Paleogenomics of Human Remains in East Asia and Yaponesia Focusing

Anthropological Science Advance Publication Review

Paleogenomics of human remains in East Asia and Yaponesia focusing on current advances and future directions Kae Koganebuchi1,2,3, Hiroki Oota1* 1Laboratory of Genome Anthropology, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan 2Advanced Medical Research Center, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan 3Department of Human Biology and Anatomy, Graduate School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan

Received 21 October 2020; accepted 30 November 2020

Abstract Ancient DNA analysis became paleogenomics once high-throughput sequencing technology was applied to ancient DNA sequencing. Paleogenomics based on whole-genome information from Ne- anderthals and Denisovans showed that small fragments of these genomes remain in the modern human genome, and corresponding studies of anatomical modern humans clarified the history of migration and expansion among Homo sapiens. Due to geographical and environmental conditions, paleogenomic studies have fallen behind in Eastern compared with Western Eurasia. Recently, however, various capture sequencing techniques, which can enrich ancient DNA, have been used in East Eurasia, and the field of paleogenomics has been further developed. This review briefly introduces the history of ancient DNA analysis leading to paleogenomics, outlines three sequencing stages (partial, draft, and complete genome sequencing) and capture methods, and discusses the necessity of high-quality sequencing for paleoge- nomes of Eastern Eurasia.

Key words: ancient DNA, Jomon, East Eurasia, capture sequencing, partial/draft/complete genome sequencing

firmed the conclusion of mtDNA D-loop sequencing. Since Introduction then, ancient DNA analysis has come to be known as paleo Ancient DNA analysis started in the mid-1980s based on genomics. the Sanger sequencing method following molecular cloning Many have expressed amazement at the subsequent using plasmids (Higuchi et al., 1984; Pääbo, 1985). With the breakthroughs made possible by paleogenomics. A draft spread of the polymerase chain reaction method, ancient whole-genome sequence of a Neanderthal revealed that the DNA analysis began to be used from the end of the 20th genomes of non-African H. sapiens today contain about century to examine many remains from various extinct or- 1–4% of the sequence inherited from Neanderthals (Green et ganisms, including archaic hominins. First of all, Neander- al., 2010). In the same year, a draft whole-genome sequence thal mitochondrial (mt) DNA sequences had a strong impact of a Denisovan was also reported: no complete skeletal re- on human evolutionary studies: the nucleotide sequence data mains had been recovered, but DNA was successfully ex- of the hypervariable region (HVR) showed that Neander- tracted from a piece of tiny bone excavated from Denisova thals (Homo neanderthalensis) were outside of modern hu- Cave in the Altai mountains. The genomic data identified mans (Homo sapiens) in terms of genetic diversity (Krings Denisovans as a sister group to the Neanderthal lineage et al., 1997). The next epoch began when high-throughput (Reich et al., 2010). Subsequently, the great achievements of sequencing technology (next-generation sequencing (NGS)) “non-draft” whole-genome sequencing of three archaic was applied for analyzing the nuclear genome from the skel- hominin individuals, Denisovan, Altai, and Vindija etal remains of a Neanderthal (Green et al., 2006). This par- Neanderthals, were reported (Meyer et al., 2012; Prüfer et tial genome sequencing (0.04% of the whole genome) con- al., 2014, 2017). The high-coverage genome sequences (30- and 52-fold coverages for Denisovans and Neanderthals from Altai, respectively) depicted a new human evolutionary * Correspondence to: Hiroki Oota, Laboratory of Genome Anthro- history, including changes in population size and gene flow pology, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113- over time among archaic hominins and H. sapiens (Browning 0033, Japan. et al., 2018; Enard and Petrov, 2018; Greenbaum et al., E-mail: [email protected] 2019; Jacobs et al., 2019; Laval et al., 2019; Meyer et al., Published online 31 March 2021 2012; Moorjani et al., 2016; Prüfer et al., 2014; Qin and in J-STAGE (www.jstage.jst.go.jp) DOI: 10.1537/ase.2011302 Stoneking, 2015; Racimo et al., 2017; Sankararaman et al.,

2012, 2014, 2016; Slon et al., 2018; Vernot et al., 2016; modern humans. Therefore, more recent paleogenomic stud- Vernot and Akey, 2014; Villanea and Schraiber, 2019). ies have been conducted using many individuals with Paleogenomic technology has also been applied to ancient low-coverage genome sequences (Allentoft et al., 2015; de H. sapiens (anatomical modern humans (AMH)) specimens, Barros Damgaard et al., 2018; Fu et al., 2016; Gamba et al., but the sequencing strategy in several research projects has 2014; González-Fortes et al., 2017; Haak et al., 2015; been slightly modified compared with those in archaic hom- Hofmanová et al., 2016; Lazaridis et al., 2014, 2016; Lipson inins. Namely, in archaic humans, complete whole-genome et al., 2017, 2018; Mathieson et al., 2015, 2018; McColl et sequencing was the aim, but in many ancient AMH speci- al., 2018; Narasimhan et al., 2019; Olalde et al., 2015, 2018, mens, complete sequencing was not achieved after draft se- 2019; Patterson et al., 2012; Raghavan et al., 2014, 2015; quencing had been reported, and analyses based on partial Reich et al., 2012; Sikora et al., 2017, 2019; Siska et al., sequencing and single-nucleotide polymorphism (SNP) 2017; Villalba-Mouco et al., 2019; Yang et al., 2020; Yu et capture became the mainstream. The whole-genome se- al., 2020). quence of Paleo-Eskimo (4000 years ago (hereafter 4.0 kya)) No exact definition currently exists for distinguishing was reported in 20-fold coverage (Rasmussen et al., 2010). partial, draft, and complete sequencing. However, in this Similarly, the whole-genome sequence of the oldest West review, we tentatively refer to 1- to 2-fold coverage whole- Eurasian individual, Ust’-Ishim (45 kya) from Russia, was genome sequencing as ‘draft sequencing,’ 30-fold coverage reported with high-quality (42.0-fold coverage) whole- and above as ‘complete whole-genome sequencing,’ and less genome sequencing (Fu et al., 2014). However, the coverag- than 1-fold coverage as ‘partial genome sequencing’ (Fig- es of the whole-genome sequences of the oldest East Eura- ure 1). We then discuss the advantages of each, including sian individual excavated from the Tianyuan cave site SNP capture sequencing (Figure 2), and what direction (41.1–39.5 kya) and the oldest individual found to date with should be considered in paleogenomic research in East Asia, a close relationship to present-day Europeans, Kostenki 14 including the Japanese archipelago (Yaponesia), in the near (38.7–36.2 kya) from Russia, were moderate (1.7–4.1- and future. 2.84-fold coverage, respectively) (Fu et al., 2013; Seguin- Orlando et al., 2014; Yang et al., 2017). The genome se- Increasing the reliability of genome sequence on quences of Goyet Q116-1 from Belgium (35.1–34.4 kya; the paleogenomics individual shows genetic affinity with both present-day Eu- ropeans and Tianyuan) and Mal’ta 1 from Russia (24 kya; The three levels of sequence quality defined in this paper the individual is closely related to Native Americans) were are shown visually in Figure 1. The top line of each row is 1.05- and 1.17-fold coverages (Fu et al., 2016; Raghavan et the human reference sequence determined by the Human al., 2014), respectively. Different sequencing strategies be- Genome Project (International Consortium Human Genome tween archaic hominins and ancient AMHs are probably Sequencing, 2004). The short pieces below are the outputs of because a reference sequence of H. sapiens is already avail- NGS analysis, each called a ‘read.’ In the partial sequencing, able and ancient AMHs are not substantially different from the reads mapped to the reference sequence are sparse, but in

