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

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 (δ13C, δ15N, δ18O) 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.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist .

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Ecological, evolutionary & environmental sciences study design All studies must disclose on these points even when the disclosure is negative.

Study description
Research sample Sampling strategy

Data collection
Stable carbon, oxygen and nitrogen isotope analysis was applied to archaeological human and animal tooth enamel and bone collagen from four archaeological sites in the Democratic Republic of the Congo. This was done to investigate dietary consumption in the Congo Basin during the Iron Age and assess human reliance on incoming domestic cereals.
Bone collagen samples were selected from humans previously excavated from the archaeological sites of Longa, Imbonga, Bolondo and Matangai Turu Northwest in the Congo Basin. !13C and !15N measurements of bone collagen were made to investigate dietary consumption, largely protein sources such as meat, from the sites of study. To provide a baseline, collagen was also analysed from a range of wild and domestic fauna from the site of Bolondo, including crocodile, antelope, goat and dog. In addition tooth enamel was samples for !18O and !13C from humans and fauna to explore the consumption of wild plant sources (C3) compared to incoming domestic crops (C4) such as pearl millet. Stable carbon and nitrogen isotope analyses were also performed on a number of charred food remains from Bolondo to further investigate the processing of different foodstuffs. For a single individual from Matangai Turu Northwest, dental calulus (calcified dental plaque) was removed and analyzed using microscopy to identify plant phytoliths and starch granules entrapped in the calculus.
Samples were selected for stable carbon and oxygen isotope analysis of tooth enamel and stable carbon and nitrogen isotope analysis of bone collagen from the available fossil material at the archaeological sites of Longa, Imbonga, Bolondo and Matangai Turu Northwest in the Congo Basin. All teeth or tooth fragments were cleaned using air-abrasion to remove any external material. Enamel powder for bulk analysis was obtained using gentle abrasion with a diamond-tipped drill along the full length of the buccal surface in order to ensure a representative measurement for the entire period of enamel formation. All bone fragments were cleaned with airabrasion to remove any soil.
Bone samples were cleaned by abrasion using a sandblaster. Samples were demineralised in 0.5M HCl for 1-7 days and rinsed three times with H2O. The residue was gelatinised in pH3 HCl at 70°C for 48 hours and the solution Ezee-filtered. Samples were lyophilised in a freeze dryer for 48hrs. Enamel was pretreated to remove organic or secondary carbonate contaminants. Samples were washed in 1.5% sodium hypochlorite for 60 minutes, followed by three rinses in purified H2O and centrifuging, before 0.1M acetic acid was added for 10 minutes, followed by another three rinses in purified H2O. Following reaction with 100% phosphoric acid, gases evolved from the samples were analyzed to stable carbon and oxygen isotopic composition using a Thermo Gas Bench 2 connected to a Thermo Delta V  Tables 7-9). Precision (u(Rw)) was determined to be ± 0.06 ‰, accuracy or systematic error (u(bias)) was ± 0.11 ‰ and the total analytical uncertainty in !13C values was estimated to be ± 0.13 ‰ using the equation presented in Supplementary material (Supplementary Table 10-13).The nitrogen contents of the samples were calculated based on the area under the N2 peak relative to the weight of the sample, calibrated using IAEA-N2. Stable nitrogen isotope values were calibrated to the AIR scale using IAEA-N-1 (!15N 0.4 ± 0.2 ‰) and IAEA-N-2 (!15N 20.3 ± 0.2 ‰). Measurement uncertainty in !15N values was monitored using three in-house standards: LEU (DL-leucine, !15N 6.5 ± 0.4 ‰), GLU (DL-glutamic acid monohydrate, !15N -1.9 ± 0.1 ‰) and MIL (millet flour from a single panicle from a plot in Senegal, !15N 3.1 ± 0.6 ‰). u(Rw) was determined to be ± 0.18 ‰, u(bias) was ± 0.59 ‰ and the total analytical uncertainty in !15N values was estimated to be ± 0.61 ‰. Analysis was performed by Amy Styring.
Dental calculus was removed from three mandibular molars: M1-M3. Images of the mineralised plaque prior to removal, as well as those from contaminant starch granules and phytoliths are published elsewhere.Microbotanical materials released from the calcified