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Isotopic and microbotanical insights into Iron Age agricultural reliance in the Central African rainforest

PMCID: PMC7591565

PMID: 33110164

Abstract

The emergence of agriculture in Central Africa has previously been associated with the migration of Bantu-speaking populations during an anthropogenic or climate-driven ‘opening’ of the rainforest. However, such models are based on assumptions of environmental requirements of key crops (e.g. Pennisetum glaucum ) and direct insights into human dietary reliance remain absent. Here, we utilise stable isotope analysis (δ 13 C, δ 15 N, δ 18 O) of human and animal remains and charred food remains, as well as plant microparticles from dental calculus, to assess the importance of incoming crops in the Congo Basin. Our data, spanning the early Iron Age to recent history, reveals variation in the adoption of cereals, with a persistent focus on forest and freshwater resources in some areas. These data provide new dietary evidence and document the longevity of mosaic subsistence strategies in the region. Bleasdale et al. examine the introduction of agricultural crops in the Congo Basin with stable isotope analysis of human and animal remains, charred food remains, and plant microparticles from dental calculus. Their findings reveal variation in the adoption of cereals from the early Iron Age, and provide long-term insights into changing human reliance on different resources.

Full Text

For the past half a century, if not longer, the processes for the dispersal of Bantu-speaking communities from Western Central Africa have been a major focus of African archaeological, linguistic, and genetic research. While there has been an increasing departure from notions of a single sweeping ‘Bantu Expansion’, the degree to which the movement of people, languages, and the emergence of farming are linked across Africa continues to be forcefully debated. Central Africa is at a key location for developing existing models for the spread of farming yet investigations of the emergence of food production, particularly in the rainforest, have been limited. Assumptions that tropical rainforests represent substantial barriers to agriculturalists have been used to rationalise a relatively late arrival of farming in the region, c. 2500 years ago, during a period of climate- or human-induced deforestation (Supplementary Note 1). However, unlike other parts of Africa, there have been few studies directly testing changes in human dietary reliance on agricultural crops, relative to local freshwater, bushmeat, and tropical forest plant resources, from the first arrival of domesticates in the region through to the present day.
Here, we present new, direct dietary information from Iron Age sites in the Democratic Republic of Congo (DRC) using the stable carbon (δ13C), nitrogen (δ15N), and oxygen (δ18O) isotope analysis of human and animal remains. Isotopic results were obtained for human burials from the sites of Imbonga (IMB; n = 1), Longa (LON; n = 1), Bolondo (BLD; n = 18), and Matangai Turu Northwest (MTNW; n = 1). In addition, bone collagen (n = 10) and enamel (n = 6) were analysed for a range of fauna from BLD to create an isotopic baseline. The sites studied represent different geographic and temporal contexts (Fig. 1). IMB is the type site for the earliest pottery tradition of the central equatorial rainforest and the individual analysed, indirectly dated to ~2050 BP (Supplementary Note 2), would have been a member of a group representing already established agriculture in the region after its initial settlement by sedentary immigrant populations a few centuries earlier. In contrast, individuals from LON and BLD represent subsistence practices during the Late Iron Age, when populations were spreading further across the Congo Basin. Finally, isotopic results from the individual from MTNW, previously identified as a likely hunter-gatherer, offers new evidence about the intricacies of subsistence, cultural, and genetic identities further to the eastern edge of the Basin (Supplementary Note 2). Collectively, the samples analysed span the period following the first arrival of food producers in this region (~2050 BP) through to relatively recent occupation (~130 BP; Supplementary Note 2, Supplementary Tables 1 and 2, and Supplementary Figs. 1, 3–7).
δ13C analysis of human tissues has long been demonstrated to provide insights into reliance on plants with different photosynthetic pathways (namely C4 versus C3) and their animal consumers (Supplementary Note 3). Significantly, in Central Africa, wild, as well as potentially domesticated (e.g., yams), forest plants are C3, while incoming cereal crops (e.g., pearl millet, sorghum, and, for later periods, maize) are C4. δ13C measurements of wild plants and animals from the rainforests of the DRC show that these forests are largely composed of C3 vegetation. Moreover, they show a recognisable ‘canopy effect’ on this C3 vegetation that results in lower δ13C among plants, and their animal consumers, living under dense canopies compared to those living in more open areas, something that has been well-documented in many other tropical regions.
The sites of IMB, LON, and BLD are located on tributaries of the Congo River in the western DRC (Fig. 1 and Supplementary Note 2) an area presently covered in dense C3-dominated evergreen and semi-deciduous forest. Stable carbon measurements of faunal tooth enamel from BLD, which largely reflect the proportions of C3/C4 plants consumed, reflect local palaeoecology, as well as providing baseline values for human diet. The final site, MTNW, is situated in the closed-canopy forest of the Ituri Region of the Northeast Congo Basin with palaeoenviromental proxies, suggesting a predominance of tropical forest tree taxa during the time of occupation.
δ15N analysis provides insights into the positions of humans within their trophic web and their potential consumption of aquatic resources, while δ18O measurements reflect water sources and environments (see “Methods” section or Supplementary Note 3 for full details). To the best of our knowledge, this is the first time that multi-tissue stable isotope analysis of prehistoric humans and animals has been applied in the Congo Basin, in order to provide long-term insights into changing human reliance on different resources. We also present results of δ13C and δ15N analysis of charred lumps interpreted as food residues from BLD, as well as microparticle analysis of dental calculus recovered from MTNW, to obtain more detailed insights into prepared and consumed foods, respectively.
Bone collagen results were assessed using established indicators of preservation, including a C/N ratio between 2.9–3.6, %C of ca.15–48%, and %N of ca. 5–17% (ref. ). Two results generated for human burials from BLD were excluded from final analysis as they produced a %N < 5 and a %C < 15 % (Supplementary Table 3).