Figure 1. Mapping by partial sequencing (less than 1-fold coverage of genome), draft genome sequencing (1- to 2-fold coverage), and complete whole-genome sequencing (more than 30-fold coverage). PALEOGENOMICS OF HUMAN REMAINS IN EAST ASIA AND YAPONESIA 3

Figure 2. Schematic of shotgun, nuclear DNA capture sequencing, and SNP capture sequencing. Images of the bone and NGS were obtained from Togo Picture Gallery (©2016 DBCLS TogoTV; https://togotv.dbcls.jp/pics.html). the draft sequencing, they ideally cover the entire reference sequencing is a method to read all of these DNA sequences sequence. Thus, a draft sequence means that the entire ge- with NGS. However, depending on the state of preservation nome sequence has been read at least once or twice on aver- of ancient human bones, more than 99% of the extracted age. Meanwhile, in the ‘complete’ whole-genome sequence, DNA is that of non-human organisms; the endogenous DNA the same region in the reference sequence is read multiple of ancient human bone is often less than 1%. Nuclear DNA times. Since reads overlap many times, this is called ‘read capture is a method of enriching only human nuclear DNA deeply.’ If a different allele from the reference sequence is from an NGS library containing the DNA of various species detected (e.g. G to A) in the partial sequence, an error may (e.g. bacteria, fungi). The method can find not only known have occurred in the NGS reaction. If it is A in the draft se- SNPs but also novel ones. SNP (e.g. 1240K) capture tech- quence, it might be a ‘real’ A allele. The deeper the read, the niques enrich reads including only known SNPs from the clearer it is. However, when the depth remains shallow, it is outputs of NGS. Only the reads containing particular SNPs unknown whether it is homozygous or heterozygous. It is are concentrated, so the depth of the reads containing the generally considered that homozygous/heterozygous status SNPs becomes deeper. However, if the original NGS library can be distinguished when reading more than 30 times, and contains too little human DNA, capture methods do not therefore in many cases it is permissible to consider 30-fold work. coverage or higher as ‘complete’ whole-genome sequencing. Difficult delivery of paleogenomics in Yaponesia Sequencing methods for paleogenomics Japanese researchers soon began to analyze ancient AMH Three sequencing methods have mainly been adopted in DNA. In the earliest analysis, mtDNA HVRs were se- paleogenomic analysis based on the state of DNA preserva- quenced in five Jomon individuals (Horai et al., 1991). Sub- tion and/or depending on the researchers’ purpose (Fig- sequently, nuclear polymorphic loci (variable number of ure 2). After extracting DNA from ancient human bone, an tandem repeats) were examined in two individuals from the artificial sequence (adapter) is attached to the end ofthe Kofun (tumulus) period (Kurosaki et al., 1993). Population DNA; this is called the ‘NGS library.’ In addition to the studies of ancient mtDNA had already begun in the 1990s DNA of the specimen, the DNA extracted from ancient hu- (Oota et al., 1995, 1999, 2001; Shinoda and Kanai, 1999; man bone contains the DNA of bacteria, virus, fungi, plants, Wang et al., 2000), and subsequently expanded into mtDNA and animals that entered from the soil postmortem. Shotgun haplogroup analysis and entire mtDNA sequencing (Adachi 4 K. KOGANEBUCHI AND H. OOTA Anthropological Science et al., 2009, 2013; Kanzawa-Kiriyama et al., 2013; Mizuno fully adopted capture sequencing techniques (Lipson et al., et al., 2014, 2017; Sato et al., 2007), even into a nuclear lo- 2018; McColl et al., 2018; Yang et al., 2020; Yu et al., 2020). cus encoding a functional gene (Sato et al., 2010). One characteristic of ancient genome analyses in East Eura- However, the application of NGS to ancient genome se- sia is that the coverage remains low even after such enrich- quencing in Japan, which was first achieved by Kanzawa- ment, and SNP capture sequencing is often slightly better Kiriyama et al. (2017), was 10 years later than its application than nucleotide DNA capture sequencing. These techniques to the Neanderthal genome. This delay was greatly influ- are based on a strategy of abandoning high-coverage se- enced by the burial environment surrounding ancient Japa- quencing because of poor DNA preservation and ‘thinly’ and nese human bones. The climate of the Japanese archipelago efficiently obtaining as much information of polymorphic (Yaponesia) is warm and humid, and the soil is acidic be- sites as possible from more individuals. The strategy is suit- cause of the volcanic nature of the region. For these reasons, able for analyzing the genomic information of ancient ‘pop- especially on Honshu (the largest island of the Japanese ar- ulations’ rather than of ‘individual’ rare specimens by phy- chipelago), it is difficult for human bones buried in the soil logenetic analysis. to survive, and even if they do, there is a high possibility that the DNA does not. Therefore, very few human remains from Comparison of sequencing coverage the paleolithic period have been found in Honshu, which is a big difference in comparison with Europe. For these geo- Low-coverage sequencing results in a small numbers of graphical and geological reasons, ancient genome research SNPs. If more than 5000 SNPs are available, it is generally in Japan has become extremely difficult. However, the situa- thought that there will be no problem summarizing the pop- tion has improved since discovering that abundant DNA ulation structure, such as with a principal-component analy- could often be extracted from the petrous part of the tempo- sis (PCA) plot (Wang et al., 2012). However, when trying to ral bone (Hansen et al., 2017; Pinhasi et al., 2015). A draft extract not only phylogeny, but also more information about whole-genome sequence of a 2500-year-old Jomon woman biological functions, from the genome sequences, the low- from the Ikawazu shell-mound site in the Atsumi peninsula, coverage genome causes difficulty in estimating the pheno- Honshu, was subsequently reported (McColl et al., 2018), types based on homozygous/heterozygous status, and the and a complete whole-genome sequencing of a Jomon indi- high-coverage genome turns out to be overwhelmingly reli- vidual from the Funadomari site in Rebun Island, Hokkaidō, able. was successfully achieved (Kanzawa-Kiriyama et al., 2019). To discuss the effects of differences in coverage, we per- The successful complete genome sequencing of the Funado- formed variant calls on four Jomon individuals whose ge- mari Jomon individual might be due to the fact that the site nome sequences have been reported so far, categorizing is located in a subarctic zone where DNA is better preserved them according to their coverages and comparing the geno- than in the temperate zones. types of the genes that have been well studied in East Asia (Table 2). There are 39 SNPs in 12 genes that we are inter- Increasing paleogenomic data in East Eurasia ested in because the derived alleles are particularly found in East Eurasian populations. We investigated genotypes and As well as the Japanese archipelago, the geographical ar- their allele depths based on the sequence data of Sangan- eas of Southeast Asia and southern China are not suitable for ji131464 (partial genome), IK002 and F5 (draft whole ge- the preservation of ancient DNA because of their hot and nome), and F23 (complete whole genome). We processed humid environments. Table 1 summarizes ancient human those data based on a method used in Sikora et al. (2017). genome studies, primarily in eastern Eurasia east of Lake Complete whole genome was called as diploid genome. Par- Baikal. There is a wide range of eras from Upper Paleolithic tial and draft whole genomes were called as pseudohaploid to Iron Age, and the most common ones are Neolithic. If we genomes which is a widely used method for low-coverage look at the coverages of each individual, most of them do not ancient genomes. For these particular SNPs, the partial exceed 1-fold coverage, and individuals with more than 10- genome sequence showed no information, even though fold coverage are rare. Because of the poor DNA preserva- phylogenetic analysis has been successfully carried out tion, strategies using capture techniques as opposed to shot- (Kanzawa-Kiriyama et al., 2017). The draft whole-genome gun sequencing have largely been standardized in the sequences of IK002 and F5 gave 79.5% and 69.2% geno genome studies of East Eurasian human remains, except for typing data, respectively. The complete whole-genome se- Jomon genomes (Table 1). In the first report of Tianyuan, the quence of F23 gave 100% genotyping at the 39 SNP sites. oldest AMH specimen in East Asia, nuclear genome se- The accuracy of these genotypings was supported by the al- quencing was restricted to a particular chromosome by tar- lele depth. For example, a variation was observed between get capture sequencing (Fu et al., 2013). Nuclear DNA cap- the rs13419896 genotypes (A/A in IK002 and G/G in F5) of ture sequencing was subsequently conducted (2.98- to the EPAS1 gene in Jomon people. The genotype involves the 4.10-fold coverage) (Yang et al., 2017). The whole genomes A/G heterozygotes shown in F23; this is based on the con- of Mal’ta, Yana RHS, and Duvanny Yar in Siberia were se- sistent results of the ancestral allele (G) and derived allele quenced by shotgun sequencing, but were not ‘complete,’ (A) determined by 10 and 8 reads, respectively. The two and those of some specimens used in the same papers had draft sequences detected only one allele on the basis of 3–4 less than 1-fold coverage (Raghavan et al., 2014; Sikora et reads and showed different genotypes being homozygous for al., 2019). A/A or G/G because of the generated pseudohaploid calls. Many studies in East/Southeast Asia have combined and However, those genotypes were called as homozygous based PALEOGENOMICS OF HUMAN REMAINS IN EAST ASIA AND YAPONESIA 5

