- Oxford, United Kingdom
Recent genomic data have revealed multiple interactions between Neanderthals and modern humans 1 , but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing... more
Recent genomic data have revealed multiple interactions between Neanderthals and modern humans 1 , but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering 2,3. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess 4 and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)—the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution. Neanderthals remain our closest known, extinct, hominin relatives, who co-existed and occasionally interbred with anatomically modern humans across Eurasia in the Late Pleistocene epoch 1. Neanderthals became extinct in Europe around 40,000 years ago (40 ka), although the extinction process across the rest of Eurasia is less clear 5. Archaeological and isotopic data from the last glacial cycle (around 120–12 ka) suggest that Neanderthals were as carnivorous as polar bears or wolves 6 with a diet heavily based on large terrestrial herbivores, such as reindeer, woolly mammoth, and woolly rhinoceros 7. By contrast, microwear analysis of tooth surfaces from Neanderthals in different ecological settings, such as wooded areas or open plains, suggests that diets were guided by local food availability 3. Analyses of phytoliths, starch granules, and proteins from calcified dental calculus also indicate that Neanderthal diets included many plants, including some that were used for medicinal purposes 8. As a result, Neanderthal diet remains a topic of considerable debate, with limited data on the specific animals and plants directly consumed or the potential effects on Neanderthal health and disease. Although genomic studies continue to reveal evidence of inter-breeding between anatomically modern humans and Neanderthals across Eurasia 9 , little is known about the health consequences of these interactions. The genetic analysis of Neanderthal dental calculus represents an opportunity to examine this issue and to reconstruct Neanderthal diet, behaviour, and disease 10. Here, we report the first genetic analysis of dental calculus from five Neanderthals (two individuals from El Sidrón cave in Spain; two individuals from Spy cave in Belgium; and one individual from Breuil Grotta in Italy) and compare these data to a historic wild-caught chimpanzee (n = 1) and modern human (n = 1), as well as to low coverage sequencing of calculus from a wide-range of ancient humans (Supplementary Table 1). To provide increased resolution of the diseases that may have affected Neanderthals, we also deeply sequenced (> 147 million reads) dental calculus from the best-preserved Neanderthal, El Sidrón 1, which suffered from a dental abscess 4. Size-based PCR-amplification biases can confound standard metabar-coding analyses (for example, sequencing of 16S ribosomal (r)RNA amplicons 11,12) of ancient dental calculus 13. Consequently, we compared metagenomic-shotgun sequencing and 16S rRNA amplicon (V4 region) analyses of the Neanderthal dental calculus specimens—by far the oldest examined to date. The 16S amplicon datasets were not representative of the biodiversity revealed by shotgun sequencing (Extended Data Figs 1, 2 and Supplementary Tables 2, 3, 7, 16), as samples clustered according to methodolgy (Fig. 1) and contained disproportionately large amounts of non-oral microorganisms that were environmental contaminants (Supplementary Tables 2, 7 and Extended Data Figs 3, 4a). As a result, the 16S amplicon datasets were excluded from downstream analysis, along with the Neanderthal sample from the Breuil Grotta, which failed to produce amplifiable sequences. The shotgun datasets consisted of short DNA fragments (< 70 bp), which complicated accurate identification of bacterial species using standard software, such as MG-RAST or DIAMOND 14 (Extended Data Fig. 4b, see Supplementary Information). To circumvent this problem, we benchmarked and used a new metagenomic alignment
