Jomon: Paleolithic Contingent in Modern Japanese

Discussion

We generated genome-wide SNP data for Izumo in the San-in region and Makurazaki in the southern Kyushu region (see Fig. 1) using Japonica array. The genetic location of Makurazaki population in the PCA plot (Fig. 3) and phylogenetic network (Fig. 5) is more or less similar to the geographical location of this city, namely, between Okinawa and the mainland. This is supported by f4(GBR, x; Kinki, Makurazaki) test showing Makurazaki having more gene flow with Okinawa and Kyushu-A than with other Yamato.

Geographically, Izumo city is facing the sea between Yaponesia and Continental East Asia (Fig. 1). The distance to the Korean Peninsula is ~350 km, and it was expected that Izumo people may be closer to continental Asians. However, f4 test showed that the Kinki population is the closest to continental Asians (f4(GBR, CHB; Okinawa, x)). This is supported by another study using genome-wide SNPs [29]. Interestingly, Izumo population has higher genetic affinity to Okinawa and Kyushu than to other populations in Honshu, as suggested by f4(GBR, x; Izumo, Kinki). This discrepancy suggests an existence of some gene flow events that are different from usually expected isolation by distance [30].

Yamaguchi-Kabata et al. [9] analyzed genome-wide SNP data of 7,000 Japanese collected from seven geographical regions (Hokkaido, Tohoku, Kanto, Tokai, Kinki, Kyushu, and Okinawa) as part of the BBJ Project. They found the two clear clusters called Hondo and Ryukyu, which we also observed (Fig. 3). We also confirmed region specific characteristics discovered by Yamaguchi-Kabata et al. [9] and later confirmed by Okada et al. [31] in which Hokkaido, Kanto, and Tokai area individuals are more or less genetically similar, whereas Tohoku individuals are slightly differentiated from those three regions people based on PCA, network and f4 tests. We also confirm that Kinki people are the closest to continental Asians as reported by Yamaguchi-Kabata et al. [9] and Watanabe et al. [29]. Kinki area includes Nara and Kyoto, past capitals of Japan and thus has a history of human migration from the Asian continent.

These results and HLA data [11] prompted Saitou [32] to propose the three-migration model. Later, Saitou and Jinam [13] and Saitou [2] proposed the “Inner-Dual Structure” model of Yaponesians, shown in Fig. 6. After the initial peopling of the Japanese Archipelago by the ancestors of Jomon and possibly including the paleolithic period people (first migration), there were possibly two (or more) human migrations from the Asian continent. Although the timing of this event is not yet determined, these people may have occupied coastal areas of the main island Honshu (“Periphery” in Fig. 6). Then the third migration from the continent mostly involved movement between the Asian Continent and six areas (Hakata in northern Kyushu, Osaka, Kyoto, Nara in Kinki, Kamakura, and Edo-Tokyo in Kanto; shown in gray circles in Fig. 6). These “Central Axis” areas were historically the cultural and political centers in Yaponesia, and are assumed to attract many new migrants from continental East Asia.



Fig. 6

Two additional data also supported the “Inner-Dual Structure” model according to Saitou [2]; five mtDNA haplogroup frequency data of 18,641 Yaponesians sampled from all the 47 prefectures provided by Genesis Healthcare (see Supplementary Fig. 6; taken from Fig 52 of Saitou [2]) and ABO blood group allele frequency data of 46 prefectures except for Okinawa by Fujita et al. [33] and those for Okinawa Prefecture [34, 35] (see Supplementary Fig. 7; taken from Figure 53 of Saitou [2]). We divided the 47 prefectures into 26 peripheral (shown in blue) and 21 central axis (shown in red) in both data. In Supplementary Fig. 6, Principal Component 1 broadly separates peripheral (right side) and central axis (left side) populations. This bias is statistically significant at the 1% level using Fisher’s exact test (see the upper right 2 × 2 table of Supplementary Fig. 6). The frequency plot of alleles A and B of the ABO blood group (Supplementary Fig. 7) tend to group peripheral prefectures (shown in blue) to the lower left of the diagonal dotted line, and this bias is also statistically significant at the 1% level using Fisher’s exact test (see the upper right 2 × 2 table of Supplementary Fig. 7).

Recently, HLA data for all 47 prefectures were analyzed [12], and a phylogenetic relationship of 15 regions was shown. Interestingly, populations in northern Kyushu and southern Kanto are phylogenetically closer to continental Asian populations, whereas northern Tohoku, Shikoku and Okinawa formed one cluster. These patterns are compatible with the “Inner-Dual Structure” model shown in Fig. 6. However, San-in (belonging to Periphery in Fig. 6) and San-yo (belonging to Central Axis in Fig. 6) populations in Chugoku region are clustered in the phylogenetic tree of Hashimito et al. [12].

