Jomon: Paleolithic Contingent in Modern Japanese

Figure 4. Phylogenetic analysis I. TreeMix tree with gene flows. A comparison of Sanganji Jomon, 1000 Genomes Project worldwide populations, Papuan, Karitiana, Mal’ta MA1, Ust’-Ishim and Denisovan based on 43 310 all sites. Denisovan was used as the outgroup, and three gene flow events were estimated. The tree was drawn by using MEGA6.38 Red colored values (only those higher than 90% are shown) are bootstrap probabilities (%) for their adjacent internal branch. Arrows were manually added to this tree, and colors of migration weight (ratio of gene flow) follow TreeMix outputs. Values inside arrows are the ratio of gene flow. Bootstrap probabilities (%) of the gene flow from Sanganji Jomon to JPT, Karitiana to the root of European and Mal’ta MA1, and LWK to European, estimated out of 1000 bootstrap replicate TreeMix outputs, are 42%, 86% and 0.4%, respectively.

TreeMix analysis We constructed maximum-likelihood population trees (ML tree) using TreeMix to investigate the phylogenetic relationship and the presence of admixture events between Sanganji Jomon and other human populations. When three migration events were assumed, the gene flow from Sanganji Jomon to JPT (modern Japanese) appeared (Figure 4) (bootstrap probability=42%). The directionality of this gene flow event was as expected, however, the inferred gene flow from Karitiana to Mal’ta MA1 (Supplementary Results S2; Supplementary Figures S16b-j and S17c-j), was in the reverse direction to what was reported by Raghavan et al.23 who used a much larger sequence data. This result might have been caused by using a relatively small SNP data set. We therefore used only modern human data, and reran TreeMix. We used a much larger 0.7 million SNP loci data. However, the resulting tree showed some anomalous gene flow directions; from Papuans to Denisovans (bootstrap probability=98%) and CEU to Papuans (bootstrap probability=86%), whereas the tree topology was consistent with that of Figure 4 (see Supplementary Figure S19). The reason for this anomaly may be that some filtering steps between our analyses and Reich et al.16 and Meyer et al.19 are different, and/or homozygous diploid genotypes in individual genome (that is, Denisovan, Papuan and so on) were used instead of their original genotype, though gene flow from Mal’ta MA1 to Karitiana correctly appeared in both all sites and transversion only when we add Mal’ta MA1, Karitiana and Ust’-Ishim to the large SNP loci data (data not shown).
Figure 4.


Figure 5. Phylogenetic analysis II. Distance-based. A total of 15 519 transversion sites were used for computing distances. (a) Neighbor-joining tree. (b) Neighbor-net network. Branches for populations starting with ‘~’ were shortened for clarity.

Distance matrix-based analysis We constructed neighbor-joining trees (Figure 5a and Supplementary Figure S20) from distance matrices, and the Native American diverged after the divergence of the Sanganji Jomon from the modern East Eurasians in these trees. This is consistent with Figure 4, although the bootstrap probability to support this branching pattern was only 64% in Figure 5a. We also drew a Neighbor-Net network using the same distance matrix (Figure 5b and Supplementary Figure S21). Major splits are consistent with the neighbor-joining tree (Figure 5a), and split X clustered the Sanganji Jomon and JPT. This split confirms the admixed nature of the mainland Japanese inferred from the PCA and TreeMix analyses. See Supplementary Results S3 for more detailed analysis using Neighbor-Net.

Ishigaki Island is one of the westernmost islands in Japan. Due to its geographical location, it is considered to have played a significant role in the migration route from Southern Asia to the Japanese archipelagos. Recently, human remains were excavated from Shiraho-Saonetabaru Cave, constituting the first physical evidence of human occupation on Ishigaki Island. In order to investigate the genetic makeup of the ancient Ishigaki people and to assess their genetic relationship with other Asian populations at a molecular level, we analysed the single nucleotide polymorphisms of the coding region of mtDNA that defines the haplogroups of these individuals. Because of the poor quality of the DNA extracted from the ancient material, it was not possible to analyse all samples. Among the 10 samples considered in this study, ancient DNA data was successfully extracted from five individuals. MtDNA haplogroups show geographic specificity within Asia; the existence of haplogroup B4e and M7a in this population hints at their linkage with Southeast Asia and the Late Pleistocene Ryukyu Islands.


Figure 3.1 The geographic distribution of the islands in the Ryukyu Archipelago, and the location of Shiraho-Saonetabaru on Ishigaki Island.

