Ancient DNA analysis of food remains in human dental calculus from the Edo period, Japan

Abstract

Although there are many methods for reconstructing diets of the past, detailed taxon identification is still challenging, and most plants hardly remain at a site. In this study, we applied DNA metabarcoding to dental calculus of premodern Japan for the taxonomic identification of food items. DNA was extracted from 13 human dental calculi from the Unko-in site (18th-19th century) of the Edo period, Japan. Polymerase chain reaction (PCR) and sequencing were performed using a primer set specific to the genus Oryza because rice (Oryza sativa) was a staple food and this was the only member of this genus present in Japan at that time. DNA metabarcoding targeting plants, animals (meat and fish), and fungi were also carried out to investigate dietary diversity. We detected amplified products of the genus Oryza from more than half of the samples using PCR and Sanger sequencing. DNA metabarcoding enabled us to identify taxa of plants and fungi, although taxa of animals were not detected, except human. Most of the plant taxonomic groups (family/genus level) are present in Japan and include candidate species consumed as food at that time, as confirmed by historical literature. The other groups featured in the lifestyle of Edo people, such as for medicinal purposes and tobacco. The results indicate that plant DNA analysis from calculus provides information about food diversity and lifestyle habits from the past and can complement other analytical methods such as microparticle analysis and stable isotope analysis.

Fig 1. Close-up views of dental calculus on the teeth from the sampled individuals of the Unko-in site.

Fig 2. PCR amplification products of dental calculus using Oryza atpE gene primer sets.

M, molecular weight markers; 1, wn2016-F01; 2, wn2016-F04; 3, wn2016-F07; 4, wn2016-F08; 5, wn2016-F10; 6, wn2016-F12; 7, wn2016-F23; 8, negative control; 9, wn2016-F24; 10, wn2016-F37; 11, wn2016-F39; 12, wn2016-F41; 13, wn2016-F43; 14, wn2016-F44; 15, soil from wn2016-F39; 16, negative control.