The “Inner-Dual Structure” model might explain why people in the Central Axis region appear closely related in the PCA and network analyzes, whereas people from the Periphery such as Makurazaki, Izumo, or Tohoku were less genetically influenced by the more recent migrations. However it is still not clear when those new and old migrants came to Yaponesia. Overall, these results appear to support the “Inner-Dual Structure” model, however other explanations such as very recent migrations might contribute to the observed patterns. The addition of samples from under-represented regions in Japan together with whole genome sequence data is needed to properly decipher the genetic make-up of Yaponesians.

Jōmon genetics
The transition from the Jōmon to the Yayoi periods marked a transition in the material culture of Japan, with the appearance and increase of tools, artefacts and archaeobotanical remains associated with agriculture. Studies have long associated this transition with the migration of Mumun people through the Korean peninsula into the closest island in the Japanese archipelago, Kyushu (Hammer & Horai, 1995; Hanihara, 1991). Early assessments using cranial and dental morphology and genetics of humans and their commensals led to the Dual Structure model (Hanihara, 1991), for which a key component is the argument that present-day Japanese are a mixture of populations indigenous to Japan since the Palaeolithic and later migrants from mainland Asia. Many studies, mostly using present-day genomes of indigenous populations in the archipelago (i.e. Ainu, Ryūkyū) as a proxy for Jōmon-related ancestry, found genetic patterns that suggest that the present-day Japanese populations are admixed, supporting that aspect of the Dual Structure model (Jeong, Nakagome, & Di Rienzo, 2016a; Jinam et al., 2015; Nakagome et al., 2015).

Advances in DNA sequencing methods have allowed access to genetic material for studying population demographic history in the Japanese archipelago. Uniparental markers were some of the earliest used to infer population history; these include mitochondrial DNA (mtDNA), which exists outside the nucleus, and the non-recombining portion of the Y-chromosome. The primary units considered when studying these markers are haplogroups, which are variants of a DNA segment that derives from the same common ancestor, and they can provide information on a population's genetic history.

Since uniparental transfer results in no recombination, it is straightforward to reconstruct phylogenetic trees and assign a haplogroup to the mtDNA or Y-chromosome region (Haber, Mezzavilla, Xue, & Tyler-Smith, 2016). Sets of haplogroups are now well annotated and easily identifiable in newly sequenced individuals using many tools and databases (e.g. ISOGG, 2019; Weissensteiner et al., 2016). If they are identified to be at high frequency in a localized area but in low frequency elsewhere, the origin of the haplogroup, or the common ancestor, can be inferred to have derived from that region. While the lack of recombination allows simple haplogroup assignation, it also means that it is a single data point representing population history, and stochastic events or sex-biased migration patterns can lead to inaccurate inference of the true population history (Nordborg, 1998; Rosenberg & Nordborg, 2002).

In contrast, the nuclear genome undergoes recombination, which means that small segments of each chromosome have effectively independent histories. Study of a single diploid genome (i.e. the 23 pairs of chromosomes in the cell nucleus) can be interpreted as study of small portions of thousands of different individuals, resulting in high statistical power for inferring the history of the population(s) represented by those individuals. Thus, sampling genome-wide is imperative for assessing the accuracy of the demographic relationships inferred from uniparental markers.

The ability to extract and sequence ancient DNA (aDNA) has revolutionized the study of human prehistory, allowing direct sampling of past populations and revealing genetic variation no longer observed today. Post-mortem degradation of DNA makes it very difficult to sequence aDNA, so the greater numbers of mtDNA makes it much easier to extract and sequence. The earliest ancient DNA studies in humans were for mtDNA (e.g. Krings et al., 1997), including from a Jōmon individual as early as 1989 (Horai et al., 1989) and Yayoi individuals as early as 1995 (Oota, Saitou, Matsushita, & Ueda, 1995). In fact, the majority of ancient DNA studies on Jōmon individuals have been on mtDNA (Schmidt & Seguchi, 2014).

Only recently have ancient nuclear genomes been retrieved from individuals from Japan. Ancient genomes have been published from human remains at different sites (Sanganji, Ikawazu, and Funadomari associated with Jōmon material culture; Shimo-Motoyama associated with Yayoi material culture; Figure 1). Two partial genomes sequenced from the Sanganji shellmound in Tōhoku were of 3000-year-old individuals (Kanzawa-Kiriyama et al., 2017). Genome-wide data have also been retrieved for a 2700-year-old individual from Ikawazu (McColl et al., 2018) and two 3900-year-old individuals from the Funadomori site in Hokkaido (Kanzawa-Kiriyama et al., 2019). Sampling of nuclear genomes is still sparse, but a rough understanding of the ancestry associated with the Jōmon is taking shape with each study.

