中濱 直之

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.

Crepidiastrum grandicollum is a critically endangered insular endemic herb found only on two oceanic islands of the Bonin Islands in Japan, namely Chichijima Island and Anijima Island. It is explicitly threatened by herbivory pressure from introduced animals. In 2009, a conservation program for C. grandicollum was begun to ensure its future persistence. To provide further information for conservation planning, we investigated the genetic diversity of C. grandicollum using 13 novel microsatellite markers in 55 individuals from four wild populations and an ex situ living collection. Two closely related woody species were also included for interspecific comparison: seven individuals of Crepidiastrum ameristophyllum and 13 of Crepidiastrum linguifolium. The 13 markers were applicable to all three species and identified 129 alleles in total. We found a clear genetic differentiation between C. grandicollum from Anijima Island and Chichijima Island. Crepidiastrum grandicollum also had low expected heterozygosity and allelic richness in populations compared to the two closely related species. Reconstructed divergence history suggested that differentiation between the islands had occurred several thousand generations ago. We suggest separate conservation units for C. grandicollum on Anijima Island and Chichijima Island given the clear (and putatively historical) genetic differentiation, which may result in speciation in the future.

Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga-Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next-generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00-1.00 and 0.00-0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.

<p>霧ヶ峰高原は多くの絶滅危惧動植物の生息地であり，国内有数の観光地でもある．しかし近年ニホンジカによるニッコウキスゲの被食被害が深刻化し，対策として複数の防鹿柵が設置されている．本研究では，亜高山帯半自然草原での防鹿柵設置による生物多様性保全効果を検証することを目的に，植物・昆虫調査およびドローン画像解析を行った．結果，植物の開花種数，開花数および絶滅危惧種の開花種数はいずれも柵内で柵外より多く，チョウおよびマルハナバチの種数・個体数も柵内で柵外より多かった．また，ドローン空撮画像をもとに防鹿柵内外のニッコウキスゲの花数を計測し，防鹿柵による保全効果をより広域で視覚化した．本成果は，防鹿柵の設置が観光資源植物および絶滅危惧動植物を保全するうえで不可欠であることを示している．</p>

© 2020, Genetics Society of Japan. All rights reserved. Table 1 on p. 135 should be replaced with the corrected Table 1 shown bellow. (Table presented).

To protect endangered and commercially important orchid species, it is important to monitor and accurately identify orchid pests. Japanagromyza tokunagai (Sasakawa) larvae are known to infest the flower buds, stems, and ovaries of various Japanese orchid species. Given that J. tokunagai can infest diverse orchid hosts, J. tokunagai has the potential to be an economically important orchid pest in greenhouses. However, damage by J. tokunagai has previously only been reported in outdoor habitats. Here we revealed that the dipteran larvae infesting the flower buds of Phalaenopsis aphrodite Rchb.f. (Orchidaceae) cultivated in a greenhouse are J. tokunagai based on DNA barcoding, using COI sequences. Infested flower buds remained closed and eventually dropped to the ground, making the P. aphrodite plants commercially worthless. Phalaenopsis aphrodite is one of the most economically important ornamental orchids. Therefore, J. tokunagai is a potential threat to orchid production and we recommend taking precautions to prevent its spread.

Natural hybridization often occurs between insular and widespread species, and investigating these hybridization patterns is important in improving our understanding of the evolution of insular plant species. To this end, we studied the floral morphology and genetic structure of the widespread species Calanthe discolor, the insular closely related species C. izu-insularis and their putative natural hybrids on Honshu Island (mainland) and the Izu Islands (oceanic islands), Japan, using microsatellite markers. Natural hybridization probably occurred on all the investigated Izu Islands in Japan, although C. izu-insularis was morphologically and genetically different from C. discolor. Bumblebees and other relatively large bees are not distributed in the Izu Islands and therefore natural hybridization would have occurred via pollination by relatively small solitary bees that are pollinators of both Calanthe spp. We also showed that in the islands more remote from the mainland, the proportions of insular C. izu-insularis increased and the floral morphologies of both species changed. The results suggested that insular C. izu-insularis evolved on the Izu Islands and the widespread C. discolor adapted to the poor pollinator fauna on oceanic islands.

