Supplementary Information for The evolution and changing ecology of the African hominid oral microbiome James A. Fellows Yates, Irina M. Velsko, Franziska Aron, Cosimo Posth, Courtney A. Hofman, Rita M. Austin, Cody E. Parker, Allison E. Mann, Kathrin Nägele, Kathryn Weedman Arthur, John W. Arthur, Catherina C. Bauer, Isabelle Crevecoeur, Christophe Cupillard, Matthew C. Curtis, Love Dalén, Marta Díaz-Zorita Bonilla, J. Carlos Díez Fernández-Lomana, Dorothée G. Drucker, Elena Escribano Escrivá, Michael Francken, Victoria E. Gibbon, Manuel Gonzalez Morales, Ana Grande Mateu, Katerina Harvati, Amanda G. Henry, Louise Humphrey, Mario Menéndez, Dušan Mihailović, Marco Peresani, Sofía Rodríguez Moroder, Mirjana Roksandic, Hélène Rougier, Sandra Sázelová, Jay T. Stock, Lawrence Guy Straus, Jiří Svoboda, Barbara Teßmann, Michael J. Walker, Robert C. Power, Cecil M. Lewis, Krithivasan Sankaranarayanan, Katerina Guschanski, Richard Wrangham, Floyd E. Dewhirst, Domingo C. Salazar- Garcia, Johannes Krause, Alexander Herbig, Christina Warinner. James A. Fellows Yates, Christina Warinner Email: [email protected], [email protected] This PDF file includes: Supplementary text Figures S1 to S14 Table S1 Legends for Datasets S1 to S3 SI References Other supplementary materials for this manuscript include the following: Datasets S1 to S3 1 Table of Contents Supplementary Information Text S1. Primate host species and subspecies included in this study 4 S1.1 Alouatta (Outgroup) 4 S1.1.1 Alouatta palliata (Mantled howler monkey) 4 S1.2 Gorilla 5 S1.2.1 Gorilla beringei (Eastern gorilla) 5 S1.2.2 Gorilla gorilla (Western lowland gorilla) 6 S1.3 Pan 6 S1.3.1 Pan troglodytes (Common chimpanzee) 7 S1.4 Homo 8 S1.4.1 Homo neanderthalensis (Neanderthal) 8 S1.4.2. Homo sapiens (Modern Human) 9 S2. Laboratory procedure 11 S2.1 Dental calculus collection 12 S2.2 DNA extraction 12 S2.3 Library preparation and sequencing 13 S2.3.1 Shallow Sequenced Dataset 13 S2.3.2 Deep Sequenced Dataset 14 S2.4 Sequencing 15 S2.4.1 Dental calculus 15 S2.4.2 Controls 15 S2.4.3 Sequence data 15 S3. Data processing and quality filtering 15 S3.1 Publicly available data 15 S3.1.1 Dental calculus 15 S3.1.2 Modern human microbiome and environment 16 S3.2 Sequencing Quality Control and Human DNA Removal 16 S3.2.1 Shallow sequenced dataset 17 S3.2.2 Deep Sequenced dataset 17 S3.3 Taxonomic binning and classification 17 S3.4 Preservation assessment and removal of low quality samples 19 S3.4.1 Cumulative percent decay 20 S3.4.2 SourceTracker 22 S3.4.3 Comparison of methods 24 S3.4.4 Abundance of eukaryotic content as a preservation indicator? 24 S3.5 Ancient DNA authentication 24 S3.6 Contamination assessment and removal 25 S4. Microbial compositional analysis 26 S4.1 Principal coordinates analysis (PCoA) 26 S4.2 PERMANOVA 27 S4.3 Hierarchical clustering 28 S4.4 Indicator analysis 30 S4.5 Are dietary practices associated with oral microbiome composition? 31 S5. Core microbiome 32 S5.1 Biofilm spatial organization and development: current state of knowledge 32 S5.1.1 Periodontal disease and associated microbial taxa 34 S5.2 Parameter selection of core microbiome analysis 36 S5.3 Comparison among African hominid core microbiomes 39 S5.4 Evolutionary and ecological relationships among core oral microbes of African hominids 40 S5.5 Evolution of oral disease virulence factors in late-colonisers 42 S5.6 Host-associated differences in early-coloniser Streptococcus species 43 S5.6.1 Streptococcus ɑ-amylase-binding proteins as an indicator of host-microbe coevolution 44 S5.7 Detection of amylase-binding protein genes abpA and abpB 46 S5.8 Salivary amylase, cooked starches, and the Expensive-Tissue Hypothesis 47 2 S5.9 Dating expansion of starch-associated streptococci using amylase binding proteins 51 S5.9.1 abpA 51 S5.9.2 abpB 52 S6. Microbial phylogenetics 52 S6.1 Super-reference construction 53 S6.2 Alignment and species selection 54 S6.3 Performance of super-reference vs. single reference mapping 55 S6.4 Single reference genome mapping statistics 56 S6.5 Variant calling and single-allelic position assessment 56 S6.6 Phylogenetic trees 57 S7. Functional and metabolic pathway analysis 60 S7.1 HUMAnN2 60 S7.1.1 Pathway abundance 61 S7.1.2 KEGG ortholog distribution 61 S7.1.3 Species contributions to KEGG orthologs 64 S7.1.4 Metabolic category PCAs 65 S7.2 AADDER 66 S7.2.1 SEED profile 66 S7.2.2 Species contributions to SEED proteins 69 S7.2.3 Metabolic category PCAs 70 Supplementary Figures Fig. S1. Primate species analysed in this study and their dental calculus 72 Fig. S2. Flowchart of study processing, authentication and analysis procedures 73 Fig. S3. Data quality and authentication filters applied to dental calculus samples in this study 74 Fig. S4. Representative ancient authenticity metrics for Neanderthals from Pesturina (PES001) and de Nadale (GDN001) caves 75 Fig. S5. Principal Coordinates Analysis (PCoA) of dental calculus and sources 76 Fig. S6. Hierarchical clustering of different Homo calculus microbiomes, comparing different lifestyles and regions 77 Fig. S7. Alluvial diagram showing effects of