The fauna from BLD fall broadly into four groups: wild browsers (antelope and duiker, n = 2), domesticated browsers (goats, n = 2), mammalian carnivores (n = 2), and aquatic species (fish and crocodile, n = 4). δ13C values from BLD mammals (n = 6) are consistent with a largely C3-based diet with measurements ranging from −23.7 to −18.2‰, although the goats, the dog, and the fox-sized carnivore could potentially have some C4 component to the diet (Fig. 2 and Supplementary Table 3). The common duiker (Sylvicapra grimmia, BLD 83/2-8) and small antelope (BLD 83/2-6 + 7) have δ15N values (5.9 and 7.3‰, respectively) consistent with herbivorous diets. In contrast, the fox-sized carnivore (13.7‰) and dog (12.3‰) display higher δ15N that is consistent with consumption of animal protein. Three of the aquatic species sampled had higher δ15N values than the catfish (δ15N 9.4‰), the crocodiles gave δ15N of 11.1 and 11.4‰, and the bichir (Polypterus sp.), a fish known to consume other fish and small vertebrates, produced a δ15N measurement of 12.7‰. The accompanying δ13C value of −24.7‰ for the bichir somewhat overlaps with that expected for C3 plants and animals, demonstrating the need to examine both δ15N and δ13C, in order to separate freshwater fish and forest resources.
The δ13C values for the bone collagen of humans from BLD (n = 11) dating to between 1426 and 1942 years cal. AD (Supplementary Figs. 3–7) are quite variable, ranging from −21.0 to −16.3‰. Alongside the individual from LON (directly dated to 1642—after 1938 cal. AD), these values are generally consistent with reliance on C3 plants, C3 plant-consuming wild and domestic animals, and freshwater resources. The average δ15N value of 14.5‰ for the human individuals from BLD in comparison to the average for the goats (9.9‰) is within the range of values reported for diet–collagen spacing, potentially indicating reliance on these domesticates. However, δ15N values ranging from 13.4 to 16.9‰, and three individuals with δ15N values higher than 15‰, as well as the riverine setting and modern and historical evidence that the site was a fishing camp (Supplementary Note 2), indicate that freshwater fish was also a major part of human diets at BLD. Nevertheless, it is still evident that all humans and domestic animals, as well as the fox-sized carnivore, have higher δ13C values than the available wild C3 or freshwater fauna, indicating the consumption of an additional resource enriched in δ13C.
A visible negative correlation between human δ15N and δ13C suggests that this was, in fact, a plant food (Fig. 2), although a Pearson’s rank test (correlation coefficient = −0.600, d.f. = 9, p = 0.05) produced results at the limit of statistical significance. This is likely a product of the small sample sizes available. The metabolic routing bias of bone collagen δ13C towards protein components of the diet (at the expense of carbohydrate and fat inputs), and importance of high protein freshwater fish in diets of the measured individuals, means that we can expect that consumption of this low-protein plant resource was actually greater than it appears in the bone collagen values of Fig. 2. Thus, although somewhat underrepresented in the present bone collagen isotope results, there is a signal indicative of some C4 plant component, which must have contributed to the diet of Late Iron Age humans and domestic animals in the Inner Congo Basin from at least the 15th century cal. AD onwards.
Tooth enamel is widely regarded as the archaeological material of choice in the tropics. Stable carbon and oxygen isotopes of tooth enamel have been shown to robustly preserve ecological variation, even in tropical regions, from the Miocene to the Late Pleistocene. Tooth enamel was sampled from human second and third molars enabling the investigation of diet during late childhood–early adolescence. Furthermore, given that tooth enamel δ13C reflects the isotopic composition of the whole diet (including carbohydrates, fats, and proteins), as opposed to the dominance of the protein signal in bone collagen, tooth enamel δ13C enables additional dietary resolution (see Supplementary Note 3).
Faunal and human δ13C enamel results from IMB, LON, BLD, and MTNW (Fig. 3 and Supplementary Table 4) reveal diverse subsistence strategies that can be divided into three broad groups: wild forest resources, forest and freshwater resources, and more open environments, including C4 food sources. The individual from the earliest site mentioned here, IMB, has a δ13C value of −14.1‰ indicating reliance on a mixture of tropical rainforest and freshwater resources, while the individual from LON shows clear evidence for reliance on tropical rainforest resources with a δ13C value of −15.1‰. Two individuals from BLD gave δ13C values of −14.7 and −13.6‰ also suggesting a heavy reliance on tropical or freshwater resources. The δ13C values of remaining individuals from BLD (−12.0 to −10.8‰) are indicative of a dominance of C3 food sources, possibly yams, plantain, or oil palm grown in slightly more open conditions, or perhaps a mixed diet of closed canopy and freshwater resources. There is no clear evidence for dietary reliance on C4 plants in any of the human tooth enamel samples analysed from IMB, LON, or BLD.
In sharp contrast to the western DRC samples, the M3 from the individual from MTNW has a δ13C enamel value of −3.2‰ that clearly indicates that C4 resources made the dominant contribution to the diet of this individual. Unfortunately, it was not possible to analyse bone collagen from this individual, but multiple teeth (M1–M3) were analysed to investigate diet throughout childhood. The results (Supplementary Fig. 8) demonstrate that this individual relied upon C4 food sources throughout childhood and as a juvenile.
Dental calculus was analysed from three mandibular molars (M1, M2, and M3) from the MTNW individual and a total of 38 starch granules and 9 phytoliths were retrieved (Supplementary Table 5). Microcharcoal is very common (Fig. 4a, aq–as). The calcium phosphate matrix was decontaminated prior to decalcification, as per a published protocol in which calculus is immersed in sodium hydroxide of 2% w/v solution for 24 h. As expected for ancient starch granules, the discovered calculus starch displays signs of damage to their semicrystalline matrix, having partially or totally lost their native birefringence. Other signs of diagenesis include fissuring, pitting, granulation, and implosion of the hilum. The taxonomic identification of starch granules depends on whether they represent unique morphometric identifiers that can be compared to published reference collections. In this respect, the mixture of polygonal, orbicular, and quadratic granules found derive from a grass seed (n = 26), but cannot specifically be assigned to pearl millet (Pennisetum glaucum), wild finger millet, (Eleusine africana), or domestic finger millet (Eleusine coracana), as they all have in common compound granules and/or markedly polygonal shapes with mean metrics <10 µm (Supplementary Fig. 9a–c).