Characterizing Jōmon-period ancestry is fundamental to understanding the genetic composition of humans that lived during the Yayoi period, who ostensibly represent various degrees of mixture between Jōmon and continental peoples. This in turn will improve our understanding of population migration on the archipelago and its role in the spread of agriculture across the archipelago. Here, we highlight some key features and emerging questions observed to date on Jōmon-related ancestry, which allow us to better understand the challenges (and rewards!) in interpreting the genetics of the Yayoi in future studies.

Analyses of both uniparental (Y and mtDNA) and nuclear DNA of present-day populations have highlighted that ancestry persists in Japan that is deeply diverged from ancestry found in mainland East Asian populations. The distribution of haplogroup frequencies across multiple populations is often used to determine where haplogroups originated. For example, those that occur in high frequency in present-day populations from Japan but low frequency elsewhere (e.g. N9b, M7a, G1b) were proposed as candidate haplogroups of Jōmon origin (Adachi, Shinoda, Umetsu, & Matsumura, 2009; Adachi et al., 2011; Kanzawa-Kiriyama, Saso, Suwa, & Saitou, 2013; Kivisild et al., 2002).

The most prominent of these is N9b, which is the most frequent mtDNA haplogroup assigned to Jōmon individuals. An estimate of the time to the most recent common ancestor (t MRCA) is ca. 22,000 years ago, which is older than the t MRCA for several other haplogroups found in East Asians (Adachi et al., 2011). The non-recombining region of the Y-chromosome has also been extensively studied in Japan. Y-haplogroup D1b is exceptionally interesting as it is found in high frequency in the Japanese archipelago and the Tibetan Plateau but not elsewhere (Hammer & Horai, 1995; Tajima et al., 2004; Watanabe et al., 2019). The estimated t MRCA for D1b is ca. 19,400 years ago (Hammer et al., 2006), similar to that estimated for N9b mtDNA. These dates, if corresponding to the expansion of Jōmon ancestry on the archipelago, suggest that Jōmon ancestry separated fairly early from mainland East Asian ancestry.

One mtDNA haplogroup found in many Jōmon-associated individuals (e.g. Kanzawa-Kiriyama et al., 2013; Adachi et al., 2011; Kim et al., 2010) as well as Yayoi-associated individuals (Igawa et al., 2009) is D4, which is widespread in northern East Asia both in past and present-day populations (e.g. Tanaka et al., 2004; Zhao et al., 2010; Hong et al., 2014; Jeong et al., 2018; Lan et al., 2019). Its widespread presence, however, suggests that it was found in the common ancestral population of all East Asians, making it difficult to evaluate its role in more recent migrations and interactions.

In genomic studies of ancient Asians, both a 40,000-year-old individual found in Tianyuan Cave, Beijing, China (Fu et al., 2013; Yang et al., 2017) and 8000-year-old individuals from Southeast Asia associated with Hòabìnhian foragers (McColl et al., 2018) show an early split time from other East Asians, including from ancient Jōmon genomes (Kanzawa-Kiriyama et al., 2017, 2019). Thus, we denote them as ‘basal Asians’. In contrast, humans living in Southeast Asia, the eastern steppes of Siberia and the southwest edges of the Tibetan Plateau from the Neolithic to historic periods (e.g. Sikora et al., 2019; Jeong et al., 2016b) share a closer relationship to present-day East Asians than the Jōmon individuals, confirming that Jōmon are distantly related to mainland East Asian ancestry (Yang, MA and Fu, Q unpublished observations).

In fact, the Jōmon sampled thus far probably separated earlier than the split time of East Asians and the early Asian population that contributed to Native Americans (Gakuhari et al., 2019; Kanzawa-Kiriyama et al., 2017, 2019). Estimates of when ancestral Native Americans separated from the East Asian lineage is ca. 36,000–25,000 years ago (Moreno-Mayar et al., 2018), and a direct estimate of the split time between the Jōmon and mainland East Asians ranges from 38,000–18,000 years ago (Kanzawa-Kiriyama et al., 2019). These estimates are broad, indicating there is still high uncertainty as to the exact date. However, studies of both uniparental and nuclear markers all highlight Jōmon ancestry separated from mainland East Asian ancestry in the Palaeolithic, with dates ranging from 38,000–18,000 years ago.