Diversity in matrilineages among the Jomon individuals of Japan

Abstract Background The Jomon period of Japan is characterised by a unique combination of sedentary and hunting/gathering lifestyles, spanning for more than 10,000 years from the final Pleistocene to the Holocene. The transition from the preceding Palaeolithic period to the Jomon period is known to have begun with the appearance of pottery usage. However, knowledge of the genetic background of the Jomon people is still limited. Aim We aimed to determine the population-scale complete mitogenome sequences of the Initial Jomon human remains and compare the occurrence of mitochondrial haplogroups in the Jomon period from temporal and regional perspectives. Subjects and methods For human remains dated to 8200–8600 cal BP, we determined their complete mitogenome sequences using target enrichment-coupled next-generation sequencing. Results We successfully obtained the complete mitogenome sequences with high depth of coverage and high concordance on consensus sequences. These sequences differed by more than three bases each, except for two individuals having completely identical sequences. Co-existence of individuals with haplogroups N9b and M7a was first observed at the same archaeological site from the Initial Jomon period. Conclusion The genetic diversity within the population was not found to be low even in the Initial Jomon period.


Introduction
The Jomon period of Japan is characterised by a unique combination of sedentary and hunting/gathering/fishing/cultivation lifestyles, spanning more than 10,000 years.This period witnessed the emergence of pottery approximately 16,500 years ago prior to the beginning of paddy field rice cultivation, which was approximately 3000 to 2400 years ago and introduced by the immigrant Yayoi people.The Jomon people had a highly developed society and culture; for example, in eastern Japan, during the Middle Jomon (around 5000 years ago), elegant and elaborate pottery was used in daily life.Jomon pottery varies temporally and spatially based on production (Taniguchi 2017).however, little is known about the genetic background of the Jomon people with respect to their temporal and regional similarities and differences.
high-throughput sequencing technologies developed in recent decades have enabled us to acquire a large number of nucleotide and genome-wide sequences.In addition, their costs have decreased dramatically.as a result, ancient genome analysis is not restricted to well-preserved ancient human samples anymore but can be used for a wide range of ancient human samples.since maternally inherited mitochondrial DNa/genome exhibits significant differences in nucleotide sequence among individuals, it has been used as a valuable index not only for individual identification of maternal relationships but also for genetic relationships among human populations.here, we aimed to determine the entire nucleotide sequences of the mitochondrial genome (mitogenome) of an ancient human individual remains excavated from an 8200-8600 cal BP Initial Jomon archaeological site, the Iyai rock shelter site in Gunma Prefecture, central honshu, Japan.We also investigated the matrilineal relationships among seven individuals.although multiple haplogroup data were determined by PcR genotyping alone, we specifically compared the occurrence of mitochondrial haplogroups in the Jomon period from temporal and regional perspectives.

Samples
In our previous study (Mizuno et al. 2020), the human remains were uncovered from individual burial pits, Iyai 1, 4, and 8, from the Iyai rock-shelter site in Gunma Prefecture, central honshu, Japan (see Kondo et al. 2018 for details).here, we analysed the following four ancient human remains from Iyai 3, 10, 12, and 15: the left femur from Iyai 3, the right petrous bone from Iyai 12, left petrous bone from Iyai 15, and left petrous bone and left femur from Iyai 10.These four human remains were uncovered from individual burial pits and were in close proximity and from the same layer as that of Iyai 1 (Figure 1).Radiocarbon dating for Iyai 1 showed a calibrated date of 8300-8200 cal BP (Kondo et al. 2018), belonging to the later part of the Initial Jomon period.
This study was approved by the ethics committee of Toho University school of Medicine (a20110_a18099_a18056).

