Road to extinction: Archival samples unveiled the process of inbreeding depression during artificial breeding in an almost extinct butterfly species

国内で最も絶滅リスクの高いチョウ、オガサワラシジミの繁殖途絶の原因を解明 --近親交配による遺伝的多様性の減少が、繁殖の失敗につながっていた-- .京都大学プレスリリース. 2024-07-12.During ex-situ conservation of endangered species, reproductive disruption risk due to inbreeding depression increases when genetic diversity is reduced due to founder effects or population declines. Elucidating the population genetic process of reproductive disruption will help understand the risks and mechanisms associated with inbreeding depression. A comprehensive understanding of these processes would require a comparison of reproductive traits and genetic diversity in the populations of endangered species that have experienced reproductive disruption. However, such previous studies, which focus on endangered species, are limited. Ex-situ conservation was initiated for the Ogasawara Holly Blue (Celastrina ogasawaraensis) (Lepidoptera, Lycaenidae), endemic to the Ogasawara Islands, Japan, in 2016 using two mated females as founders. The wild populations might have already been extinct since 2020. All the ex-situ conserved populations were also disrupted in their 20th generation in 2020. Archival samples have been preserved for genetic analysis for the last 20 years. Here, we examined the population genetic background behind reproductive disruption in this ex-situ conserved species for these samples. We used ISSR genotyping by sequencing (MIG-seq) to analyse the individuals from the wild and ex-situ conserved populations collected during 2001–2020. Genetic diversity, hatchability and the number of sperm decreased after the start of progressive breeding in ex-situ conservation. At least 26 individuals were required to retain the genetic diversity of the wild populations at the start of ex-situ conservation. This study highlights the significant risk of failure in ex-situ conservation efforts due to the small number of founders and breeding individuals

Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing

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

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study

Background and Aims Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades

現生個体と標本サンプルの遺伝解析を用いた半自然生態系における絶滅危惧種の保全生態学的研究

京都大学0048新制・課程博士博士(農学)甲第20428号農博第2213号新制||農||1048(附属図書館)学位論文||H29||N5049(農学部図書室)京都大学大学院農学研究科森林科学専攻(主査)教授 井鷺 裕司, 教授 柴田 昌三, 教授 神﨑 護学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Methods for retaining well-preserved DNA with dried specimens of insects

遺伝情報を長期保存できる昆虫標本の作製方法を新たに開発 --「遺伝資源」としての昆虫標本を次世代に--. 京都大学プレスリリース. 2019-12-26.Dried specimens of insects are increasingly seen as genetic resources. However, genetic analysis of dried specimens of insects is hampered by the deterioration of the DNA. In this study, we developed methods for preparing dried specimens of insects with well-preserved DNA, mainly for PCR-based genetic analysis. First, we compared the effects of either exposure to ethyl acetate vapour for from 10 min to 6 h or by freezing on the fragmentation of DNA in order to determine optimal length of time needed for killing insects using the above methods. Second, we compared the fragmentation of DNA after preservation by drying or immersion of legs in 99.5% ethanol or 99% propylene glycol in 0.2-ml tubes. We assessed degrees of fragmentation of DNA by determining polymerase chain reaction (PCR) success rates with primers for 313-, 710- and 1555-bp fragments using DNA that was collected immediately, and at one, six and 12 months after preparing the specimens. Differing times taken to kill insects did not affect the fragmentation of DNA. In dried specimens, DNA was seriously fragmented after one month, whereas that in legs prepared by immersion in 99.5% ethanol or 99% propylene glycol contained long fragments of DNA (1555 bp~) after 12 months. Propylene glycol was more suitable for preservation than ethanol, because the latter evaporates. Thus, to preserve insect DNA we suggest inserting the pin on which an insect is impaled into the hinged lid of a 0.2-ml tube containing 99% propylene glycol so that when the lid is closed the legs of the insect are preserved in the solution

Timing of mowing influences genetic diversity and reproductive success in endangered semi-natural grassland plants

