH3 solution in a heater block at 75°C for 20h, following Longin et al., [116]. The gelatin was then filtered in an Ezee-FilterTM (Elkay Laboratory Products (UK) Ltd.) to remove small (<8 μm) particles, and then ultra-filtered with Sartorius “Vivaspin 15” 30 KDa ultra-filters [117]. Prior to use, the filter was cleaned to remove carbon containing humectants [118]. The sample was then lyophilized for 48 hours. C:N ratios, %C, %N, δ13C and δ15N values were measured at the MPI-EVA using a Thermo Finnigan Flash EA coupled to a Delta V isotope ratio mass spectrometer. For acceptable quality collagen, the atomic C:N ratio should be between 2.9 and 3.4 and a collagen yield of more than 1% of weight [119–121]. For Burial 26, the isotopic results, C:N ratios and collagen values are well within the accepted ranges (Table 1). The samples provided enough collagen for radiocarbon dating and were sent to the Klaus-Tschira-AMS facility of the Curt-Engelhorn Centre in Mannheim (MAMS), Germany, where they were graphitized and dated [122]. The resulting date was corrected for a residual preparation background estimated from pretreated 14C-free bone samples, kindly provided by the Oxford Radiocarbon Accelerator Unit (ORAU). The radiocarbon dates were calibrated using OxCal 4.1 [123] and SHcal13 [124] (Table 1). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Table 1. Isotopic values, C:N ratios, amount of collagen extracted (%Coll) refer to the >30 kDa fraction. δ13C values are reported relative to the vPDB standard and δ15N values are reported relative to the AIR. https://doi.org/10.1371/journal.pone.0137456.t001 In addition to the date obtained at the MPI-EVA, another date was obtained from Beta Analytic. Despite the excellent preservation of Burial 26, small fragments of bone from the nasal cavity and sphenoid could not be reassembled to the cranium. A portion of 8.707 grams of this highly fragmented material was sent to Beta Analytic Laboratories in Miami in December 2008 (Beta# 253511). The final age result was 8540±50 14C BP, the calibration age range was obtained with OxCal 4.1 [123] and SH13 [124] which resulted in an interval between 9.47 and 9.54 cal kyBP (68.2%) and between 9.43 and 9.55 cal kyBP (95.4%). Since the date from the Beta Analytic did not follow the same quality control parameters we adopted for bones at the MPI-EVA, we consider the latter as more accurate for dating Burial 26.