Table 1. Ancient nuclear genome studies in Eastern Eurasia (East of Lake Baikal) Approximate No. of dating Nuclear genome Citation Period Local area/site/population Method individuals (years before coverage (fold) present) Targeted capture Fu et al. (2013) 1.75 (Chr. 21) Near the Zhoukoudian site in sequencing Upper Paleolithic 1 40000 northern China (Tianyuan) Nuclear DNA Yang et al. (2017) 1.71–4.09 capture Raghavan et al. Upper Paleolithic 1 MA-1 24000 1.17 Shotgun sequencing (2014) 1 Yana RHS (Yana1) 31600 25 1 Yana RHS (Yana2) 7 1 Duvanny Yar (Kolyma1) 9800 14 Upper Paleolithic Sikora et al. (2019) 14 Ekven, Uelen, Magadan 3000–2000 0.003–1.681 Shotgun sequencing –Post Iron Age 6 Devil’s Gate Cave 7600 0.105–6.56 6 Ust’Belaya 6500–600 0.058–1.835 1 Yana (Young Yana) 800 1.947 1 Himalayan arc (Chokhopani) 3150–2400 7.253 Jeong et al. (2016) Prehistoric 3 Himalayan arc (Mebrak) 2400–1850 0.044–1.048 Shotgun sequencing 4 Himalayan arc (Samdzong) 1750–1250 0.090–3.493 Fukushima Prefecture (Sanganji 1 3000 0.01* Kanzawa-Kiriyama 131421-3) Neolithic Shotgun sequencing et al. (2017) Fukushima Prefecture (Sanganji 1 3100 0.02* 131464) Siska et al. (2017) Neolithic 5 Devil’s Gate Cave 9400–7200 0.001–0.059 Shotgun sequencing 3 Laos 8000–2400 0.603–1.341 3 Malaysia 3200–450 0.012–1.729 9 Vietnam 4300–300 0.103–0.257 1 Indonesia 2300 0.143 Shotgun sequencing 3 Thailand 1800–1700 0.161–0.422 1 Japan 2500 1.85 McColl et al. Neolithic 0 Philippines — — (2018) –Iron Age 1 Laos 2400 0.942 3 Malaysia 3900–400 0.021–0.343 3 Vietnam 3800 0.006–0.009 Nuclear DNA 1 Indonesia 1900 0.105 capture 5 Thailand 1800–1700 0.008–0.163 0 Japan — — 2 Philippines 1900–1800 0.004–0.029 8 Vietnam (Man Bac) 4100–3600 0.005–0.106 2 Vietnam (Nui Nap) 2100–1900 0.042–0.373 Lipson et al. Neolithic 1240K capture (2018) –Iron Age 2 Myanmar (Oakaie 1) 3200–2700 0.011–0.178 5 Thailand (Ban Chiang) 3500–2400 0.005–0.030 1 Cambodia (Vat Komnou) 1900–1700 0.047 Kanzawa-Kiriyama 1 Rebun Island (F23) 3500–3800 48 (peak of depth) Neolithic Shotgun sequencing et al. (2019) 1 Rebun Island (F5) 3500–3800 1 (peak of depth) 1 Innner Mongolia (Yumin) 8400–8300 7.25 1 Shandong (Bianbian) 9500 2.21 1 Shandong (Xiaogao) 8800–8600 7.6 1 Shandong (Boshan) 8300–8000 7.36 3 Shandong (Xiaojingshan) 7900–7700 0.22–0.64 1 Fujan (Qihe) 8400 0.45 Nuclear DNA Yang et al. (2020) Neolithic–Historic 1 Liang Island (Liangdao1) 8300–8100 2.72 capture 1 Liang Island (Liangdao2) 7600 1.68 2 Fujan (Suogang) 4800–4300 0.03–0.04 8 Fujan (Xitoucun) 4600–4200 0.01–0.66 5 Fujan (Tanshian) 4500–4200 0.01–0.41 1 Fujan (Chuanyun) 300 0.38 6 K. KOGANEBUCHI AND H. OOTA Anthropological Science

Table 1. (continued) Approximate No. of dating Nuclear genome Citation Period Local area/site/population Method individuals (years before coverage (fold) present) Amur River Basin (early 2 7500–7300 0.239–0.287 Neolithic) 1 Amur River Basin (Iron Age) 2200–2100 0.068 Amur River Basin (Xianbei_ 3 2300–2100 0.095–0.241 Iron Age) West Liao River (Hamin- 1 5700 1.431 mangha_Middle Neolithic) West Liao River (Banlashan_ 3 5600–5100 0.048–5.878 Middle Neolithic) West Liao River (Late Neolith- 3 4100–3300 0.212–2.398 ic) 2 West Liao River (Bronze Age) 3100–2400 0.574–0.579 Neolithic Ning et al. (2020) 1 West Liao River (Bronze Age) 3100–2400 1.653 Shotgun sequencing –Iron Age Inner Mongolia (Middle 3 5600–5100 0.065–0.348 Neolithic) 3 Shaanxi (Late Neolithic) 4300–4000 0.026–4.478 Upper Yellow River Basin (Late 7 4100–3900 0.043–2.416 Neolithic) Upper Yellow River Basin (Late 4 1900–1800 0.034–2.439 Neolithic) 8 Yellow River Basin (Iron Age) 6300–5100 0.047–6.824 Yellow River Basin (Late 8 4300–3800 0.202–7.533 Neolithic) Yellow River Basin (later 6 2600–2100 0.033–2.048 Bronze/Iron Age) 0 Angara River — — 0 Yenisei River — — 1 Irkutsk City 4400 0.449 0 Lena River — — Shotgun sequencing 4 Upper Lena River 4000–3800 0.149–0.349 2 Lower Lena River 4600–6600 0.487–0.579 Upper Paleolithic 1 Eastern Siberia 14000 1.991 Yu et al. (2020) –Bronze Age 1 Angara River 7300 0.037 1 Yenisei River 4700 1.628 3 Irkutsk City 4400 0.465–1.518 9 Lena River 6900–3700 0.220–1.511 1240K capture 2 Upper Lena River 4000–3800 0.044–2.034 2 Lower Lena River 4600–6600 1.665–2.067 1 Eastern Siberia 14000 1.656 * Estimated value based on the percentage of bases covering hg19.

