倉田 正観

Aquatic coleopteran and hemipteran insects primarily inhabit lentic waters, many of which are at risk of extinction due to development, agriculture, and invasive alien species. Environmental DNA (eDNA) analysis has recently emerged as a powerful tool for conducting comprehensive distribution surveys. The cytochrome c oxidase subunit I (COI) universal primers are conventionally used for DNA barcoding but they often result in non-specific amplification and frequent amplication failures. Primers in the mitochondrial DNA (mtDNA) 16S rRNA region that alleviate these issues have been developed and are considered helpful for eDNA analysis. It is necessary to accumulate reference sequences of the mtDNA 16S rRNA region in aquatic coleopteran and hemipteran insects. However, molecular identification at the genus or species level remains challenging, as only a few of these insect groups in Japan have registered reference DNA sequences for both the mtDNACOI and 16S rRNA. Therefore, we constructed a comprehensive dataset of the mtDNA 16S rRNA region for these insects distributed in Japan. As a result of this study, we were able to obtain partial sequences of the mtDNA 16S rRNA region from 140 coleopteran taxa (35.5% of Japanese aquatic species or subspecies) and 58 hemipteran taxa (45.3% of Japanese aquatic species or subspecies). These voucher specimens were deposited in four research institutions. The DNA sequence datasets are expected to significantly contribute as an essential database for eDNA analysis and other DNA metabarcoding studies.

Abstract Adonis is a genus popularly known as ornamental plants called fukujusō, belonging to the Ranunculaceae, and distributed from Europe to West and Central Asia to East Asia. In Japan, four species are recognized, including diploid and tetraploid lineages. In the past decades, some genetic studies have raised questions about lineage boundaries, between Japan and Korea. In this study, we used genome-wide sequencing of a large sampling dataset of Japanese and Korean Adonis species to address phylogenetic relationships between species. We also compared samples according to their distribution, morphological traits, and chromosome numbers. As a result, we identified six phylogenetic lineages that can be distinguished as species, four of these distributed in Japan. Based on the molecular and morphological data, we suggest that specimens from Japan and Korea previously identified as A. amurensis belong to different lineages and should be treated as distinct species. We point out differences between those lineages and suggest a new combination of the name Adonis puberula to describe the Hokkaido (Japan) specimens. We obtained evidence suggesting that Japanese A. multiflora is a sister to A. shikokuensis, including both Japanese and Korean samples with morphological differences that can be explored in future studies. Finally, A. ramosa (fukujusō), the Japan endemic tetraploid species, showed great genetic and morphological variation, as previously reported by other studies.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Abstract Plant species with overlapping distributions are expected to experience the same historical events. In contrast, species with different preferred habitat types may exhibit incongruent phylogeographic patterns because of their habitat‐dependent demographic changes in spite of their overlapping distributions. On the western side of the Japanese Archipelago, boreal forest and non‐forest vegetation were dominant during the last glacial period; subsequently, temperate forests expanded, and open vegetation (e.g., grasslands) decreased. Such vegetation shifts can result in incongruent phylogeographic patterns among temperate forest and grassland species; however, few phylogeographic studies have investigated this hypothesis. Here, we evaluated the phylogeographic structure of two related Geranium species that occur in grassland (Geranium krameri) and temperate forest (Geranium shikokianum) habitats using chloroplast genome sequencing and nuclear genome‐wide single‐nucleotide polymorphisms genotyping. The phylogeographic structure was stronger for the temperate forest species than the grassland species, despite its currently scattered distribution for grassland species. The observed patterns indicate that the phylogeographic histories of these species were influenced by their habitat configurations since the last glacial period. During the ice age, grasslands in Japan were much more widespread than today; this would have enabled the grassland species to form a continuous distribution, leading to low divergence among regional populations. In contrast, the significant genetic divergence within the forest species can be attributed to the glacial isolation of regional populations. Our genetic results suggest that vegetation transitions have variously controlled the population dynamics of two species with overlapping distributions.

Abstract Recent phylogeographical studies have revealed that refugia sometimes retain high levels of genetic heterogeneity due to multiple colonization events, a phenomenon defined as ‘refugia within refugium’. In previous research, we reported a complex genetic structure within the Geranium yesoense complex, an alpine plant found in an interglacial refugium at high elevation in Central Japan, probably resulting from multiple colonization and hybridization events. However, we were unable to evaluate instances of introgression due to limited sample size. In the present study, we performed additional chloroplast genome sequencing, along with Sanger sequencing of selected chloroplast DNA regions, to elucidate the phylogenetic relationships among the refugial populations. The chloroplast genome sequence of a sample from Mt. Asama (an important refugium) was nested within the northern lineage (i.e. var. yesoense and var. pseudopratense), and haplotypes from Mt. Asama and Mt. Ibuki were also grouped with those of the northern lineage. Although our previous study suggested hybridization events between northern and southern lineages (i.e. var. nipponicum) at Mt. Asama, haplotypes from the southern lineage were not detected at range margins. This suggests that directional introgression occurred in these regions. Overall, our results further support that genetic heterogeneity within these refugia was amplified by recolonization and hybridization during past climate oscillations.