In contrast, starch granules from Sorghum bicolor are polymorphic with roughly polygonal, orbicular, and quadratic shapes, a centric hilum often creased or slit (Supplementary Fig. 9d–h), and can be uniquely identified by prismatic–polygonal shapes with mean maximum length 18–30 μm (ref. ). Another common starch granule type identified is parabolic and/or oblong elongate (n = 11; Fig. 4). In our reference collection, the best possible match for this cohort is in the Dioscoreaceae, which in sub-Saharan Africa produces the highest number of unique identifiers in granule morphometrics and overwhelmingly associates with wild yams.The remaining type (ovate, n = 1; Fig. 4) is also tentatively associated with an underground storage organ, and similar granules occur in the Asphodelaceae family. With regards to phytoliths, all phytoliths (n = 9) come from one tooth (M1) and they are characterised as medium to large, brown globular bodies, with tuberculate to echinate projections. These large, brown phytoliths with variably tuberculated to echinated spines are referenced in the nutshell of Elaeis guineensis (Supplementary Fig. 9i–n), whose charred remains were also discovered at the site.
The charred food fragments recovered from contexts at BLD (n = 8) fall into three groups based on their δ13C values: those with δ13C values < −27‰; a single food fragment with an intermediate δ13C value of −24‰; and two food fragments with δ13C values around −9‰ (Supplementary Table 6). δ13C values ranging from < −27 to −24‰ are likely indicative of food fragments consisting of C3 or aquatic resources, while δ13C values of −9‰ indicate that the primary content was likely C4 resources. The charred food fragments from BLD contained between 0.6 and 2.5% N, which means that for all samples apart from BE06, the N2 peak was too small for reliable determination of their δ15N values. BE06, a sample with a high δ13C value (−9.3‰), has a relatively high δ15N value (7.8‰) compared to those of herbivores from the site, perhaps indicating that C4 plant resources like pearl millet and sorghum consumed by humans were growing in different soil conditions compared to the plants eaten by wild and domestic herbivores.
The discovery of pearl millet (Pennisetum glaucum) in Iron Age pits in Southern Cameroon was one of the most significant findings of the past two decades in Central African archaeology. The discovery sparked debate over the environmental context for early agriculture, contributing to an increasingly complex narrative for the settlement of the Central Africa rainforest. Yet direct assessments about the degree to which these early farming communities, particularly in the DRC, relied on C4 resources are limited. Our data enable direct exploration of the adoption of agriculture at different points during the Iron Age in the DRC and highlight substantial regional variability, particularly with regards to uptake of C4 crops into the diet. The IMB individual, indirectly dated to ~2050 BP (Supplementary Note 2), provides a snapshot of potentially early agricultural groups entering the region. While this period is commonly associated with the arrival of cereal cultivation, this individual shows a heavy reliance on C3 closed rainforest or freshwater resources rather than C4 crops. Moving to the Late Iron Age and ongoing ‘Bantu expansion’ into a number of tributaries of the Congo River, individuals from LON and BLD show no clear evidence for a dietary reliance on C4 plants in tooth enamel samples. Collagen values, however, do suggest some degree of C4 consumption in addition to a core reliance on C3 closed rainforest and more open C3 resources, perhaps including plantain, oil palm and yams or manioc, and freshwater resources. This interpretation is supported by zooarchaeological research at BLD that highlights a dominance of fish, as well as the apparent importance of riverine locations for these settlements.
Four Iron Age sites were selected for study from across the DRC (Fig. 1 and Supplementary Note 2): IMB, LON, BLD, and MTNW. Bulk bone collagen δ13C and δ15N measurements were obtained for human burials from LON (n = 1), BLD (n = 11), and MTNW (n = 1). Totals reflect results taken forward for interpretation. For BLD, two results were excluded from final analysis due to poor preservation (for full details see: “Results” section and Supplementary Table 3). Bone collagen was also analysed from a range of faunal remains from BLD (n = 10) to establish a dietary baseline, including domestic browsers (goats), wild browsers (duiker), and fish. To further explore dietary intake, human tooth enamel was sampled from IMB (n = 1), LON (n = 1), BLD (n = 11), and MTNW (n = 1). Due to differential preservation, it was only possible to generate both a bulk collagen and tooth enamel results for four human burials from BLD, and the single individual from LON. In addition, animal tooth enamel from BLD (n = 6) was analysed for δ13C and δ18O to aid the interpretation of human values.
BLD is a c. 660 BP to present site located in the western Interior Congo Basin on the floodplain of the Tshuapa River. The site was first excavated in 1983 with the most recent field season taking place in 2016, with financial support of the Deutsche Forschungsgemeinschaft. Owing to partly waterlogged conditions, the level of organic preservation at BLD is far higher than at sites located above the floodplain, and a number of human burials have been excavated (Supplementary Fig. 2). In addition, tooth enamel samples were analysed from one human individual from each of the sites of IMB and LON. Both sites were excavated by Manfred Eggert in the 1980s. IMB is the type site for the oldest pottery of the equatorial forest and is located on the Momboyo River, and LON is located on the Ruki River.
Finally, a single individual dating to 813 ± 35 BP was analysed from MTNW, a Later Stone Age rockshelter located in the Ituri rainforest of the Eastern Congo Basin.This individual was previously identified as a likely hunter-gatherer based on morphological evidence, associated lithics, presence of wild fauna, and absence of domesticated plants. However, the presence of a large assemblage of ceramics, iron slag, and iron objects means it is impossible to say definitively whether the individual was from a primarily foraging group or associated with Bantu-speaking groups. Unfortunately, it was not possible to sample any associated fauna from MTNW for this study, but a range of forest taxa were present at the site, including porcupines, antelopes, primates, small bovids, and snails. While there are clear geographical and ecological differences between MTNW and BLD the fauna at both sites are indicative of a closed forest environment. For the individual from Matangai Turu, it was possible to sample all three permanent molars to track dietary consumption throughout childhood. The tooth enamel δ13C of human molars is influenced by dietary intake during the time of tooth formation with the first molar forming between 2 months prior to birth to 4 years after birth, the second molar between 4 and 7 years, and third molar between 9 and 16 years (Supplementary Note 3).