DNA extraction, next-generation sequencing (NGS) library preparation, enrichment of the ancient mitogenome, and sequencing
DNa was extracted from the bones, and the NGs library was constructed as described in our previous study (Mizuno et al. 2021).While performing DNa extraction, purification, and NGs library construction, all possible precautions were taken to avoid contamination.experiments were performed in a laboratory that was exclusively dedicated to ancient DNa research and physically isolated from other molecular research laboratories.all experiments were performed in a laminar flow cabinet that was routinely irradiated with UV light.Frequent surface cleaning was routinely performed before and after the experiments.Facemasks, head caps, and clean laboratory coats were always worn, and gloves were frequently replaced.all the procedures were performed using sterilised disposable tubes and filter pipette tips.all non-disposable glass and metallic materials were dry-heat sterilised at 160 °c for 2-4 h.
after exhaustive brushing to eliminate dirt and exogenous contaminants, the outer bone surface was mechanically removed using a sanding machine (Dremel) to remove surface contaminants.The clean bone was cut into small pieces of approximately 0.5-1 cm 3 in size, using an electric drill cutter (Dremel).The bone fragments were cooled with liquid nitrogen, and finely powdered bone was obtained by grinding the bone fragments in a mill (Multi-Beads shocker MB601U, Yasui Kikai).
The powdered samples (100-500 mg) were decalcified in 0.5 M eDTa (ph 8.0) for 2 h at 56 °c in a rotating hybridisation oven, and the supernatant was removed by centrifugation.The decalcification step was performed thrice.DNa was extracted using phenol: chloroform: isoamyl alcohol (25:24:1) followed by extraction with an equal volume of chloroform.after centrifugation, the aqueous solution was removed and concentrated by centrifugation dialysis using the amicon Ultra-15-30 kDa centrifugal filter (Merck Millipore) to a final volume of 200 μl.The DNa solution was purified using silica-based Minielute spin columns (Qiagen), according to the manufacturer's protocol.The obtained DNa was quantified using the Quant-iT dsDNa hs assay kit (Invitrogen), according to the manufacturer's protocol.Using the DNa, we prepared single-and double-stranded NGs libraries.For the double-stranded libraries, we treated the DNa with PrecR Repair Mix (New england Biolabs).We carried out in-solution target enrichment using the sureselect kit (agilent Technologies), as described in a previous study (Kihana et al. 2013, Mizuno et al. 2020, 2021).The library was sequenced on the Miseq platform (Illumina, Usa) using the Miseq Reagent Kit v3 150 cycles.

Data processing
Raw sequencing reads were trimmed by removing adapter sequences and low-complexity sequences using fastp ver.0.20.0 (chen et al. 2018).The trimmed reads were mapped against the human mitogenome reference sequence rcRs (andrews et al. 1999) using the Burrows-Wheeler alignment tool (li and Durbin 2009) with optimal parameters for ancient DNa (schubert et al. 2012).Reads with a mapping quality < 30 were filtered out, and high-quality mapped reads were retained using saMtools ver.1.9 (li et al. 2009).To minimise the effects of nucleotide mis-incorporations on building a consensus mitogenome sequence, the first two bases on each end of the read were clipped with BamUtil ver.1.0.14 (Jun et al. 2015).concordance percentages were estimated using Mitosuite ver.1.0.9 (Ishiya and Ueda 2017).Finally, consensus sequences were built using Mitosuite, and their mitochondrial haplogroup assignments were called haploGrep2 (https://haplogrep.uibk.ac.at/) (Weissensteiner et al. 2016).Variants were checked using

Complete mitogenome sequences of Iyai individuals
In this study, using combined target enrichment and NGs analysis, we obtained the complete nucleotide sequence information for four mitogenomes of the Initial Jomon individuals, with sufficient depth.Table 1 shows the results obtained, with high accuracy, for the seven individuals, in the study by Mizuno et al. (2020) and this study, with Iyai 10 having a depth of 61, while Iyai 3, Iyai 12, and Iyai 15 having depths of over 200.sufficient depth and full-length mitogenome sequences were obtained for all the individuals.Their concordance on consensus sequences was 0.989, 0.988, 0.986, and 0.988 for Iyai 3, 10, 12, and 15, respectively.Figure s1 shows the deamination pattern, an indicator of ancient DNa, observed in all the individuals analysed in this study, similar to the results obtained in our previous studies (Mizuno et al. 2020(Mizuno et al. , 2021)).