on the pseudohaploid calling method for low-coverage an- gotes) leads to some analytical difficulties. First, the pheno- cient genomes, and could actually be heterozygous. In F23, type is unclear when the homozygote has a trait that differs 10 of 39 SNPs were heterozygotes, whereas in IK002 and from the heterozygote. For example, the G/G homozygote is F5, all SNP sites were typed as homozygotes. Therefore, it is alcohol-tolerant at rs671 in the ALDH2 gene, whereas the difficult to distinguish between homozygotes and heterozy- A/G heterozygote causes flushes and makes one feel sick gotes in draft genome sequencing, and a complete genome after drinking alcohol (Harada et al., 1981; Oota et al., sequence is required to distinguish between them. 2004). We found no derived allele in the three Jomon individuals (Table 2), which was consistent with our prediction The need for high-coverage sequencing based on a survey of Okinawa and Sakishima islanders (Koganebuchi et al., 2017). We also predicted that the de- Current trends in paleogenomics in East Eurasia include rived allele at rs671 must have started arriving in the Japa- the reconstruction of population history using low-coverage nese archipelago from the East Eurasian continent around sequences with many individuals (Table 1). However, a lack 3000 years ago, but found it difficult to find individual(s) of correct genotype information (homozygotes or heterozy- with the derived allele in recently published ancient genome PALEOGENOMICS OF HUMAN REMAINS IN EAST ASIA AND YAPONESIA 7 - Citation Nishimura et al. (2017) Kanzawa-Kiriyama et al. (2019), et al. (2016) Yang Nakagome et al. (2017) Kanzawa-Kiriyama et al. (2019), et al. (2006) Yoshiura Kanzawa-Kiriyama et al. (2019) Yamaguchi et al. (2012), Yamaguchi Motokawa et al. (2007) Hanaoka et al. (2012), Beall et al. (2010) Kanzawa-Kiriyama et al. (2019), Kimura et al. (2009) Kanzawa-Kiriyama et al. (2019), Kimura et al. (2015) Akiyama et al. (2017) Koganebuchi et al. (2017) Koganebuchi et al. (under review) † † † † † † † † 4/6 8/6 6/5 F23 7/0 8/0 1/3 0/9 0/1 5/0 3/3 2/0 11/0 11/2 10/0 0/21 13/0 20/0 13/0 0/15 15/0 7/13 15/7 22/0 17/2 17/0 26/0 14/0 10/8 23/0 15/0 0/13 19/0 0/16 0/15 20/0 19/0 23/1 12/0 14/14 whole genome Complete F5 3/0 0/2 0/0 0/2 0/0 0/0 4/0 0/0 0/3 2/0 1/0 0/0 0/1 1/0 0/0 0/1 0/0 0/1 0/0 2/0 2/0 2/0 0/0 0/0 0/3 4/0 2/0 3/0 3/0 0/5 3/0 0/0 0/1 1/0 1/0 1/0 3/0 0/0 2/1* 1/0 0/0 0/2 1/0 1/0 3/0 4/0 0/0 1/0 0/0 2/0 0/2 1/0 0/2 0/0 1/0 0/0 0/0 0/0 2/0 2/0 1/0 3/0 2/0 0/3 3/0 3/0 4/0 3/0 4/0 0/0 0/6 1/0 1/0 4/0 3/0 1/0 1/2* 3/1* IK002 Draft whole genome Allele depth (reference/alternative) 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Partial genome Sanganji131464 T/T T/T T/T T/T T/T T/T C/T C/T T/C T/C F23 T/C T/C T/C C/C C/C C/C C/C C/C C/C C/C C/C C/C A/A A/A A/A G/A G/A A/A G/G G/G A/G G/G G/G G/G A/G G/G G/G G/G G/G whole genome Complete F5 T/T T/T T/T T/T T/T T/T T/T C/C C/C C/C C/C C/C C/C C/C A/A A/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— Genotype T/T T/T T/T T/T T/T T/T C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C C/C A/A A/A A/A A/A G/G G/G G/G G/G G/G G/G G/G G/G G/G G/G —/— —/— —/— —/— —/— —/— —/— —/— IK002 Draft whole genome Comparison between sequencing coverages —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— Partial genome 2. Table Sanganji131464 T T T T T T T T T T T T T T T T T T T A C C C C A A A A A A A C G G G G G G G Derived T T T A A C C C C C C C A C C C A C A C A A C C C C A C C G G G G G G G G G G Ancestral Position 28197037 48258198 46556345 89985940 89986154 78358945 46575388 46713201 112211833 112241766 117563687 117579457 112228849 117569046 112222788 117558703 112204427 117552885 100266112 117547772 141475837 141501338 141481581 100239319 141485134 141493961 141515213 100268856 100260789 109513601 219746561 239155053 239177073 100229017 239187042 239187679 239187680 239203761 100238413 4 9 9 9 9 4 4 9 4 4 4 4 4 4 2 4 9 2 2 2 2 2 2 4 2 2 2 2 4 12 12 12 12 12 15 16 16 16 17 Chromosome rs number rs3113195 rs671 rs4372078 rs10817678 rs441 rs7848647 rs2238151 rs4648328 rs6478108 rs4956451 rs886205 rs6536991 rs1800414 rs17822931 rs3810936 rs1229984 rs1693425 rs12502572 rs1800592 rs9995751 rs1789920 rs13419896 rs2228479 rs885479 rs112735431 rs698 rs10114470 rs4953354 rs3827760 rs4953388 rs10177996 rs934945 rs11894535 rs2066702 rs142175638 rs190386281 rs147573126 rs4663302 rs2066701 Gene Data processing after bam (file name to store the alignment information obtained after mapping) followed Sikora et al. (2017). Processed as low coverage: IK002, F5, Sanganji. Processed as high coverage: F23. *Sites that found two UCP1 ALDH2 OCA2 ABCC11 EPAS1 MC1R RNF213 ADH1C TNFSF15 EDAR WNT10A PERIOD2 ADH1B low-coverage (draft) genome sequence; 4.0×–10× = mid sequence; genome (draft) 1.0×–4.0× = low-coverage sequence; genome <1.0× = partial review: this in coverage of Definition genome. high-coverage a in 10× than less were that †Sites genome. low-coverage a in alleles dle-coverage whole-genome sequence; >10× = high-coverage sequence. 