The δ13C and δ15N ratios of the bone collagen were determined using a Thermo Scientific Flash 2000 Elemental Analyser coupled to a Thermo Delta V Advantage mass spectrometer at the Isotope Laboratory, MPI-SHH, Jena. Isotopic values are reported as the ratio of the heavier isotope to the lighter isotope (13C/12C or 15N/14N) as δ values in parts per mill (‰) relative to international standards, VPDB for δ13C and atmospheric N2 (AIR) for δ15N. Results were calibrated against international standards of (IAEA-CH-6: δ13C = −10.80 ± 0.47‰, IAEA-N-2: δ15N = 20.3 ± 0.2‰, and USGS40: δ13C = −26.38 ± 0.042‰, δ15N = 4.5 ± 0.1‰) and a laboratory standard (fish gelatin: δ13C = ~−15.1‰, δ15N = ~14.3‰). Based on replicate analyses long-term machine error over a year is ±0.2‰ for δ13C and ±0.2‰ for δ15N. Overall measurement precision was studied through the measurement of repeats of fish gelatin (n = 80, ±0.2‰ for δ13C and ±0.2‰ for δ15N). The faunal (n = 8) and human (n = 12) bone collagen results from BLD and LON are presented in Supplementary Table 3. Samples with a C/N ratio between 2.9–3.6, %C of ca.15–48, and %N of ca.5–17% were carried forward for interpretation.
Dental calculus was processed from three mandibular molars (M1, M2, and M3) from the MTNW individual. Images of the mineralised plaque prior to removal from the teeth, as well as those from contaminant starch granules and phytoliths are published elsewhere (see ref. ; Fig. 2). The elemental breakdown includes carbon, oxygen, calcium, and phosphorus, with small quantities of aluminium, silicon, nitrogen, sodium, and chlorine, and the Ca:P ratio was 2:1–1:7 indicating hydroxyapatite. We present microbotanical materials released from the calcified matrix after thorough decontamination protocols and decalcification in a cleanroom laboratory, as well as microbotanicals still trapped in the calculus matrix, but visible enough to have their two dimensional morphology identified. Identifications were made according to published morphometric classification criteria for the identification of ancient starch from sub-Saharan plants.
Charred fragments classified as prepared food remains during archaeobotanical analysis at BLD were retrieved from flotation samples after sorting under a binocular microscope. A total of 2–3 mg of each sample was weighed into tin capsules for stable carbon and nitrogen isotope analysis. The δ13C and δ15N ratios of the charred food fragments were determined, using a Thermo MAT 253 continuous flow isotope ratio mass spectrometer coupled to a Thermo Flash 1112 Series elemental analyser in the Institut für Geowissenschaften, Goethe-Universität, Frankfurt am Main, Germany. Isotopic data are provided in Supplementary Table 6.
The carbon contents of the samples were calculated based on the area under the CO2 peak relative to the weight of the sample, calibrated using IAEA-CH-7. Stable carbon isotope values were calibrated to the VPDB scale using IAEA-C-7 (δ13C −32.15 ± 0.05‰) and IAEA-USGS24 (δ13C –16.05 ± 0.04‰). Measurement uncertainty in δ13C values was monitored using three in-house standards: LEU (DL-leucine, δ13C −28.3 ± 0.1‰), GLU (DL-glutamic acid monohydrate, δ13C −10.4 ± 0.1‰), and MIL (millet flour from a single panicle from a plot in Senegal, δ13C −10.2 ± 0.1‰; Supplementary Data 1). Precision (u(R)) was determined to be ±0.06‰, accuracy or systematic error (u(bias)) was ±0.11‰, and the total analytical uncertainty in δ13C values was estimated to be ±0.13‰, using the equation presented in Supplementary material (Supplementary Data 1).
Bone samples from five individuals from BLD, and the single individual from LON were sent for radiocarbon dating at the Scottish Universities Environmental Research Centre AMS Laboratory, Glasgow (SUERC, Lab ID: GU), in order to improve understanding of their chronology. Samples were pretreated using previously published methods. Radiocarbon ages were calibrated to calendar timescale using OxCal 4 (ref. ) and IntCal13 atmospheric calibration curve (Supplementary Note 2 and Supplementary Figs. 1, 3–7).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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"[{\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Introduction\", \"text\": \"For the past half a century, if not longer, the processes for the dispersal of Bantu-speaking communities from Western Central Africa have been a major focus of African archaeological, linguistic, and genetic research. While there has been an increasing departure from notions of a single sweeping \\u2018Bantu Expansion\\u2019, the degree to which the movement of people, languages, and the emergence of farming are linked across Africa continues to be forcefully debated. Central Africa is at a key location for developing existing models for the spread of farming yet investigations of the emergence of food production, particularly in the rainforest, have been limited. Assumptions that tropical rainforests represent substantial barriers to agriculturalists have been used to rationalise a relatively late arrival of farming in the region, c. 2500 years ago, during a period of climate- or human-induced deforestation (Supplementary Note\\u00a01). However, unlike other parts of Africa, there have been few studies directly testing changes in human dietary reliance on agricultural crops, relative to local freshwater, bushmeat, and tropical forest plant resources, from the first arrival of domesticates in the region through to the present day.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig1\", \"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\"], \"section\": \"Introduction\", \"text\": \"Here, we present new, direct dietary information from Iron Age sites in the Democratic Republic of Congo (DRC) using the stable carbon (\\u03b413C), nitrogen (\\u03b415N), and oxygen (\\u03b418O) isotope analysis of human and animal remains. Isotopic results were obtained for human burials from the sites of Imbonga (IMB; n\\u2009=\\u20091), Longa (LON; n\\u2009=\\u20091), Bolondo (BLD; n\\u2009=\\u200918), and Matangai Turu Northwest (MTNW; n\\u2009=\\u20091). In addition, bone collagen (n\\u2009=\\u200910) and enamel (n\\u2009=\\u20096) were analysed for a range of fauna from BLD to create an isotopic baseline. The sites studied represent different geographic and temporal contexts (Fig.