Discussion
In this study, for the first time to the best of our knowledge, haplogroups N9b and M7a were observed at the same archaeological site from the Initial Jomon period.sequence analysis using complete mitogenome sequences makes it possible to obtain precise haplotype definitions, including information on the singleton (s).The haplogroup M7a individual, Iyai 3, was not classified into known sub-haplogroups but had some singletons.among haplogroup N9b individuals, Iyai 4 and 12 were classified as known sub-haplogroup N9b3.The remaining four (Iyai 1, 8, 10, and 15) had an unidentified new haplotype that did not belong to any known sub-haplogroup of N9b.Notably, Iyai 8 and 10 had a completely identical haplogroup N9b sequence throughout the entire mitogenome.This finding reveals that Iyai 8 and 10 are matrilineally related.as they are  roughly contemporaneous in age and the excavated human bones were located next to each other, the two individuals were probably close relatives.The Jomon period is divided into Incipient, Initial, early, Middle, late, and Final based on the pottery-type chronology.The Jomon mitogenome haplogroup data, according to the chronological division, are listed in Table 2. Out of 94 data points in total, haplogroup identification based on full-length mitogenome sequences is limited to 28 in this as well as previous studies (Mccoll et al. 2018;Kanzawa-Kiriyama et al. 2019;cooke et al. 2021;Mizuno et al. 2021;adachi et al. 2021;Waku et al. 2022).however, haplogroup identification for the remaining 66 is inferred from limited sequence information from PcR-based haplogroup typing data (adachi et al. 2011, 2013;Takahashi et al. 2019;Kanzawa-Kiriyama et al. 2013).The Initial Jomon was reported from four sites, including the site focussed on in this study in which both N9b and M7a are distributed; the others were D4b2 from Yugura cave, and M80"D and N9a2a from higashimyou shell midden.The early Jomon was reported from six sites, and M7a was found at sites from north to south, while N9b was not found at any site south of Kyushu.In Middle Jomon, both N9b and M7a were observed at the sites, all of which were located in chiba Prefecture.The late Jomon was reported from six sites, located in hokkaido in the north and Okinawa in the south, and M7a was detected both at hokkaido in the north and Okinawa in the south.N9b was observed in large numbers at Funadomari; the others were D4b2 from the shomyoji shell midden and D4h2 from Funadomari.In the Final Jomon, N9b was observed in large numbers at hokkaido sites.Both N9b and M7a were observed at both the sites in honshu.The others in hokkaido were mostly observed at Usu-Moshiri: G1b and D4h2 at Usu-Moshiri, G1b at Minami-Usu 6, and D4h2 at Onkoromanai.
Using these data, we summarised the Jomon-period human data reported to date for the entire Japanese archipelago (Figure 4).
1. haplogroup M7a is widely distributed throughout the Japanese archipelago.2. haplogroup N9b is widely distributed throughout the Japanese archipelago, except in Kyushu and southwards.3. haplogroups G1b and D4h are distributed only in hokkaido.4. although regional differences were observed, no period differences were observed (at present).5. haplogroups M7a and N9b coexist at the same sites throughout the Japanese archipelago, except for westward.
To the best of our knowledge, this is the first study to determine the full-length mitogenome sequences of several individuals from the same archaeological site, Iyai, belonging to the Initial Jomon period.We observed that even among individuals having the same haplogroup, nucleotide sequences are highly diverse at the full-length level.Furthermore, the genetic diversity within the population was not low in the Initial Jomon period, suggesting active genetic interaction with other groups, at least on matrilineage.

Figure 1 .
Figure 1.Buried human skeletons at the iyai rock shelter site; seven individuals analysed in this study.

Table 1 .
mapping results of ngs data for the iyai initial Jomon individuals.

Table 2 .
mitogenome haplogroups of the Jomon individuals reported till date.

Table 2 .
Dating information follows the notation of the original papers.Continued. #