ObjectivesThe Ainu, the indigenous people living on the northernmost island of Japan, Hokkaido, have long been a focus of anthropological interest because of their cultural, linguistic, and physical identity. A major problem with genetic studies on the Ainu is that the previously published data stemmed almost exclusively from only 51 modern‐day individuals living in Biratori Town, central Hokkaido. To clarify the actual genetic characteristics of the Ainu, individuals who are less influenced by mainland Japanese, who started large‐scale immigration into Hokkaido about 150 years ago, should be examined. Moreover, the samples should be collected from all over Hokkaido.Materials and methodsMitochondrial DNA haplogroups of 94 Ainu individuals from the Edo era were successfully determined by analyzing haplogroup‐defining polymorphisms in the hypervariable and coding regions. Thereafter, their frequencies were compared to those of other populations.ResultsOur findings indicate that the Ainu still retain the matrilineage of the Hokkaido Jomon people. However, the Siberian influence on this population is far greater than previously recognized. Moreover, the influence of mainland Japanese is evident, especially in the southwestern part of Hokkaido that is adjacent to Honshu, the main island of Japan.DiscussionOur results suggest that the Ainu were formed from the Hokkaido Jomon people, but subsequently underwent considerable admixture with adjacent populations. The present study strongly recommends revision of the widely accepted dual‐structure model for the population history of the Japanese, in which the Ainu are assumed to be the direct descendants of the Jomon people.

The Austronesian language is spread from Madagascar in the west, Island Southeast Asia (ISEA) in the east (e.g. the Philippines and Indonesian archipelagoes) and throughout the Pacific, as far east as Easter Island. While it seems clear that the remote ancestors of Austronesian speakers originated in Southern China, and migrated to Taiwan with the development of rice farming by c. 5500 BP and onto the northern Philippines by c. 4000 BP (the Austronesian Dispersal Hypothesis or ADH), we know very little about the origins and emergence of Austronesian speakers in the Indonesian Archipelago. Using a combination of cranial morphometric and ancient mtDNA analyses on a new dataset from Gua Hairmau, that spans the pre-Neolithic through to Metal Period (5712—5591cal BP to 1864—1719 cal BP), we rigorously test the validity of the ADH in ISEA. A morphometric analysis of 23 adult male crania, using 16 of Martin’s standard measurements, was carried out with results compared to an East and Southeast Asian dataset of 30 sample populations spanning the Late Pleistocene through to Metal Period, in addition to 39 modern samples from East and Southeast Asia, near Oceania and Australia. Further, 20 samples were analyzed for ancient mtDNA and assigned to identified haplogroups. We demonstrate that the archaeological human remains from Gua Harimau cave, Sumatra, Indonesia provide clear evidence for at least two (cranio-morphometrically defined) and perhaps even three (in the context of the ancient mtDNA results) distinct populations from two separate time periods. The results of these analyses provide substantive support for the ADH model in explaining the origins and population history of ISEA peoples.

Starting 16000 years ago, the Neolithic lifestyle known as the Jomon culture spread across the Japanese archipelago. Although extensively studied by archaeology and physical anthropology, little is known about the genetic characteristics of the Jomon people. Here, we report the entire mitogenome and partial nuclear genome of skeletal remains from the initial Jomon period that were excavated from the Higashimyo shell midden site at Saga City, Kyushu Island, Japan. This is the first genome analysis of the initial Jomon people of Kyushu Island. These results provide important data for understanding the temporal transition and regional differences of the Jomon people. The mitochondrial DNA and Y-chromosome haplogroups were similar to those found in the previously reported later Jomon people. Moreover, comparison of three nuclear genomes from the initial to final Jomon periods indicated genetic continuity throughout the Jomon period within the Japanese archipelago with no significant evidence of admixture. This indicates that the genetic differentiation found among the Jomon people was promoted by the progression of regionalization throughout the Jomon period. Further accumulation of high-quality Jomon genome data spanning a wide range of regions and ages will clarify both intimate regional and temporal differences of the Jomon people and details of their admixture history with rice farmers, as suggested by Jomon mitochondrial genome data. The results obtained from this study provide important information for further analysis.

To investigate the genetic changes in the indigenous population of northern Kyushu Island from the early Jomon to Yayoi periods, mitochondrial DNA from nine human skeletal remains excavated from the Iwashita Cave and Shimo-motoyama rock shelter sites located in Sasebo city was examined using the amplified product length polymorphism (APLP) method and next-generation sequencing analyses. Although mitochondrial DNA could be extracted from two samples, we could not determine the genetic characteristics of the population in this area and their changes from the Jomon to Yayoi periods accurately. However, the average sequencing depths for these samples were 178.73 and 61.02, which indicates the validity of the DNA capture methods used in this study. The individuals who were successfully analyzed belonged to the northwestern Kyushu Yayoi population, which is thought to be a descendant of the Jomon people. Therefore, the current data are expected to be used to speculate the characteristics of the mitochondrial DNA of the Kyushu Jomon population. However, mitochondrial haplogroups were assigned as M7a and D4a1 by APLP analysis, and based on the whole mitochondrial DNA genome sequencing data, the ancestral types were found to be M7a1a4 and D4a1. Haplogroup M7a has been detected at several Jomon sites in Honshu and Hokkaido and is thought to be a typical Jomon haplogroup. On the other hand, the existence of haplogroup D4a, considered to be derived from immigrant Yayoi population at the site suggested that the segregation of the indigenous Jomon people and immigrants in this region was not clear.

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.