When ancient DNA is analysed, it is necessary to exclude false-positive results caused by contamination with contemporary DNA (Sampietro et al. 2006). In order to prevent contamination during excavation, the remains were handled using gloves and were not touched with bare hands. Bone samples were wrapped in aluminium foil and stored in a refrigerator at 4°C until DNA extraction. Standard precautions were practised to avoid contamination, such as separation of pre- and post-polymerase chain reaction (PCR) experimental setups, use of disposable laboratory gear and filter-plugged pipette tips, treatment with DNA contamination removal solution (DNA Away; Molecular Bio Products, San Diego, CA, USA), ultraviolet (UV) irradiation of equipment and benches, and negative extraction and PCR controls (Shinoda et al. 2006).

DNA was extracted from the skeletal samples, according to previously published protocols (Adachi et al. 2009). The tooth and bone samples were dipped in DNA contamination removal solution for 15 min, rinsed with DNase-/RNase-free distilled water, and allowed to air-dry. The outer surface of the samples was removed using a dental drill, and the samples were again rinsed with DNase-/RNase-free distilled water and allowed to air-dry under UV irradiation. Then the tooth samples were encased in silicone rubber and the dentin around the cavitas dentis and the dental pulp was powdered and extracted through the cut plane of the root tip, as described by Gilbert et al. (2003). The bone samples were pulverised using a mill (Multi-beads shocker; Yasui Kikai, Osaka, Japan).


Figure 3.2 Results of PCR-luminex analysis. Number of each lane shows the sample number. Lane 1 and 2 are positive controls and lane 9 is negative control.
A: 63 base pair amplified products indicates haplogroup M7a.
B: 54 base pair products indicates haplogroup M7a1.
M: size marker.

Though small in number, the distribution of mtDNA haplogroups in this period provides insights into regional population history. Haplogroups B4 and R are the most prevalent in Southeast Asia, especially in the coastal region (Trejaut et al. 2005), indicating that this haplogroup may have been introduced to Japan from Southeast Asia. Interestingly, haplogroup B was also found in ancient Chinese samples (Fu et al. 2012). It seems that the ancestral population of coastal East Asia and Island Southeast Asia was enriched by the founder lineages of haplogroup B4, and the Ryukyu Islands may be one of the northernmost regions where this population arrived in the Palaeolithic period. This finding indicates that the southeast influx into the ancient Ryukyu population affected their genetic makeup and that the ancestors of the Aboriginal Taiwanese or Asian coastal region populations might be the main source of this haplogroup in the Ryukyu Islands.

The geographic specificity of haplogroup M7a is the most intriguing result of this study. Its ancestral haplogroup M7, although a characteristic of East Asian populations, was not found in the northeast region of the continent (Torroni et al. 1993; Derenko et al. 2007). Haplogroup M7a is absent or scarce in the East and Southeast Asian populations outside Japan. Moreover, M7a is one of the prevailing haplogroups not only among modern Japanese, including Honshu, Okinawa islanders, and Ainu populations (Tanaka et al. 2004), but also in the Jomon population (Adachi et al. 2011). The frequency of haplogroup M7a among modern Japanese is highest in the Okinawa islanders (23.3 per cent; Umetsu et al. 2005) – gradually decreasing towards the northern part of Honshu (Shinoda 2007). This finding indicates that this haplogroup may have a southern origin. Moreover, the age of haplogroup M7a was calculated to be ca. 23,000 BP, and the age likely falls within the onset of the Last Glacial Maximum (LGM; Adachi et al. 2011).


Figure 3.3 Results of amplified product length polymorphism (APLP) analysis. Number of each lane shows the sample number.

Our results confirm that the haplogroup M7a entered Japan, with the earliest settlers more than 20,000 years ago from Southeast Asia or the southern region of the Asian continent. The fact that positive proof of human occupation by haplogroup M7a on Ishigaki Island appears about 30,000–20,000 BP fits this scenario.

The peopling in Japan can be seen as a complex process, as the earliest settlements and recent migrations affected the resident populations differently. Although we can draw limited conclusions from this study, our results of ancient mtDNA analysis help to shed light on late Palaeolithic human migrations to, and within, the Japanese archipelagos from Southeast Asia.

Since hot and humid conditions are unfavourable for DNA preservation, there is a low possibility of finding well-preserved DNA in regions with a tropical climate, like the Ryukyu Islands. However, the present study shows that sufficient amounts of DNA were available in the human skeletal samples from the late Palaeolithic period obtained from the Ryukyu Islands because the remains were protected within caves. It seems that caves are favourable burial sites from the viewpoint of DNA preservation (Fehren-Schmitz et al. 2011). However, further studies are necessary to obtain more details on the human skeletal remains excavated from this region.

Shinoda, Ken-ichi, and Noboru Adachi. “Ancient DNA Analysis of Palaeolithic Ryukyu Islanders.” New Perspectives in Southeast Asian and Pacific Prehistory, edited by Philip J. Piper et al., vol. 45, ANU Press, Acton ACT, Australia, 2017, pp. 51–60. JSTOR, www.jstor.org/stable/j.ctt1pwtd26.10.