Tsuneo Kakuda

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

A Unique Primer with an Inosine Chain at the 5'-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method.

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.

Pamphilius ishikawai feeds on Astilbe : the first record of Pamphiliidae (Hymenoptera) associated with Saxifragaceae

Based on field observations and rearing experiments in Nagano and Tochigi prefectures in central Honshu, Japan, host plants and life history of a leaf-rolling sawfly, Pamphilius ishikawai Shinohara, 1979, are recorded for the first time. The larva is a solitary leaf-roller on Astilbe. This is the first record of the Saxifragaceae as a host plant of the Pamphiliidae. We made molecular phylogenetic analysis using mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences for nine individuals of P. ishikawai feeding on three different species of Astilbe in two localities with different climates and for six outgroup pamphiliine species. It revealed very small genetic variability within P. ishikawai and suggested probable monophyly of the P. sulphureipes group.

DNA Barcodes Identify the Larvae and Unassociated Male of Three Onycholyda Sawflies (Hymenoptera, Pamphiliidae) from China

A molecular analysis based on mitochondrial cytochrome oxidase subunit 1 (COI) gene sequences has indicated that larvae collected in Sichuan and Zhejiang Provinces, China, belong to Onycholyda xanthogaster Shinohara, 1999, and O. fulvicornis Shinohara, in Shinohara Wei, 2016 (Hymenoptera, Pamphiliidae), and that a male Onycholyda specimen from Mt. Tianmushan, Zhejiang Province is the hitherto unknown male of O. tianmushana Shinohara Xiao, 2006. The first host plant records are Rubus inopertus (Focke) Focke (Rosaceae) for O. xanthogaster and Rubus hirsutus Thunb. for O. fulvicornis. The larvae of O. xanthogaster and O. fulvicornis are briefly described and O. xanthogaster is newly recorded from Sichuan Province. The male of O. tianmushana is described for the first time.

Cranio-morphometric and aDNA corroboration of the Austronesian dispersal model in ancient Island Southeast Asia: Support from Gua Harimau, Indonesia

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.

Ethnic derivation of the Ainu inferred from ancient mitochondrial DNA data

Abstract Objectives The 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 methods Mitochondrial 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. Results Our 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. Discussion Our 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.