\\u00a01). IMB is the type site for the earliest pottery tradition of the central equatorial rainforest and the individual analysed, indirectly dated to ~2050 BP (Supplementary Note\\u00a02), would have been a member of a group representing already established agriculture in the region after its initial settlement by sedentary immigrant populations a few centuries earlier. In contrast, individuals from LON and BLD represent subsistence practices during the Late Iron Age, when populations were spreading further across the Congo Basin. Finally, isotopic results from the individual from MTNW, previously identified as a likely hunter-gatherer, offers new evidence about the intricacies of subsistence, cultural, and genetic identities further to the eastern edge of the Basin (Supplementary Note\\u00a02). Collectively, the samples analysed span the period following the first arrival of food producers in this region (~2050 BP) through to relatively recent occupation (~130 BP; Supplementary Note\\u00a02, Supplementary Tables\\u00a01 and 2, and Supplementary Figs.\\u00a01, 3\\u20137).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Introduction\", \"text\": \"\\u03b413C analysis of human tissues has long been demonstrated to provide insights into reliance on plants with different photosynthetic pathways (namely C4 versus C3) and their animal consumers (Supplementary Note\\u00a03). Significantly, in Central Africa, wild, as well as potentially domesticated (e.g., yams), forest plants are C3, while incoming cereal crops (e.g., pearl millet, sorghum, and, for later periods, maize) are C4. \\u03b413C measurements of wild plants and animals from the rainforests of the DRC show that these forests are largely composed of C3 vegetation. Moreover, they show a recognisable \\u2018canopy effect\\u2019 on this C3 vegetation that results in lower \\u03b413C among plants, and their animal consumers, living under dense canopies compared to those living in more open areas, something that has been well-documented in many other tropical regions.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig1\", \"MOESM1\"], \"section\": \"Introduction\", \"text\": \"The sites of IMB, LON, and BLD are located on tributaries of the Congo River in the western DRC (Fig. 1 and Supplementary Note\\u00a02) an area presently covered in dense C3-dominated evergreen and semi-deciduous forest. Stable carbon measurements of faunal tooth enamel from BLD, which largely reflect the proportions of C3/C4 plants consumed, reflect local palaeoecology, as well as providing baseline values for human diet. The final site, MTNW, is situated in the closed-canopy forest of the Ituri Region of the Northeast Congo Basin with palaeoenviromental proxies, suggesting a predominance of tropical forest tree taxa during the time of occupation.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Introduction\", \"text\": \"\\u03b415N analysis provides insights into the positions of humans within their trophic web and their potential consumption of aquatic resources, while \\u03b418O measurements reflect water sources and environments (see \\u201cMethods\\u201d section or Supplementary Note\\u00a03 for full details). To the best of our knowledge, this is the first time that multi-tissue stable isotope analysis of prehistoric humans and animals has been applied in the Congo Basin, in order to provide long-term insights into changing human reliance on different resources. We also present results of \\u03b413C and \\u03b415N analysis of charred lumps interpreted as food residues from BLD, as well as microparticle analysis of dental calculus recovered from MTNW, to obtain more detailed insights into prepared and consumed foods, respectively.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Faunal and human bone collagen\", \"text\": \"Bone collagen results were assessed using established indicators of preservation, including a C/N ratio between 2.9\\u20133.6, %C of ca.15\\u201348%, and %N of ca. 5\\u201317% (ref. ). Two results generated for human burials from BLD were excluded from final analysis as they produced a %N\\u2009<\\u20095 and a %C\\u2009<\\u200915 % (Supplementary Table\\u00a03).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig2\", \"MOESM1\"], \"section\": \"Faunal and human bone collagen\", \"text\": \"The fauna from BLD fall broadly into four groups: wild browsers (antelope and duiker, n\\u2009=\\u20092), domesticated browsers (goats, n\\u2009=\\u20092), mammalian carnivores (n\\u2009=\\u20092), and aquatic species (fish and crocodile, n\\u2009=\\u20094). \\u03b413C values from BLD mammals (n\\u2009=\\u20096) are consistent with a largely C3-based diet with measurements ranging from \\u221223.7 to \\u221218.2\\u2030, although the goats, the dog, and the fox-sized carnivore could potentially have some C4 component to the diet (Fig.\\u00a02 and Supplementary Table\\u00a03). The common duiker (Sylvicapra grimmia, BLD 83/2-8) and small antelope (BLD 83/2-6\\u2009+\\u20097) have \\u03b415N values (5.9 and 7.3\\u2030, respectively) consistent with herbivorous diets. In contrast, the fox-sized carnivore (13.7\\u2030) and dog (12.3\\u2030) display higher \\u03b415N that is consistent with consumption of animal protein. Three of the aquatic species sampled had higher \\u03b415N values than the catfish (\\u03b415N 9.4\\u2030), the crocodiles gave \\u03b415N of 11.1 and 11.4\\u2030, and the bichir (Polypterus sp.), a fish known to consume other fish and small vertebrates, produced a \\u03b415N measurement of 12.7\\u2030. The accompanying \\u03b413C value of \\u221224.7\\u2030 for the bichir somewhat overlaps with that expected for C3 plants and animals, demonstrating the need to examine both \\u03b415N and \\u03b413C, in order to separate freshwater fish and forest resources.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\", \"MOESM1\", \"MOESM1\"], \"section\": \"Faunal and human bone collagen\", \"text\": \"The \\u03b413C values for the bone collagen of humans from BLD (n\\u2009=\\u200911) dating to between 1426 and 1942 years cal. AD (Supplementary Figs.\\u00a03\\u20137) are quite variable, ranging from \\u221221.0 to \\u221216.3\\u2030. Alongside the individual from LON (directly dated to 1642\\u2014after 1938 cal. AD), these values are generally consistent with reliance on C3 plants, C3 plant-consuming wild and domestic animals, and freshwater resources. The average \\u03b415N value of 14.5\\u2030 for the human individuals from BLD in comparison to the average for the goats (9.9\\u2030) is within the range of values reported for diet\\u2013collagen spacing, potentially indicating reliance on these domesticates. However, \\u03b415N values ranging from 13.4 to 16.9\\u2030, and three individuals with \\u03b415N values higher than 15\\u2030, as well as the riverine setting and modern and historical evidence that the site was a fishing camp (Supplementary Note\\u00a02), indicate that freshwater fish was also a major part of human diets at BLD. Nevertheless, it is still evident that all humans and domestic animals, as well as the fox-sized carnivore, have higher \\u03b413C values than the available wild C3 or freshwater fauna, indicating the consumption of an additional resource enriched in \\u03b413C.