半自然草地性絶滅危惧植物の保全に好適な草刈り時期を解明 --開花結実期の草刈りが繁殖と遺伝的多様性を低下させる--. 京都大学プレスリリース. 2016-02-16.Recent global land-use changes have led to reductions in many herbaceous plant species in semi-natural grassland landscapes. Changes in management frequency and intensity are known to cause declines in plant populations. However, little is known about the impact of changes in the timing of management practices on the genetic diversity as well as the reproductive success of rare semi-natural grassland species. We determined the suitable management (mowing) timing for Vincetoxicum pycnostelma Kitag. (Apocynaceae; Asclepiadoideae), an endangered summer- and autumn-blooming semi-natural grassland herb. We examined 15 V. pycnostelma populations to assess the effects of mowing timing on the genetic diversity of each population using nine microsatellite markers and on pollination and reproductive success. Pollination success was not affected by flowering timing. Mowing during the mid- to late flowering and fruiting periods of V. pycnostelma (July–September) had a significant negative effect on the number of inflorescences and total fruits produced, whereas mowing before flowering and growing periods (April, May and November–March) had positive effects on the number of inflorescences and fruits, respectively. Furthermore, mowing during the mid- to late flowering and fruiting periods also caused a significant decrease in genetic diversity. Our results demonstrated that mowing events during the mid- to late flowering and fruiting periods caused significant declines in the genetic diversity and/or reproductive success of V. pycnostelma. By contrast, mowing before flowering periods significantly enhanced reproductive success. To conserve semi-natural grassland herb diversity, mowing should be avoided during seasons when the flowering and fruiting periods of many endangered species overlap

Habitat diversification associated with urban development has a little effect on genetic structure in the annual native plant Commelina communis in an East Asian megacity

Abstract Urban development greatly alters the natural and semi‐natural habitats of native plants. Urbanisation results in a range of diverse habitats including remnant agricultural lands, urban parks, and roadside habitats. This habitat diversity often promotes trait divergence within urban areas. However, the mechanisms by which diverse urban habitats influence the population genetic structure of individual plant species remain poorly understood. We investigated the effects of urbanisation on genetic diversity and structure within 24 Commelina communis populations across diverse habitat types (rural agricultural land, urban agricultural land, urban park land, and urban roadsides) within the Kyoto–Osaka–Kobe megacity in Japan. We conducted multiplexed inter‐simple sequence repeat genotyping to compare genetic diversity among populations in different habitats. We also examined the correlation between Nei's genetic distance and geographic and environmental distances and performed principal coordinate analysis (PCoA) to evaluate genetic differentiation among urban habitats. There were no significant differences in genetic diversity indices between urban and rural populations and among urban habitat types. Although we detected no isolation‐by‐distance structure in population pairs of the same habitat type and in those of different habitats, the difference in surrounding landscape facilitated genetic differentiation not only between urban and rural habitats but also between different urban habitats. PCoA revealed no clear genetic differentiation among rural and urban habitat populations. Our findings indicate that the establishment of diverse habitat types through urbanisation has no and little impact on genetic diversity and structure, respectively, in C. communis, likely due to its high selfing rate and ability to adapt to urban conditions

Integrative approach clarifies the distinct taxonomic account of gryloblattids endemic to Hokkaido, Japan, with a description of two new species (Insecta, Grylloblattodea)

Galloisiana yezoensis Asahina, 1961 was described on the basis of female specimens from the Ishikari Mountains, Hokkaido, Japan, and has been known as the only species of Grylloblattidae in Hokkaido. In preliminary research, several species closely related to G. yezoensis were collected from various sites in Hokkaido. In the present study, the taxonomic status of G. yezoensis and its relatives was reexamined through molecular and morphological analyses approaches. As a result, the phylogenetic tree of the COII region, and mitochondrial DNA, suggest that G. yezoensis and its relatives form a unique clade distinct from the Galloisiana clade. We therefore establish a new genus, Arctigalloisiana gen. nov., with two new species, A. poropnetopa sp. nov. and A. yubariensis sp. nov. The type species is Arctigalloisiana yezoensis comb. nov., and its male is described here for the first time