Jomon: Paleolithic Contingent in Modern Japanese

Mitochondrial DNA analysis of the human skeleton of the initial Jomon phase excavated at the Yugura cave site, Nagano, Japan
NOBORU ADACHI, JUNMEI SAWADA, MINORU YONEDA, KOICHI KOBAYASHI, SHIGERU ITOH

Abstract
Obtaining genetic information about early humans is indispensable to our understanding of the demographic history of mankind. In the present study, we performed a detailed mitochondrial DNA (mtDNA) analysis of a skeleton of the initial Jomon era unearthed from the Yugura cave site in Nagano, Japan, which was dated to 7920–7795 calBP by direct 14C dating. mtDNA of the Yugura skeleton was designated to haplogroup D4b2, which is widely observed in present-day East Asians, including the Japanese, but is absent in Hokkaido Jomon people. This finding indicates that the basal population of Japan was heterogeneous with respect to their mtDNA lineage. This is the first report on the genotype of the people from the initial phase of the Jomon period.




Figure 1
Map of the Yugura cave site, located in Nagano prefecture, central Japan.


Introduction
Mitochondrial DNA (mtDNA) studies of the modern Japanese have revealed that this population is rich in matrilineal diversity (e.g. Maruyama et al., 2003; Tanaka et al., 2004), and that there is considerable regional difference in haplogroup frequencies (e.g. Umetsu et al., 2005; Matsukusa et al., 2010). Although Jomon people are generally considered to be homogeneous from the morphological viewpoint (e.g. Hanihara, 1991), mtDNA analyses of the Jomon people suggest that maternal heterogeneity and its regional differences were observed as long ago as the Jomon era (Horai et al., 1989; Shinoda and Kanai, 1999; Shinoda, 2003; Adachi et al., 2011).

Moreover, studies on lithic industries indicate that regional differences in their styles can be extended back to the Upper Paleolithic era (reviewed by Oda, 2003). This cultural variation may reflect the difference of the genetic background of the people.

To clarify the origin of this heterogeneity, obtaining genetic information about the early (if at all possible, Paleolithic) humans is indispensable. However, currently, the oldest genetic data of humans in Japan are from early Jomon skeletons (about 6000 years before present) (Horai et al., 1989; Adachi et al., 2011).

In the present study, we performed a detailed mtDNA and radiocarbon analyses of the Jomon skeleton unearthed at the Yugura cave site in Nagano, Japan. This skeleton belongs to the initial Jomon phase, and it is known to be one of the oldest Jomon skeletons in Japan.

Figure 5
Picture of the skeleton excavated from the Yugura cave site.

Radiocarbon dating
Collagen was extracted by the gelatinization method (Longin, 1971; Yoneda et al., 2002), and its purity collagen estimated based on yield, carbon and nitrogen concentrations (Van Klinken, 1999). Radiocarbon dating was conducted on a graphite target produced from extracted collagen (Kitagawa et al., 1993) using the accelerator mass spectrometry (AMS) facility of Paleo Labo Co., Ltd. (Kobayashi et al., 2007).

Extraction and purification of DNA
Intensive precautionary measures to avoid modern DNA contamination were carried out as described previously (Adachi et al., 2011). DNA was extracted from the left lower third molar and from the left lower first molar independently, in order to validate the results.

Tooth samples were dipped in a 13% bleach solution (Nacalai Tesque Inc., Kyoto, Japan) for 15 min, rinsed several times with DNase/RNase-free distilled water (Invitrogen, Carlsbad, CA), and allowed to air-dry. Furthermore, the outer surface of the samples was removed to a depth of 1 mm with a dental drill. Next, the samples were rinsed with DNase/RNase-free distilled water once again and allowed to air-dry under irradiation in a UV crosslinker for 90 min. The samples were then encased in Exafine silicone rubber (GC, Tokyo, Japan) following the protocol described by Gilbert et al. (2003). After approximately 10 min, or once the rubber was hard, the tip of the root of the tooth sample was removed with a horizontal cut and then powdered using a mill (Multi-beads shocker®; Yasui Kikai, Osaka, Japan). Thereafter, the dentin around the cavitas dentis and the dental pulp was powdered and removed through the root tip by using a dental drill. The powdered samples were decalcified with 0.5 M EDTA (pH 8.0) (Invitrogen) at 56°C overnight. The EDTA buffer was then replaced with a fresh solution, and the samples were decalcified for a further 36 h at 56°C. Decalcified samples were lysed in 1000 μl of Genomic Lyse buffer (Genetic ID, Fairfield, IA) with 50 μl of 20 mg/ml proteinase K (Invitrogen) overnight at 56°C. Finally, the lysate was extracted twice with 1200 μl of phenol/chloroform/isoamyl alcohol (25:24:1) and 1200 μl of chloroform. DNA was extracted from the lysate by using a FAST ID DNA Extraction Kit (Genetic ID) in accordance with the manufacturer’s instructions.

Sequencing and single nucleotide polymorphisms typing of mtDNA
Segments of mtDNA that covered parts of the tRNAPro gene, the hypervariable segments (HVS) 1 (nucleotide position (np) 15999–16141, 16121–16238, and 16209–16366, relative to the revised Cambridge reference sequence (rCRS: Andrews et al., 1999), HVS 2 (np 128–256), and a segment of the coding region that covered parts of the NADH dehydrogenase 3 and tRNAArg genes (np 10287–10425) were amplified and sequenced using primers that have been described previously (Adachi et al., 2009). In this regard, the –21M13 and M13RVN sequences were removed from these primers to maximize the robustness of the polymerase chain reaction (PCR). To exclude the possibility of modern DNA contamination, candidates for contamination were analyzed for the segments described above. Informed consent was obtained from the DNA donors. This study was approved by the ethics committee of the University of Yamanashi.

Amplifications were carried out in a total reaction volume of 20 μl, containing a 2 μl aliquot of DNA extract, 0.2 μM of each primer, and reagents of the Multiplex PCR kit (QIAGEN, Hilden, Germany). The conditions for PCR included incubation at 95°C for 15 min, followed by 36 cycles at 94°C for 20 s, 54°C for 90 s, and 72°C for 15 s, and a final extension at 72°C for 10 min. Following PCR, the products were subjected to direct sequencing using the same primers and the BigDye® Terminators v. 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on a 310 DNA Sequencer (Applied Biosystems) equipped with Ridom TraceEdit software (Ridom, Würzburg, Germany).

To securely assign mtDNAs to relevant haplogroups, 36 haplogroup-diagnostic single nucleotide polymorphisms (SNPs) and a 9 bp repeat variation in the non-coding cytochrome oxidase II/tRNALys intergenic region (9 bp) were analyzed by the multiplex amplified product-length polymorphism (APLP) method. Protocols were identical to those described previously (Adachi et al., 2009), except for the PCR condition: incubation at 95°C for 15 min, followed by 40 cycles at 94°C for 30 s, 56°C for 90 s, and 72°C for 10 s, and a final extension at 72°C for 10 min.

Thereafter, we assigned Yugura mtDNAs to the relevant haplogroup according to the HVS and coding region data, by using the classification tree built by Van Oven and Kayser (2009).