increasing the minimum abundance threshold to the MALT OTU table-based core microbiome calculations 78 Fig. S8. Alluvial diagram showing effects of dropping and retaining a single-individual Gorilla population in core microbiome calculations at genus and species taxonomic levels 79 Fig. S9. Core microbiome strategy and virulence genes 80 Fig. S10. Comparison of the number of multi-allelic single nucleotide polymorphisms (SNPs) identified when using a single representative genome mapping strategy versus a multi-reference genome (super-reference) mapping approach 81 Fig. S11. Neighbour joining trees of eight well-supported core calculus taxa from single representative mappings of the deep sequenced dataset 82 Fig. S12. Replication of production dataset phylogenies with low-coverage and damage-containing screening dataset with additional European individuals 83 Fig. S13. Principal Components Analysis (PCA) biplots of functional annotations with the top 10 positive and negative loadings separating Homo from the other hosts 84 Fig. S14. Principal Components Analysis (PCA) using KEGG orthologs or SEED-classified proteins belonging to specific metabolic pathway categories 85 Supplementary Tables Table S1. Summary of host species and dental calculus samples included in study 86 Legends for Datasets S1 to S3 Data S1. Laboratory metadata: detailed sample, laboratory and analytical metadata (.xlsx) Data S2. Metagenomic taxa tables: OTU tables from MALT taxonomic assignment (.xlsx) Data S3. Core microbiome taxa (.xlsx) References 88 3 S1. Primate host species and subspecies included in this study To better understand oral microbiome evolution and ecology in primates, dental calculus from six host species spanning three hominid genera and one New World howler monkey outgroup were collected and analysed in this study (Table S1). These taxa were selected to focus on African members of the family Hominidae (great apes including ‘archaic’ and modern humans) and using the New World howler monkey species Alouatta palliata as an outgroup. A cladogram illustrating the relationships between these host species is provided in Fig. S1A. Dental calculus samples in this study were obtained from primate research stations, museums, dental clinics, and archaeological collections originating from 19 countries in Africa, Europe, and Central America (Fig. 1a; Data S1). For present-day modern humans, dental calculus was obtained under informed consent during routine dental cleanings, and research protocols for analyzing de-identified samples were approved by the Institutional Review Board for Human Research Participant Protection at the University of Oklahoma (IRB#4543). Archaeological dental calculus within the genus Homo was obtained from three groups chosen to represent distinctive phases in human history: 1) pre-antibiotic era modern humans, 2) pre-agricultural modern humans, and 3) extinct Homo. All non-human primate dental calculus was obtained postmortem from wild populations. To avoid potential group- or collection-specific batch effects, dental calculus samples for each host species were drawn from at least two different populations, with the exception of the howler monkey outgroup, which were all obtained from a single island in Nicaragua. A total of 115 new dental calculus samples from 109 individuals were initially screened for this study, of which 75 individuals passed genetic quality filters. In addition, we also analysed previously published calculus data from four Neanderthals, one chimpanzee (1), and ten present-day modern humans (2), of which all but one Neanderthal sample passed our genetic quality filters. Our combined total sample size was thus 124 individuals, of which 89 passed genetic quality filters. An overview of each host species, contributing institution, and collection details are provided below and are summarised in Data S1. Below we describe each species, their dietary habits, and known factors affecting their oral environment (e.g., saliva production, salivary pH, salivary protein composition, and antifeedant intake), which may influence their oral microbiome. Please note that in the following sections we use the following temporal terminology: ‘Ma’ for million years ago; ‘ka’ for thousand years ago. S1.1 Alouatta (Outgroup) Alouatta is a genus of New World monkeys in the family Atelidae native to tropical forests in Central and South America. New World monkeys (Platyrrhini) are estimated to have diverged from Old World monkeys and apes (Catarrhini) approximately 40 Ma (3, 4). S1.1.1 Alouatta palliata (Mantled howler monkey) Mantled howler monkeys are among the largest Central American monkeys. They consume a seasonal diet mostly of young leaves and ripe fruits, focusing primarily on trees in the family Moraceae, especially Ficus spp., Brosimum alicastrum, and Poulsenia armata, but also the trees Cecropia obtusifolia, Spondias radlkoferi, and Dipholis minutiflora (5). Microwear analysis of A. palliata dentition shows a high degree of anisotropy (6) reflecting the relative toughness of their diet and repetitive masticatory abrasion by plant phytoliths.