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig2\", \"Fig2\"], \"section\": \"Faunal and human bone collagen\", \"text\": \"A visible negative correlation between human \\u03b415N and \\u03b413C suggests that this was, in fact, a plant food (Fig.\\u00a02), although a Pearson\\u2019s rank test (correlation coefficient\\u2009=\\u2009\\u22120.600, d.f.\\u2009=\\u20099, p\\u2009=\\u20090.05) produced results at the limit of statistical significance. This is likely a product of the small sample sizes available. The metabolic routing bias of bone collagen \\u03b413C towards protein components of the diet (at the expense of carbohydrate and fat inputs), and importance of high protein freshwater fish in diets of the measured individuals, means that we can expect that consumption of this low-protein plant resource was actually greater than it appears in the bone collagen values of Fig.\\u00a02. Thus, although somewhat underrepresented in the present bone collagen isotope results, there is a signal indicative of some C4 plant component, which must have contributed to the diet of Late Iron Age humans and domestic animals in the Inner Congo Basin from at least the 15th century cal. AD onwards.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Faunal and human tooth enamel\", \"text\": \"Tooth enamel is widely regarded as the archaeological material of choice in the tropics. Stable carbon and oxygen isotopes of tooth enamel have been shown to robustly preserve ecological variation, even in tropical regions, from the Miocene to the Late Pleistocene. Tooth enamel was sampled from human second and third molars enabling the investigation of diet during late childhood\\u2013early adolescence. Furthermore, given that tooth enamel \\u03b413C reflects the isotopic composition of the whole diet (including carbohydrates, fats, and proteins), as opposed to the dominance of the protein signal in bone collagen, tooth enamel \\u03b413C enables additional dietary resolution (see Supplementary Note\\u00a03).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig3\", \"MOESM1\"], \"section\": \"Faunal and human tooth enamel\", \"text\": \"Faunal and human \\u03b413C enamel results from IMB, LON, BLD, and MTNW (Fig.\\u00a03 and Supplementary Table\\u00a04) reveal diverse subsistence strategies that can be divided into three broad groups: wild forest resources, forest and freshwater resources, and more open environments, including C4 food sources. The individual from the earliest site mentioned here, IMB, has a \\u03b413C value of \\u221214.1\\u2030 indicating reliance on a mixture of tropical rainforest and freshwater resources, while the individual from LON shows clear evidence for reliance on tropical rainforest resources with a \\u03b413C value of \\u221215.1\\u2030. Two individuals from BLD gave \\u03b413C values of \\u221214.7 and \\u221213.6\\u2030 also suggesting a heavy reliance on tropical or freshwater resources. The \\u03b413C values of remaining individuals from BLD (\\u221212.0 to \\u221210.8\\u2030) are indicative of a dominance of C3 food sources, possibly yams, plantain, or oil palm grown in slightly more open conditions, or perhaps a mixed diet of closed canopy and freshwater resources. There is no clear evidence for dietary reliance on C4 plants in any of the human tooth enamel samples analysed from IMB, LON, or BLD.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Faunal and human tooth enamel\", \"text\": \"In sharp contrast to the western DRC samples, the M3 from the individual from MTNW has a \\u03b413C enamel value of \\u22123.2\\u2030 that clearly indicates that C4 resources made the dominant contribution to the diet of this individual. Unfortunately, it was not possible to analyse bone collagen from this individual, but multiple teeth (M1\\u2013M3) were analysed to investigate diet throughout childhood. The results (Supplementary Fig.\\u00a08) demonstrate that this individual relied upon C4 food sources throughout childhood and as a juvenile.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\", \"Fig4\", \"MOESM1\"], \"section\": \"Microbotanical remains dental calculus\", \"text\": \"Dental calculus was analysed from three mandibular molars (M1, M2, and M3) from the MTNW individual and a total of 38 starch granules and 9 phytoliths were retrieved (Supplementary Table\\u00a05). Microcharcoal is very common (Fig.\\u00a04a, aq\\u2013as). The calcium phosphate matrix was decontaminated prior to decalcification, as per a published protocol in which calculus is immersed in sodium hydroxide of 2% w/v solution for 24\\u2009h. As expected for ancient starch granules, the discovered calculus starch displays signs of damage to their semicrystalline matrix, having partially or totally lost their native birefringence. Other signs of diagenesis include fissuring, pitting, granulation, and implosion of the hilum. The taxonomic identification of starch granules depends on whether they represent unique morphometric identifiers that can be compared to published reference collections. In this respect, the mixture of polygonal, orbicular, and quadratic granules found derive from a grass seed (n\\u2009=\\u200926), but cannot specifically be assigned to pearl millet (Pennisetum glaucum), wild finger millet, (Eleusine africana), or domestic finger millet (Eleusine coracana), as they all have in common compound granules and/or markedly polygonal shapes with mean metrics <10\\u2009\\u00b5m (Supplementary Fig.\\u00a09a\\u2013c).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\", \"Fig4\", \"Fig4\", \"MOESM1\"], \"section\": \"Microbotanical remains dental calculus\", \"text\": \"In contrast, starch granules from Sorghum bicolor are polymorphic with roughly polygonal, orbicular, and quadratic shapes, a centric hilum often creased or slit (Supplementary Fig.\\u00a09d\\u2013h), and can be uniquely identified by prismatic\\u2013polygonal shapes with mean maximum length 18\\u201330\\u2009\\u03bcm (ref. ). Another common starch granule type identified is parabolic and/or oblong elongate (n\\u2009=\\u200911; Fig.\\u00a04). In our reference collection, the best possible match for this cohort is in the Dioscoreaceae, which in sub-Saharan Africa produces the highest number of unique identifiers in granule morphometrics and overwhelmingly associates with wild yams.The remaining type (ovate, n\\u2009=\\u20091; Fig.