Rifkin, Riaan F.Vikram, SurendraRamond, Jean-BaptisteRey-Iglesia, AlbaBrand, Tina B.Porraz, GuillaumeVal, AuroreHall, GrantWoodborne, Stephan M.Le Bailly, MatthieuPotgieter, MarnieUnderdown, Simon J.Koopman, Jessica E.Cowan, Don A.Van de Peer, YvesWillerslev, EskeHansen, Anders J.2021-05-252021-05-252020-05-06Rifkin, R.F., Vikrarn, S., Ramond, J.B. et al. 2020, 'Multi-proxy analyses of a mid-15th century middle iron age Bantu-speaker palaeo-faecal specimen elucidates the configuration of the ‘ancestral’ sub-Saharan African intestinal microbiome', Microbiome, vol. 8, art. 62, pp. 1-23.2049-2618 (online)10.1186/s40168-020-00832-xhttp://hdl.handle.net/2263/80097Additional file 1: Figure S1. Additional information concerning the archaeological provenance of the BRS faecal specimen. Figure S2. Processing of the faecal specimen at the Centre for GeoGenetics, Copenhagen, Denmark. Figure S3. Dot-plot indicating the occurrence of statistically-significant C-T p-values. Figure S4. Biplot of δ13C and δ15N stable isotope values obtained for the BRS specimen. Figure S5. SEM analysis detected bacterial cells, plant fragments and saprophytic organisms. Figure S6. Heat-map indicating differences in taxonomic community structure for IM datasets. Figure S7. Comparing ‘relative abundance’ and ‘presence-absence’ as taxonomic representation. Figure S8. Comparison of the incidence of the twenty-four authenticated ancient IM taxa. Figure S9. Heat-map indicating the presence of fifteen functional ARGs identified.Additional file 2: Table S1. Sequence reads for environmental- and subsistence-related taxa detected. Table S2. Information concerning 14C Accelerator Mass Spectrometry (AMS) dating. Table S3a. Processing protocol and results for isotope analyses. Table S3b. Results for isotope analyses (Merck standard). Table S3c. Results for isotope analyses (DLValine standard). Table S4. Abundance of bacterial taxonomic categories in the IM datasets. Table S5. Sequence read-length distribution for taxa identified in this study. Table S6. Significant KEGG pathways in the comparative IM datasets analysed. Table S7. Relative abundance of eighteen significant KEGG pathways in the IM cohorts. Table S8. Enrichment and depletion of KO metabolic gene categories in the comparative IM sample cohorts based on p-value (p=<0.05) designation. Table S9. Enrichment and depletion of KO metabolic gene categories in the comparative IM sample cohorts based on false discovery rate (FDR) corrected p-values (q=<0.05). Table S10. Enrichment and depletion of KO metabolic gene categories in the ancient and modern comparative IM sample cohort as calculated for the twenty-four authenticated ancient IM taxa. Table S11. Comparison of relative abundance of antibiotic resistance genes in the comparative IM cohorts. Table S12. Raw and filtered high-quality sequence read counts as related to the comparative IM datasets. TableS13. Information concerning the comparative NCBI genomes used during this study.BACKGROUND : The archaeological incidence of ancient human faecal material provides a rare opportunity to explore the taxonomic composition and metabolic capacity of the ancestral human intestinal microbiome (IM). Here, we report the results of the shotgun metagenomic analyses of an ancient South African palaeo-faecal specimen. METHODS : Following the recovery of a single desiccated palaeo-faecal specimen from Bushman Rock Shelter in Limpopo Province, South Africa, we applied a multi-proxy analytical protocol to the sample. The extraction of ancient DNA from the specimen and its subsequent shotgun metagenomic sequencing facilitated the taxonomic and metabolic characterisation of this ancient human IM. RESULTS : Our results indicate that the distal IM of the Neolithic ‘Middle Iron Age’ (c. AD 1460) Bantu-speaking individual exhibits features indicative of a largely mixed forager-agro-pastoralist diet. Subsequent comparison with the IMs of the Tyrolean Iceman (Ötzi) and contemporary Hadza hunter-gatherers, Malawian agro-pastoralists and Italians reveals that this IM precedes recent adaptation to ‘Western’ diets, including the consumption of coffee, tea, chocolate, citrus and soy, and the use of antibiotics, analgesics and also exposure to various toxic environmental pollutants. CONCLUSIONS : Our analyses reveal some of the causes and means by which current human IMs are likely to have responded to recent dietary changes, prescription medications and environmental pollutants, providing rare insight into human IM evolution following the advent of the Neolithic c. 12,000 years ago.en© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License.Ancient DNAHuman evolutionMolecular ecologyIntestinal microbiomeTaxonomic compositionMetabolic capacityArticle