8 K. KOGANEBUCHI AND H. OOTA Anthropological Science data from continental specimens because of their very low ia, Southeast/East Asia, and Siberia (Browning et al., 2018; coverage. Second, if homozygous/heterozygous status is Jacobs et al., 2019). Thus, the genomes of archaic hominins, unclear in regions including SNPs on the genome, it is diffi- especially those of Denisovans, are very important for un- cult to conduct phasing and define haplotypes. Hence, all derstanding the population history in East Eurasia. Also population genomic analyses based on haplotype frequency from this perspective, as linkage disequilibrium in rare vari- and/or linkage between SNPs (e.g. fineSTRUCTURE) are ants is often used to detect introgression from archaic homi- prone to error and/or not available when the homozygous/ nins (e.g. Plagnol and Wall, 2006), high-coverage deep se- heterozygous status is unknown because of low-coverage quencing is also required. sequencing (Parks and Lambert, 2015). Therefore, we conclude that the number of Jomon ge- SNP capture sequencing with sufficient depth to deter- nomes needs to be increased with high-coverage as op- mine homozygous/heterozygous status would be useful, but posed to low-coverage or SNP capture sequencing. The raises some issues: every genotyping array has ascertain- Jomon genomes of Sanganji (131464), Ikawazu (IK002), ment biases. It has been reported that standard f4 statistics in and Funadomari (F5 and F23) make a cluster in a PCA plot population genomics sometimes shows opposite results in (Kanzawa-Kiriyama et al., 2019); however, only F23 has SNP array and sequencing data for the same multiple popu- sufficiently high coverage. Because the Jomon period lasted lations (Bergström et al., 2020). Similar problems might for more than 10000 years, from 16000 to 3000 years ago, occur with SNP capture sequencing because of ascertain- whether people of all ages and regions were homogeneous ment biases. In addition, in principle, SNP array and capture should be investigated further; this would not only help re- sequencing ignore rare variants; information on these rare veal the history of human populations in the Japanese archi- variants is very useful for clarifying local population differ- pelago, but also clarify the human population history in East entiation. Therefore, high-coverage sequencing is required Eurasia. for phenotypic prediction and local population history studies that make full use of genomic information. Acknowledgments Jomon genomes and Denisovan introgression into We thank Dr. Takashi Gakuhari, Dr. Shigeki Nakagome, East Eurasia Dr. Daisuke Waku, Dr. Yusuke Watanabe, and Dr. Jun Ohashi for the many discussions on our research. This work All the Jomon genomes from Sanganji (131464), Ikawazu was supported by JSPS KAKENHI grant no. 19H05350 to (IK002), and Funadomari (F5 and F23) reported thus far in- H.O. triguingly show that the Jomon lineage diverged before the diversification of present-day East Eurasian populations; References this was initially indicated in the Sanganji Jomon (Kanzawa- Adachi N., Shinoda K., Umetsu K., and Matsumura H. (2009) Mi- Kiriyama et al., 2017). The Ikawazu Jomon (IK002) was tochondrial DNA analysis of Jomon skeletons from the Fu- closely related to an 8000-year-old hunter-gatherer in Laos nadomari site, Hokkaido, and its implication for the origins of (McColl et al., 2018). The Funadomari Jomon (F23) was Native American. American Journal of Physical Anthropolo- likely to have diverged from East Eurasians before the diver- gy, 138: 255–265. DOI: 10.1002/ajpa.20923 gence of present-day Native Americans, and after that of the Adachi N., Sawada J., Yoneda M., Kobayashi K., and Itoh S. 40000-year-old Tianyuan (Kanzawa-Kiriyama et al., 2019). (2013) Mitochondrial DNA analysis of the human skeleton of the initial Jomon phase excavated at the Yugura cave site, Detailed analyses of the IK002 genome showed that the Ika- Nagano, Japan. Anthropological Science, 121: 137–143. DOI: wazu Jomon lineage diverged before 26 kya from basal East 10.1537/ase.130313 Eurasians involving the ancestorial population of present- Akiyama T., Katsumura T., Nakagome S., Lee S. Il, Joh K., et al. day East Asians, Northeast Asians (East Siberians), and Na- (2017) An ancestral haplotype of the human PERIOD2 gene tive Americans, without gene flow from Mal’ta (MA-1) associates with reduced sensitivity to light-induced melatonin (Gakuhari et al., 2020), which the ancestors of Northeast suppression. PLoS ONE, 12: e0178373 1–19. DOI: 10.1371/ journal.pone.0178373 Asians and Native Americans received (Raghavan et al., Allentoft M.E., Sikora M., Sjögren K.G., Rasmussen S., Rasmussen 2014). Thus, the Jomon would be one of the oldest lineages M., et al. (2015) Population genomics of Bronze Age Eurasia. in East Eurasia, and this is highly important for understand- Nature, 522: 167–172. DOI: 10.1038/nature14507 ing human genome diversity in East Eurasian populations. Beall C.M., Cavalleri G.L., Deng L., Elston R.C., Gao Y., et al. Another important issue in East Eurasian population his- (2010) Natural selection on EPAS1 (HIF2α) associated with tory is Denisovan introgression. Initially, Denisovan intro- low hemoglobin concentration in Tibetan highlanders. Pro- ceedings of the National Academy of Sciences of the United gression into Papua New Guinean people was described States of America, 107: 11459–11464. DOI: 10.1073/ (Reich et al., 2010), but no such introgression was present in pnas.1002443107 continental Southeast/East Asia (Reich et al., 2011). Howev- Bergström A., McCarthy S.A., Hui R., Almarri M.A., Ayub Q., et er, Denisovan alleles were found in Southeast/East Asians al. (2020) Insights into human genetic variation and popula- afterwards (Qin and Stoneking, 2015; Skoglund and tion history from 929 diverse genomes. Science, 367: Jakobsson, 2011). Both Neanderthal and Denisovan ge- eaay5012. DOI: 10.1126/science.aay5012 Browning S.R., Browning B.L., Zhou Y., Tucci S., and Akey J.M. nomes were found in Melanesian individuals at various ra- (2018) Analysis of human sequence data reveals two pulses of tios (Vernot et al., 2016), and the Denisovan genome was archaic Denisovan admixture. Cell, 173: 53–61. DOI: found in Philippine Negritos (Jinam et al., 2017). More re- 10.1016/j.cell.2018.02.031 cently, multiple Denisovan lineages were detected in Ocean- de Barros Damgaard P., Marchi N., Rasmussen S., Peyrot M., PALEOGENOMICS OF HUMAN REMAINS IN EAST ASIA AND YAPONESIA 9

