Mitochondrial DNA 16S region and voucher specimen collection of Japanese aquatic Coleoptera and Hemiptera for environmental DNA metabarcoding analyses
Naoyuki Nakahama, Kei Hirasawa, Masaya Kato, Kohei Watanabe, Seikan Kurata, Masakazu Hayashi
ZooKeys, 1253, 103, 119, Pensoft Publishers, 2025年09月23日
研究論文（学術雑誌）, 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.

Genome-wide analysis confirms the independent origin of the Japanese and Korean Adonis amurensis (Ranunculaceae)
Diego Tavares Vasques, Daiki Takahashi, Naoko Ishikawa, Seikan Kurata, Satoshi Nanami, Hiroshi Ikeda
Plant Systematics and Evolution, 311, 3, Springer Science and Business Media LLC, 2025年05月07日
研究論文（学術雑誌）, 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.

Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing
Seikan Kurata, Shota Mano, Naoyuki Nakahama, Shun Hirota, Yoshihisa Suyama, Motomi Ito
Biodiversity Data Journal, 12, Pensoft Publishers, 2024年06月18日, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）, 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.

Phylogeographic incongruence between two related Geranium species with divergent habitat preferences in East Asia
Seikan Kurata, Shota Sakaguchi, Shun K. Hirota, Osamu Kurashima, Yoshihisa Suyama, Motomi Ito
Ecological Research, Wiley, 2024年03月07日, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）, 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.

Refugia within refugium of Geranium yesoense varieties: a follow-up study using chloroplast genome sequencing data of specimens from Mt. Asama, Japan
Seikan Kurata, Shota Sakaguchi, Osamu Kurashima, Risa Ogawa, Yoshihisa Suyama, Sachiko Nishida, Motomi Ito
Biological Journal of the Linnean Society, Oxford University Press (OUP), 2024年01月02日, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）, 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.

Contribution of genetic analyses to semi‐natural grassland biodiversity conservation in Japan
Naoyuki Nakahama, Seikan Kurata, Atushi Ushimaru
Plant Species Biology, Wiley, 2023年05月31日, ［査読有り］
研究論文（学術雑誌）

From East Asia to Beringia: reconstructed range dynamics of Geranium erianthum (Geraniaceae) during the last glacial period in the northern Pacific region
Seikan Kurata, Shota Sakaguchi, Hajime Ikeda, Shun K. Hirota, Osamu Kurashima, Yoshihisa Suyama, Motomi Ito
Plant Systematics and Evolution, 308, 4, Springer Science and Business Media LLC, 2022年08月, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）

Phylogenetics of Japanese Geranium (Geraniaceae) using chloroplast genome sequences and genome-wide single nucleotide polymorphisms
Seikan Kurata, Diego Tavares Vasques, Shota Sakaguchi, Shun K. Hirota, Osamu Kurashima, Yoshihisa Suyama, Sachiko Nishida, Motomi Ito
PLANT SYSTEMATICS AND EVOLUTION, 307, 6, 2021年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Flora of herbaceous and arboreous plants in Komaba Campus of the University of Tokyo, Japan
Seikan Kurata, Naoko Ishikawa, Diego Vasques, Masayuki Saito, Osamu Kurashima, Motomi Ito
Biodiversity Data Journal, 9, Pensoft Publishers, 2021年09月27日, ［査読有り］
研究論文（学術雑誌）, Recent studies revealed that green spaces in urban areas are critical for conservation of native biodiversity and that assessment of the present flora of green spaces in urban areas is critical for protection of the native biodiversity. The Komaba Campus of the University of Tokyo embraces a relevant green area, located in a highly urbanised area in Tokyo Metropolis (35.66 N 139.68 E, Japan). The total area of this Campus is 25.4 ha, from which, 4.5 ha are covered by vegetation. Although intense urbanisation can be observed around the Campus, new insect species had been reported for the Campus area, suggesting that the biodiversity on the Campus still demands some attention. Differently from fauna surveys, no flora survey has been done for more than 30 years on the Campus. In this study, we have extensively surveyed the plants diversity on the Komaba Campus of the University of Tokyo, aiming for an update of the plants list on this green urban area in Tokyo.


The survey covered all herbaceous and arboreous plants growing wild on the Campus. Garden plants were excluded in this survey because these plants were supposed to be cultivated. The final dataset contained, in total, 324 taxa, from which 234 were herbaceous plants and 90 were arboreous plants. The top three taxa are as follows: Poaceae (38 taxa), Asteraceae (34 taxa) and Rosaceae (14 taxa), respectively. This is the first update to the Flora of the Komaba Campus of the University of Tokyo in 30 years and represent an important contribution to conservation of native species in the Tokyo metropolis.