\\u00a04) is also tentatively associated with an underground storage organ, and similar granules occur in the Asphodelaceae family. With regards to phytoliths, all phytoliths (n\\u2009=\\u20099) come from one tooth (M1) and they are characterised as medium to large, brown globular bodies, with tuberculate to echinate projections. These large, brown phytoliths with variably tuberculated to echinated spines are referenced in the nutshell of Elaeis guineensis (Supplementary Fig.\\u00a09i\\u2013n), whose charred remains were also discovered at the site.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Charred food fragments from Bolondo\", \"text\": \"The charred food fragments recovered from contexts at BLD (n\\u2009=\\u20098) fall into three groups based on their \\u03b413C values: those with \\u03b413C values\\u2009<\\u2009\\u221227\\u2030; a single food fragment with an intermediate \\u03b413C value of \\u221224\\u2030; and two food fragments with \\u03b413C values around \\u22129\\u2030 (Supplementary Table\\u00a06). \\u03b413C values ranging from <\\u2009\\u221227 to \\u221224\\u2030 are likely indicative of food fragments consisting of C3 or aquatic resources, while \\u03b413C values of \\u22129\\u2030 indicate that the primary content was likely C4 resources. The charred food fragments from BLD contained between 0.6 and 2.5% N, which means that for all samples apart from BE06, the N2 peak was too small for reliable determination of their \\u03b415N values. BE06, a sample with a high \\u03b413C value (\\u22129.3\\u2030), has a relatively high \\u03b415N value (7.8\\u2030) compared to those of herbivores from the site, perhaps indicating that C4 plant resources like pearl millet and sorghum consumed by humans were growing in different soil conditions compared to the plants eaten by wild and domestic herbivores.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Discussion\", \"text\": \"The discovery of pearl millet (Pennisetum glaucum) in Iron Age pits in Southern Cameroon was one of the most significant findings of the past two decades in Central African archaeology. The discovery sparked debate over the environmental context for early agriculture, contributing to an increasingly complex narrative for the settlement of the Central Africa rainforest. Yet direct assessments about the degree to which these early farming communities, particularly in the DRC, relied on C4 resources are limited. Our data enable direct exploration of the adoption of agriculture at different points during the Iron Age in the DRC and highlight substantial regional variability, particularly with regards to uptake of C4 crops into the diet. The IMB individual, indirectly dated to ~2050 BP (Supplementary Note\\u00a02), provides a snapshot of potentially early agricultural groups entering the region. While this period is commonly associated with the arrival of cereal cultivation, this individual shows a heavy reliance on C3 closed rainforest or freshwater resources rather than C4 crops. Moving to the Late Iron Age and ongoing \\u2018Bantu expansion\\u2019 into a number of tributaries of the Congo River, individuals from LON and BLD show no clear evidence for a dietary reliance on C4 plants in tooth enamel samples. Collagen values, however, do suggest some degree of C4 consumption in addition to a core reliance on C3 closed rainforest and more open C3 resources, perhaps including plantain, oil palm and yams or manioc, and freshwater resources. This interpretation is supported by zooarchaeological research at BLD that highlights a dominance of fish, as well as the apparent importance of riverine locations for these settlements.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig1\", \"MOESM1\", \"MOESM1\"], \"section\": \"Methods\", \"text\": \"Four Iron Age sites were selected for study from across the DRC (Fig.\\u00a01 and Supplementary Note\\u00a02): IMB, LON, BLD, and MTNW. Bulk bone collagen \\u03b413C and \\u03b415N measurements were obtained for human burials from LON (n\\u2009=\\u20091), BLD (n\\u2009=\\u200911), and MTNW (n\\u2009=\\u20091). Totals reflect results taken forward for interpretation. For BLD, two results were excluded from final analysis due to poor preservation (for full details see: \\u201cResults\\u201d section and Supplementary Table\\u00a03). Bone collagen was also analysed from a range of faunal remains from BLD (n\\u2009=\\u200910) to establish a dietary baseline, including domestic browsers (goats), wild browsers (duiker), and fish. To further explore dietary intake, human tooth enamel was sampled from IMB (n\\u2009=\\u20091), LON (n\\u2009=\\u20091), BLD (n\\u2009=\\u200911), and MTNW (n\\u2009=\\u20091). Due to differential preservation, it was only possible to generate both a bulk collagen and tooth enamel results for four human burials from BLD, and the single individual from LON. In addition, animal tooth enamel from BLD (n\\u2009=\\u20096) was analysed for \\u03b413C and \\u03b418O to aid the interpretation of human values.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Methods\", \"text\": \"BLD is a c. 660 BP to present site located in the western Interior Congo Basin on the floodplain of the Tshuapa River. The site was first excavated in 1983 with the most recent field season taking place in 2016, with financial support of the Deutsche Forschungsgemeinschaft. Owing to partly waterlogged conditions, the level of organic preservation at BLD is far higher than at sites located above the floodplain, and a number of human burials have been excavated (Supplementary Fig.\\u00a02). In addition, tooth enamel samples were analysed from one human individual from each of the sites of IMB and LON. Both sites were excavated by Manfred Eggert in the 1980s. IMB is the type site for the oldest pottery of the equatorial forest and is located on the Momboyo River, and LON is located on the Ruki River.