Renaud G., et al. (2018) 137 ancient human genomes from Transactions of the Royal Society of London. Series B, Bio- across the Eurasian steppes. Nature, 557: 369–374. DOI: logical Sciences, 333: 409–416. DOI: 10.1098/rstb.1991.0091 10.1038/s41586-018-0094-2 International Consortium Human Genome Sequencing (2004) Fin- Enard D. and Petrov D.A. (2018) Evidence that RNA viruses drove ishing the euchromatic sequence of the human genome. Na- adaptive introgression between Neanderthals and modern hu- ture, 431: 931–945. DOI: 10.1038/nature03001 mans. Cell, 175: 360–371. DOI: 10.1016/j.cell.2018.08.034 Jacobs G.S., Hudjashov G., Saag L., Kusuma P., Darusallam C.C., Fu Q., Meyer M., Gao X., Stenzel U., Burbano H.A., et al. (2013) et al. (2019) Multiple deeply divergent Denisovan ancestries DNA analysis of an early modern human from Tianyuan Cave, in Papuans. Cell, 177: 1010–1021. DOI: 10.1016/j. China. Proceedings of the National Academy of Sciences of cell.2019.02.035 the United States of America, 110: 2223–2227. DOI: 10.1073/ Jeong C., Ozga A.T., Witonsky D.B., Malmström H., Edlund H., et pnas.1221359110 al. (2016) Long-term genetic stability and a high-altitude East Fu Q., Li H., Moorjani P., Jay F., Slepchenko S.M., et al. (2014) Asian origin for the peoples of the high valleys of the Himala- Genome sequence of a 45,000-year-old modern human from yan arc. Proceedings of the National Academy of Sciences of western Siberia. Nature, 514: 445–449. DOI: 10.1038/ the United States of America, 113: 7485–7490. DOI: 10.1073/ nature13810 pnas.1520844113 Fu Q., Posth C., Hajdinjak M., Petr M., Mallick S., et al. (2016) The Jinam T.A., Phipps M.E., Aghakhanian F., Majumder P.P., Datar F., genetic history of Ice Age Europe. Nature, 534: 200–205. et al. (2017) Discerning the origins of the Negritos, First Sun- https://doi.org/10.1038/nature17993 daland People: deep divergence and archaic admixture. Ge- Gakuhari T., Nakagome S., Rasmussen S., Allentoft M.E., Sato T., nome Biology and Evolution, 9: 2013–2022. DOI: 10.1093/ et al. (2020). Ancient Jomon genome sequence analysis sheds gbe/evx118 light on migration patterns of early East Asian. Communica- Kanzawa-Kiriyama H., Saso A., Suwa G., and Saitou N. (2013) tions Biology, 3: 437. DOI: 10.1038/s42003-020-01162-2 Ancient mitochondrial DNA sequences of Jomon teeth sam- Gamba C., Jones E.R., Teasdale, M.D., McLaughlin, R.L., ples from Sanganji, Tohoku district, Japan. Anthropological Gonzalez-Fortes G., et al. (2014). Genome flux and stasis in a Science, 121: 89–103. DOI: 10.1537/ase.121113 five millennium transect of European prehistory. Nature Com- Kanzawa-Kiriyama H., Kryukov K., Jinam T.A., Hosomichi K., munications, 5: 5257. DOI: 10.1038/ncomms6257 Saso A., et al. (2017) A partial nuclear genome of the Jomons González-Fortes G., Jones E.R., Lightfoot E., Bonsall C., Lazar C., who lived 3000 years ago in Fukushima, Japan. Journal of et al. (2017) Paleogenomic evidence for multi-generational Human Genetics, 62: 213–221. DOI: 10.1038/jhg.2016.110 mixing between Neolithic farmers and Mesolithic hunter- Kanzawa-Kiriyama H., Jinam T.A., Kawai Y., Sato T., Hosomichi gatherers in the lower Danube Basin. Current Biology, 27: K., et al. (2019) Late Jomon male and female genome se- 1801–1810. DOI: 10.1016/j.cub.2017.05.023 quences from the Funadomari site in Hokkaido, Japan. An- Green R.E., Krause J., Ptak S.E., Briggs A.W., Ronan M.T., et al. thropological Science, 127: 83–108. DOI: 10.1537/ase.190415 (2006) Analysis of one million base pairs of Neanderthal Kimura R., Yamaguchi T., Takeda M., Kondo O., Toma T., et al. DNA. Nature, 444: 330–336. DOI: 10.1038/nature05336 (2009) A common variation in EDAR is a genetic determinant Green R.E., Krause J., Briggs A.W., Maricic T., Stenzel U., Kircher of shovel-shaped incisors. American Journal of Human Genet- M., et al. (2010) A draft sequence of the Neandertal genome. ics, 85: 528–535. DOI: 10.1016/j.ajhg.2009.09.006 Science, 328: 710–722. DOI: 10.1126/science.1188021 Kimura R., Watanabe C., Kawaguchi A., Kim Y. Il, Park S.B., et al. Greenbaum G., Getz W.M., Rosenberg N.A., Feldman M.W., (2015) Common polymorphisms in WNT10A affect tooth Hovers E., et al. (2019) Disease transmission and introgres- morphology as well as hair shape. Human Molecular Genet- sion can explain the long-lasting contact zone of modern hu- ics, 24: 2673–2680. DOI: 10.1093/hmg/ddv014 mans and Neanderthals. Nature Communications, 10: 5003. Koganebuchi K., Haneji K., Toma T., Joh K., Soejima H., et al. DOI: 10.1038/s41467-019-12862-7 (2017) The allele frequency of ALDH2*Glu504Lys and Haak W., Lazaridis I., Patterson N., Rohland N., Mallick S., et al. ADH1B*Arg47His for the Ryukyu islanders and their history (2015) Massive migration from the steppe was a source for of expansion among East Asians. American Journal of Human Indo-European languages in Europe. Nature, 522: 207–211. Biology, 29: e22933. DOI: 10.1002/ajhb.22933 DOI: 10.1038/nature14317 Krings M., Stone A., Schmitz R.W., Krainitzki H., Stoneking M., Hanaoka M., Droma Y., Basnyat B., Ito M., Kobayashi N., et al. et al. (1997) Neandertal DNA sequences and the origin of (2012) Genetic variants in EPAS1 contribute to adaptation to modern humans. Cell, 90: 19–30. DOI: 10.1016/S0092-8674 high-altitude hypoxia in Sherpas. PLoS ONE, 7: e50566 DOI: (00)80310-4 10.1371/journal.pone.0050566 Kurosaki K., Matsushita T., and Ueda S. (1993) Individual DNA Hansen H.B., Damgaard P.B., Margaryan A., Stenderup J., identification from ancient human remains. American Journal Lynnerup N., et al. (2017) Comparing ancient DNA preserva- of Human Genetics, 53: 638–643. tion in petrous bone and tooth cementum. PLoS ONE, 12: Laval G., Peyrégne S., Zidane N., Harmant C., Renaud F., et al. e0170940. DOI: 10.1371/journal.pone.0170940 (2019) Recent adaptive acquisition by African rainforest Harada S., Agarwal D.P., and Goedde H.W. (1981) Aldehyde dehy- hunter-gatherers of the Late Pleistocene sickle-cell mutation drogenase deficiency as cause of facial flushing reaction to suggests past differences in malaria exposure. American Jour- alcohol in Japanese. The Lancet, 318(8253): 982. DOI: nal of Human Genetics, 104: 553–561. DOI: 10.1016/j.ajhg. 10.1016/S0140-6736(81)91172-7 2019.02.007 Higuchi R., Bowman B., Freiberger M., Ryder O.A., and Wilson Lazaridis I., Patterson N., Mittnik A., Renaud G., Mallick S., et al. A.C. (1984) DNA sequences from the quagga, an extinct (2014) Ancient human genomes suggest three ancestral popu- member of the horse family. Nature, 312: 282–284. DOI: lations for present-day Europeans. Nature, 513: 409–413. 10.1038/312282a0 DOI: 10.1038/nature13673 Hofmanová Z., Kreutzer S., Hellenthal G., Sell C., Diekmann Y., et Lazaridis I., Nadel D., Rollefson G., Merrett D.C., Rohland N., et al. (2016) Early farmers from across Europe directly descend- al. (2016) Genomic insights into the origin of farming in the ed from Neolithic Aegeans. Proceedings of the National Acad- ancient Near East. Nature, 536: 419–424. DOI: 10.1038/ emy of Sciences of the United States of America, 113: 6886– nature19310 6891. DOI: 10.1073/pnas.1523951113 Lipson M., Szécsényi-Nagy A., Mallick S., Pósa A., Stégmár B., et Horai S., Kondo R., Murayama K., Hayashi S., Koike H., et al. al. (2017) Parallel palaeogenomic transects reveal complex (1991) Phylogenetic affiliation of ancient and contemporary genetic history of early European farmers. Nature, 551: 368– humans inferred from mitochondrial DNA. Philosophical 372. DOI: 10.1038/nature24476 10 K. KOGANEBUCHI AND H. OOTA Anthropological Science