Development and characterization of nuclear microsatellite markers to reveal the neutral demographic background of flower color polymorphism in Geranium thunbergii (Geraniaceae)
Seikan Kurata, Shota Sakaguchi, Hitomi Mishima, Takashi Tsuchimatsu, Motomi Ito
GENES & GENETIC SYSTEMS, 96, 2, 99, 104, 2021年04月, ［査読有り］
英語, 研究論文（学術雑誌）

Refugia within refugium of Geranium yesoense (Geraniaceae) in Japan were driven by recolonization into the southern interglacial refugium
Seikan Kurata, Shota Sakaguchi, Shun K. Hirota, Osamu Kurashima, Yoshihisa Suyama, Sachiko Nishida, Motomi Ito
BIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY, 132, 3, 552, 572, 2021年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Genetic diversity and population demography of Geranium soboliferum var. kiusianum: a glacial relict plant in the wetlands of Japan
Seikan Kurata, Shota Sakaguchi, Motomi Ito
CONSERVATION GENETICS, 20, 3, 431, 445, 2019年06月, ［査読有り］
英語, 研究論文（学術雑誌）

Development of nuclear microsatellite markers for the threatened wetland plant Geranium soboliferum var. kiusianum (Geraniaceae)
Seikan Kurata, Shota Sakaguchi, Motomi Ito
PLANT SPECIES BIOLOGY, 32, 4, 466, 470, 2017年10月, ［査読有り］
英語

DEVELOPMENT OF NUCLEAR AND CHLOROPLAST MICROSATELLITE MARKERS FOR THE ENDANGERED CONIFER CALLITRIS SULCATA (CUPRESSACEAE)
Shota Sakaguchi, Guillaume Lannuzel, Bruno Fogliani, Adrien S. Wulff, Laurent L'Huillier, Seikan Kurata, Saneyoshi Ueno, Yuji Isagi, Yoshihiko Tsumura, Motomi Ito
APPLICATIONS IN PLANT SCIENCES, 3, 8, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）

日本人の撹乱は生物多様性維持に貢献してきたか―草地性植物の集団動態から探る
科学研究費助成事業
2022年04月01日 - 2025年03月31日
倉田 正観
日本学術振興会, 若手研究, 秋田県立大学, 22K14976

侵略的外来種の侵略的性質獲得の原因解明と抑制に関する研究
科学研究費助成事業
2021年10月07日 - 2025年03月31日
上原 浩一, 梅木 清, 加藤 顕, タヴァレス・ヴァスケス ジエーゴ, 倉田 正観
本研究は、国家や大陸を超えて蔓延し、生物多様性に深刻な悪影響を及ぼす侵略的外来種の侵略のメカニズムを解明する国際共同研究である。対象とするのは日本と近隣のアジア地域に自生し、19世紀以降欧米に人為的に導入され悪質な侵略的外来種として問題になっているイタドリ(Fallopiajaponica, 英名 Japanese knotweed)、オオイタドリ(Fallopia sachalinensis,英名 Giant knotweed)である。これら原産地では他の植物に埋もれた、めだたない植物が、新たな地域に導入後、どのようにして強い侵略性を 獲得し分布を拡大したか、イタドリをモデルとして侵略的外来種の侵 略的性質の形成メカニズムを解明し、今後の侵略的外来種抑制のため の基盤情報を得ることを目的としている。そのためイタドリの原産国である日本と、侵入 地域である英国・米国の研究者が共同で日本の自生地域と欧米の侵入地域の詳細な調査を 行い、それらの植物体と周囲の生物相や環境を比較解析することで侵略的外来植物の侵略 的性質獲得の法則性を見いだし、その抑制法を検討することで地球規模の生物多様性の保全に貢献したい。
本年度は英国およびアメリカの海外研究協力者と連携し海外調査のため、新型コロナ感染症の蔓延状況などの情報も含め調査計画を立てた。また英国の侵略的イタドリ・オオイタドリについて、DNAサンプル採集用の歯を送ってもらい、これまで日本各地とお逢い街で得られたイタドリと合わせ予備的解析を行なった。
日本学術振興会, 国際共同研究加速基金(国際共同研究強化(B)), 千葉大学, 21KK0189