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Methods\", \"text\": \"Finally, a single individual dating to 813\\u2009\\u00b1\\u200935 BP was analysed from MTNW, a Later Stone Age rockshelter located in the Ituri rainforest of the Eastern Congo Basin.This individual was previously identified as a likely hunter-gatherer based on morphological evidence, associated lithics, presence of wild fauna, and absence of domesticated plants. However, the presence of a large assemblage of ceramics, iron slag, and iron objects means it is impossible to say definitively whether the individual was from a primarily foraging group or associated with Bantu-speaking groups. Unfortunately, it was not possible to sample any associated fauna from MTNW for this study, but a range of forest taxa were present at the site, including porcupines, antelopes, primates, small bovids, and snails. While there are clear geographical and ecological differences between MTNW and BLD the fauna at both sites are indicative of a closed forest environment. For the individual from Matangai Turu, it was possible to sample all three permanent molars to track dietary consumption throughout childhood. The tooth enamel \\u03b413C of human molars is influenced by dietary intake during the time of tooth formation with the first molar forming between 2 months prior to birth to 4 years after birth, the second molar between 4 and 7 years, and third molar between 9 and 16 years (Supplementary Note\\u00a03).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Stable isotope analysis of bone collagen\", \"text\": \"The \\u03b413C and \\u03b415N ratios of the bone collagen were determined using a Thermo Scientific Flash 2000 Elemental Analyser coupled to a Thermo Delta V Advantage mass spectrometer at the Isotope Laboratory, MPI-SHH, Jena. Isotopic values are reported as the ratio of the heavier isotope to the lighter isotope (13C/12C or 15N/14N) as \\u03b4 values in parts per mill (\\u2030) relative to international standards, VPDB for \\u03b413C and atmospheric N2 (AIR) for \\u03b415N. Results were calibrated against international standards of (IAEA-CH-6: \\u03b413C\\u2009=\\u2009\\u221210.80\\u2009\\u00b1\\u20090.47\\u2030, IAEA-N-2: \\u03b415N\\u2009=\\u200920.3\\u2009\\u00b1\\u20090.2\\u2030, and USGS40: \\u03b413C\\u2009=\\u2009\\u221226.38\\u2009\\u00b1\\u20090.042\\u2030, \\u03b415N\\u2009=\\u20094.5\\u2009\\u00b1\\u20090.1\\u2030) and a laboratory standard (fish gelatin: \\u03b413C\\u2009=\\u2009~\\u221215.1\\u2030, \\u03b415N\\u2009=\\u2009~14.3\\u2030). Based on replicate analyses long-term machine error over a year is \\u00b10.2\\u2030 for \\u03b413C and \\u00b10.2\\u2030 for \\u03b415N. Overall measurement precision was studied through the measurement of repeats of fish gelatin (n\\u2009=\\u200980, \\u00b10.2\\u2030 for \\u03b413C and \\u00b10.2\\u2030 for \\u03b415N). The faunal (n\\u2009=\\u20098) and human (n\\u2009=\\u200912) bone collagen results from BLD and LON are presented in Supplementary Table\\u00a03. Samples with a C/N ratio between 2.9\\u20133.6, %C of ca.15\\u201348, and %N of ca.5\\u201317% were carried forward for interpretation.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"Fig2\"], \"section\": \"Microparticle analysis of dental calculus from MTNW\", \"text\": \"Dental calculus was processed from three mandibular molars (M1, M2, and M3) from the MTNW individual. Images of the mineralised plaque prior to removal from the teeth, as well as those from contaminant starch granules and phytoliths are published elsewhere (see ref. ; Fig.\\u00a02). The elemental breakdown includes carbon, oxygen, calcium, and phosphorus, with small quantities of aluminium, silicon, nitrogen, sodium, and chlorine, and the Ca:P ratio was 2:1\\u20131:7 indicating hydroxyapatite. We present microbotanical materials released from the calcified matrix after thorough decontamination protocols and decalcification in a cleanroom laboratory, as well as microbotanicals still trapped in the calculus matrix, but visible enough to have their two dimensional morphology identified. Identifications were made according to published morphometric classification criteria for the identification of ancient starch from sub-Saharan plants.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\"], \"section\": \"Stable isotope analysis of charred food fragments\", \"text\": \"Charred fragments classified as prepared food remains during archaeobotanical analysis at BLD were retrieved from flotation samples after sorting under a binocular microscope. A total of 2\\u20133\\u2009mg of each sample was weighed into tin capsules for stable carbon and nitrogen isotope analysis. The \\u03b413C and \\u03b415N ratios of the charred food fragments were determined, using a Thermo MAT 253 continuous flow isotope ratio mass spectrometer coupled to a Thermo Flash 1112 Series elemental analyser in the Institut f\\u00fcr Geowissenschaften, Goethe-Universit\\u00e4t, Frankfurt am Main, Germany. Isotopic data are provided in Supplementary Table\\u00a06.\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM4\", \"MOESM4\"], \"section\": \"Stable isotope analysis of charred food fragments\", \"text\": \"The carbon contents of the samples were calculated based on the area under the CO2 peak relative to the weight of the sample, calibrated using IAEA-CH-7. Stable carbon isotope values were calibrated to the VPDB scale using IAEA-C-7 (\\u03b413C \\u221232.15\\u2009\\u00b1\\u20090.05\\u2030) and IAEA-USGS24 (\\u03b413C \\u201316.05\\u2009\\u00b1\\u20090.04\\u2030). Measurement uncertainty in \\u03b413C values was monitored using three in-house standards: LEU (DL-leucine, \\u03b413C \\u221228.3\\u2009\\u00b1\\u20090.1\\u2030), GLU (DL-glutamic acid monohydrate, \\u03b413C \\u221210.4\\u2009\\u00b1\\u20090.1\\u2030), and MIL (millet flour from a single panicle from a plot in Senegal, \\u03b413C \\u221210.2\\u2009\\u00b1\\u20090.1\\u2030; Supplementary Data\\u00a01). Precision (u(R)) was determined to be \\u00b10.06\\u2030, accuracy or systematic error (u(bias)) was \\u00b10.11\\u2030, and the total analytical uncertainty in \\u03b413C values was estimated to be \\u00b10.13\\u2030, using the equation presented in Supplementary material (Supplementary Data\\u00a01).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM1\", \"MOESM1\", \"MOESM1\", \"MOESM1\"], \"section\": \"AMS dating\", \"text\": \"Bone samples from five individuals from BLD, and the single individual from LON were sent for radiocarbon dating at the Scottish Universities Environmental Research Centre AMS Laboratory, Glasgow (SUERC, Lab ID: GU), in order to improve understanding of their chronology. Samples were pretreated using previously published methods. Radiocarbon ages were calibrated to calendar timescale using OxCal 4 (ref. ) and IntCal13 atmospheric calibration curve (Supplementary Note\\u00a02 and Supplementary Figs.\\u00a01, 3\\u20137).\"}, {\"pmc\": \"PMC7591565\", \"pmid\": \"33110164\", \"reference_ids\": [\"MOESM5\"], \"section\": \"Reporting summary\", \"text\": \"Further information on research design is available in the\\u00a0Nature Research Reporting Summary linked to this article.\"}]"

Metadata

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