Lipson M., Cheronet O., Mallick S., Rohland N., Oxenham M., et Oota H., Saitou N., Matsushita T., and Ueda S. (1995) A genetic al. (2018) Ancient genomes document multiple waves of mi- study of 2,000-year-old human remains from Japan using migration in Southeast Asian prehistory. Science, 361: 92–95. tochondrial DNA sequences. American Journal of Physical DOI: 10.1126/science.aat3188 Anthropology, 98: 133–145. DOI: 10.1002/ajpa.1330980204 Mathieson I., Lazaridis I., Rohland N., Mallick S., Patterson N., et Oota H., Saitou N., Matsushita T., and Ueda S. (1999) Molecular al. (2015) Genome-wide patterns of selection in 230 ancient genetic analysis of remains of a 2,000-year-old human popu- Eurasians. Nature, 528: 499–503. DOI: 10.1038/nature16152 lation in China—and its relevance for the origin of the modern Mathieson I., Alpaslan-Roodenberg S., Posth C., Szécsényi-Nagy Japanese population. American Journal of Human Genetics, A., Rohland N., et al. (2018) The genomic history of south- 64: 250–258. DOI: 10.1086/302197 eastern Europe. Nature, 555: 197–203. DOI: 10.1038/nature Oota H., Kurosaki K., Pookajorn, S., Ishida T., and Ueda S. (2001) 25778 Genetic study of the Paleolithic and Neolithic Southeast McColl H., Racimo F., Vinner L., Demeter F., Gakuhari T., et al. Asians. Human Biology, 73: 225–231. DOI: 10.1353/ (2018) The prehistoric peopling of Southeast Asia. Science, hub.2001.0023 361: 88–92. DOI: 10.1126/science.aat3628 Oota H., Pakstis A.J., Bonne-Tamir B., Goldman D., Grigorenko Meyer M., Kircher M., Gansauge M.T., Li H., Racimo F., et al. E., et al. (2004) The evolution and population genetics of the (2012) A high-coverage genome sequence from an archaic ALDH2 locus: random genetic drift, selection, and low levels Denisovan individual. Science, 338: 222–226. DOI: 10.1126/ of recombination. Annals of Human Genetics, 68: 93–109. science.1224344 DOI: 10.1046/j.1529-8817.2003.00060.x Mizuno F., Gojobori J., Wang L., Onishi K., Sugiyama S., et al. Pääbo S. (1985) Molecular cloning of ancient Egyptian mummy (2014) Complete mitogenome analysis of indigenous popula- DNA. Nature, 314: 644–645. DOI: 10.1038/314644a0 tions in Mexico: its relevance for the origin of Mesoameri- Parks M. and Lambert D. (2015) Impacts of low coverage depths cans. Journal of Human Genetics, 59: 359–367. DOI: 10.1038/ and post-mortem DNA damage on variant calling: a simula- jhg.2014.35 tion study. BMC Genomics, 16: 19. DOI: 10.1186/s12864- Mizuno F., Kumagai M., Kurosaki K., Hayashi M., Sugiyama S., et 015-1219-8 al. (2017) Imputation approach for deducing a complete mi- Patterson N., Moorjani P., Luo Y., Mallick S., Rohland N., et al. togenome sequence from low-depth-coverage next-generation (2012) Ancient admixture in human history. Genetics, 192: sequencing data: application to ancient remains from the 1065–1093. DOI: 10.1534/genetics.112.145037 Moon Pyramid, Mexico. Journal of Human Genetics, 62: Pinhasi R., Fernandes D., Sirak K., Novak M., Connell S., et al. 631–635. DOI: 10.1038/jhg.2017.14 (2015) Optimal ancient DNA yields from the inner ear part of Moorjani P., Sankararaman S., Fu Q., Przeworski M., Patterson N., the human petrous bone. PLoS ONE, 10: e0129102. DOI: and Reich D. (2016) A genetic method for dating ancient ge- 10.1371/journal.pone.0129102 nomes provides a direct estimate of human generation interval Plagnol V. and Wall J.D. (2006) Possible ancestral structure in hu- in the last 45,000 years. Proceedings of the National Academy man populations. PLoS Genetics, 2: e105. DOI: 10.1371/ of Sciences of the United States of America, 113: 5652–5657. journal.pgen.0020105 DOI: 10.1073/pnas.1514696113 Prüfer K., Racimo F., Patterson N., Jay F., Sankararaman S., et al. Motokawa T., Kato T., Hashimoto Y., and Katagiri T. (2007) Effect (2014) The complete genome sequence of a Neanderthal from of Val92Met and Arg163Gln variants of the MC1R gene on the Altai Mountains. Nature, 505: 43–49. DOI: 10.1038/ freckles and solar lentigines in Japanese. Pigment Cell Re- nature12886 search, 20: 140–143. DOI: 10.1111/j.1600-0749.2007.00364.x Prüfer K., Filippo C. de, Grote S., Mafessoni F., Korlević P., et al. Nakagome S., Chinen H., Iraha A., Hokama A., Takeyama Y., et al. (2017) A high-coverage Neandertal genome from Vindija (2017) Confounding effects of microbiome on the susceptibil- Cave in Croatia. Science, 358: 655–658. DOI: 10.1126/ ity of TNFSF15 to Crohn’s disease in the Ryukyu Islands. SCIENCE.AAO1887 Human Genetics, 136: 387–397. DOI: 10.1007/s00439-017- Qin P. and Stoneking M. (2015) Denisovan ancestry in East Eura- 1764-0 sian and Native American populations. Molecular Biology Narasimhan V.M., Patterson N., Moorjani P., Rohland N., Bernardos and Evolution, 32: 2665–2674. DOI: 10.1093/molbev/msv141 R., et al. (2019) The formation of human populations in South Racimo F., Marnetto D., and Huerta-Sánchez E. (2017) Signatures and Central Asia. Science, 365: eaat7487 DOI: 10.1126/ of archaic adaptive introgression in present-day human popu- science.aat7487 lations. Molecular Biology and Evolution, 34: 296–317. DOI: Ning C., Li T., Wang K., Zhang F., Li T., et al. (2020) Ancient ge- 10.1093/molbev/msw216 nomes from northern China suggest links between subsistence Raghavan M., DeGiorgio M., Albrechtsen A., Moltke I., Skoglund changes and human migration. Nature Communications, 11: P., et al. (2014) The genetic prehistory of the New World Arc- 2700. https://doi.org/10.1038/s41467-020-16557-2 tic. Science, 345: 1255832. DOI: 10.1126/science.1255832 Nishimura T., Katsumura T., Motoi M., Oota H., and Watanuki S. Raghavan M., Steinrucken M., Harris K., Schiffels S., Rasmussen (2017) Experimental evidence reveals the UCP1 genotype S., et al. (2015) Genomic evidence for the Pleistocene and re- changes the oxygen consumption attributed to non-shivering cent population history of Native Americans. Science, 349: thermogenesis in humans. Scientific Reports, 7: 5570. DOI: aab3884. DOI: 10.1126/science.aab3884 10.1038/s41598-017-05766-3 Rasmussen M., Li Y., LindGreen S., Pedersen J.S., Albrechtsen A., Olalde I., Schroeder H., Sandoval-Velasco M., Vinner L., Lobón I., Moltke I., et al. (2010) Ancient human genome sequence of an et al. (2015) A common genetic origin for early farmers from extinct Palaeo-Eskimo. Nature, 463: 757–762. DOI: 10.1038/ Mediterranean Cardial and Central European LBK cultures. nature08835 Molecular Biology and Evolution, 32: 3132–3142. DOI: Reich D., Green R.E., Kircher M., Krause J., Patterson N., et al. 10.1093/molbev/msv181 (2010) Genetic history of an archaic hominin group from Den- Olalde I., Brace S., Allentoft M.E., Armit I., Kristiansen K., et al. isova cave in Siberia. Nature, 468: 1053–1060. DOI: 10.1038/ (2018) The Beaker phenomenon and the genomic transforma- nature09710 tion of northwest Europe. Nature, 555: 190–196. DOI: Reich D., Patterson N., Kircher M., Delfin F., Nandineni M.R., et 10.1038/nature25738 al. (2011) Denisova admixture and the first modern human Olalde I., Mallick S., Patterson N., Rohland N., Villalba-Mouco V., dispersals into Southeast Asia and Oceania. American Journal et al. (2019) The genomic history of the Iberian peninsula over of Human Genetics, 89: 516–528. DOI: 10.1016/j.ajhg. the past 8000 years. Science, 363: 1230–1234. DOI: 10.1126/ 2011.09.005 science.aav4040 Reich D., Patterson N., Campbell D., Tandon A., Mazieres S., et al. PALEOGENOMICS OF HUMAN REMAINS IN EAST ASIA AND YAPONESIA 11

(2012) Reconstructing Native American population history. s41586-018-0455-x Nature, 488: 370–374. DOI: 10.1038/nature11258 Vernot B. and Akey J.M. (2014). Resurrecting surviving Neander- Sankararaman S., Patterson N., Li H., Pääbo S., and Reich D. tal lineages from modern human genomes. Science, 343: (2012) The date of interbreeding between Neandertals and 1017–1021. DOI: 10.1126/science.1245938 modern Humans. PLoS Genetics, 8: e1002947. DOI: 10.1371/ Vernot B., Tucci S., Kelso J., Schraiber J.G., Wolf A.B., et al. journal.pgen.1002947 (2016) Excavating Neandertal and Denisovan DNA from the Sankararaman S., Mallick S., Dannemann M., Prüfer K., Kelso J., genomes of Melanesian individuals. Science, 352: 235–239. et al. (2014) The genomic landscape of Neanderthal ancestry DOI: 10.1126/science.aad9416 in present-day humans. Nature, 507: 354–357. DOI: 10.1038/ Villalba-Mouco V., van de Loosdrecht M.S., Posth C., Mora R., nature12961 Martínez-Moreno J., et al. (2019) Survival of Late Pleistocene Sankararaman S., Mallick S., Patterson N., and Reich D. (2016) hunter-gatherer ancestry in the Iberian peninsula. Current Bi- The combined landscape of Denisovan and Neanderthal an- ology, 29: 1169–1177. DOI: 10.1016/j.cub.2019.02.006 cestry in present-day humans. Current Biology, 26: 1241– Villanea F.A. and Schraiber J.G. (2019) Multiple episodes of inter- 1247. DOI: 10.1016/j.cub.2016.03.037 breeding between Neanderthal and modern humans. Nature Sato T., Amano T., Ono H., Ishida H., Kodera H., et al. (2007) Ori- Ecology and Evolution, 3: 39–44. DOI: 10.1038/s41559-018- gins and genetic features of the Okhotsk people, revealed by 0735-8 ancient mitochondrial DNA analysis. Journal of Human Ge- Wang C., Zöllner S., and Rosenberg N.A. (2012) A quantitative netics, 52: 618–627. DOI: 10.1007/s10038-007-0164-z comparison of the similarity between genes and geography in Sato T., Kazuta H., Amano T., Ono H., Ishida H., et al. (2010) Pol- worldwide human populations. PLoS Genetics, 8: e1002886. ymorphisms and allele frequencies of the ABO blood group DOI: 10.1371/journal.pgen.1002886 gene among the Jomon, Epi-Jomon and Okhotsk people in Wang L., Oota H., Saitou N., Jin F., Matsushita T., et al. (2000) Hokkaido, northern Japan, revealed by ancient DNA analysis. Genetic structure of a 2,500-year-old human population in Journal of Human Genetics, 55: 691–696. DOI: 10.1038/ China and its spatiotemporal changes. Molecular Biology and jhg.2010.90 Evolution, 17: 1396–1400. DOI: 10.1093/oxfordjournals. Seguin-Orlando A., Korneliussen T.S., Sikora M., Malaspinas A.S., molbev.a026422 Manica A., et al. (2014) Genomic structure in Europeans dat- Yamaguchi K., Watanabe C., Kawaguchi A., Sato T., Naka I., et al. ing back at least 36,200 years. Science, 346: 1113–1118. DOI: (2012) Association of melanocortin 1 receptor gene (MC1R) 10.1126/science.aaa0114 polymorphisms with skin reflectance and freckles in Japanese. Shinoda K.I. and Kanai S. (1999) Intracemetery genetic analysis at Journal of Human Genetics, 57: 700–708. DOI: 10.1038/ the Nakazuma Jomon site in Japan by mitochondrial DNA jhg.2012.96 sequencing. Anthropological Science, 107: 129–140. DOI: Yang M.A., Gao X., Theunert C., Tong H., Aximu-Petri A., et al. 10.1537/ase.107.129 (2017) 40,000-year-old individual from Asia provides insight Sikora M., Seguin-Orlando A., Sousa V.C., Albrechtsen A., Kor- into early population structure in Eurasia. Current Biology, neliussen T., et al. (2017) Ancient genomes show social and 27: 3202–3208. DOI: 10.1016/j.cub.2017.09.030 reproductive behavior of early Upper Paleolithic foragers. Yang M.A., Fan X., Sun B., Chen C., Lang J., et al. (2020) Ancient Science, 358: 659–662. DOI: 10.1126/science.aao1807 DNA indicates human population shifts and admixture in Sikora M., Pitulko V.V., Sousa V.C., Allentoft M.E., Vinner L., et northern and southern China. Science, 369: 282–288. DOI: al. (2019) The population history of northeastern Siberia since 10.1126/science.aba0909 the Pleistocene. Nature, 570: 182–188. DOI: 10.1038/s41586- Yang Z., Zhong H., Chen J., Zhang X., Zhang H., et al. (2016) A 019-1279-z genetic mechanism for convergent skin lightening during re- Siska V., Jones E.R., Jeon S., Bhak Y., Kim H.M., et al. (2017) cent human evolution. Molecular Biology and Evolution, 33: Genome-wide data from two early Neolithic East Asian indi- 1177–1187. DOI: 10.1093/molbev/msw003 viduals dating to 7700 years ago. Science Advances, 3: Yoshiura K., Kinoshita A., Ishida T., Ninokata A., Ishikawa T., et al. e1601877. DOI: 10.1126/sciadv.1601877 (2006) A SNP in the ABCC11 gene is the determinant of hu- Skoglund P. and Jakobsson M. (2011) Archaic human ancestry in man earwax type. Nature Genetics, 38: 324–330. DOI: East Asia. Proceedings of the National Academy of Sciences 10.1038/ng1733 of the United States of America, 108: 18301–18306. DOI: Yu H., Spyrou M.A., Karapetian M., Shnaider S., Radzevičiūtė R., 10.1073/pnas.1108181108 et al. (2020) Paleolithic to Bronze Age Siberians reveal con- Slon V., Mafessoni F., Vernot B., de Filippo C., Grote S., et al. nections with First Americans and across Eurasia. Cell, 181: (2018) The genome of the offspring of a Neanderthal mother 1232–1245. DOI: 10.1016/j.cell.2020.04.037 and a Denisovan father. Nature, 561: 113–116. DOI: 10.1038/