type:Departmental Bulletin Paper Herbarium specimens hold their DNA information at the time of collection from fields. This DNA information provides us with the opportunity to directly compare the past and present. However, it is difficult to analyze specimen DNA in the usual way because the DNA has been fragmented due to the effects of ultraviolet light and chemical fumigation. Therefore, in this study, we examined possibilities and methods of MIG-seq analysis using specimens of six plant species. As the results, several thousand or more loci were sequenced on average in all species, indicating that MIG-seq analysis is also useful for plant specimen-derived DNA. The number of loci and number of sequence reads obtained by the next-generation sequencer were significantly positively correlated, and the number of loci continues to increase without convergence even if the number of leads exceeds 300,000. Although the number of loci after correction of the number of reads decreased in old specimen samples of three species, a relatively large number of loci could be obtained in many samples in the case of specimens prepared around 1980. Also a sufficient number of loci were also sequenced in some samples of older specimens. Therefore, as long as the specimen condition is good, MIG-seq analysis should be useful even for a fairly old sample. Moreover, when the sequence data from multiple independent library constructions were integrated, the number of loci continued to rise even when the number of reads exceeded about 800,000. In conclusion, the most effective way to perform MIG-seq analysis for plant specimen-derived DNA would be to carry out multiple independent library preparations and obtain as many reads as possible. 原著 2018年度神奈川大学総合理学研究所共同研究助成論文

Commelina communis f. ciliata (Commelinaceae), a newly distinguished taxon, is an annual andromonoecious herb exhibiting a mixed mating system, the details of which remain unclear. We developed microsatellite markers for use in exploring the evolution of andromonoecy and mixed mating in the species. Fifteen micro satellite loci were developed using next-generation sequencing. The primer sets were used to evaluate 65 C. communis f. ciliata individuals from three populations in Japan; we found 1-13 alleles per locus and the expected heterozygosity ranged from 0.00 to 0.76. The markers are potentially useful to examine intra- and inter-species genetic structure and the mixed mating strategy of Commelina species via paternity analysis.

Heterostyly is a genetic polymorphism of anther and stigma heights, which are reciprocally placed among morphs. The maintenance of heterostyly has often been ascribed to disassortative mating, but alternative explanations are also possible, which can be examined by analyzing mating patterns. However, parental analysis with genetic markers has only been applied to a limited number of taxa, despite the phylogenetic diversity of heterostylous species. Here, we examined the mating patterns of a distylous primrose, Primula kisoana (Primulaceae), in its natural habitat, using 11 microsatellite markers. We also examined other fitness components. We found no differences in seed production, vegetative growth, or survival between morphs. However, the siring success of thrum fathers was much higher than that of pin fathers; outcrossing occurred unilaterally between thrum fathers and pin mothers, and selfing was more frequent in thrum plants than in pin plants. These findings are consistent with the explanation that distyly in this species is maintained either by overdominance due to previously reported recessive lethal effects of the thrum allele or by effectively dioecious mating if inbreeding depression is strong. We built a mathematical model of the overdominance hypothesis to confirm that it is logically sound. These results warrant further investigations into the possible roles of overdominance and dioecy in the maintenance of heterostyly.

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.

Semi-natural grassland areas expanded worldwide several thousand years ago following an increase in anthropogenic activities. However, semi-natural grassland habitat areas have been declining in recent decades due to changes in landuse, which have caused a loss of grassland biodiversity. Reconstructing historical and recent demographic changes in semi-natural grassland species will help clarify the factors affecting their population decline. Here we quantified past and recent demographic histories of Melitaea ambigua (Lepidoptera; Nymphalidae), an endangered grassland butterfly species in Japan. We examined changes in demography over the past 10,000 years based on 1378 bp of mitochondrial COI gene. We then examined changes in its genetic diversity and structure during the last 30 years using nine microsatellite DNA markers. The effective population size of M. ambigua increased about 3000-6000 years ago. In contrast, the genetic diversity and effective population sizes of many populations significantly declined from the 1980s to 2010s, which is consistent with a recent decline in the species population size. Our data suggest that the M. ambigua demography can be traced to changes in area covered by semi-natural grasslands throughout the Holocene.

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

The monitoring of genetic characters and definition of effective conservation units are important for conservation of endangered semi-natural grassland species.Using molecular markers, we elucidated the recent transitions in genetic diversity for the endangered grassland butterfly Melitaea protomedia (Lepidoptera; Nymphalidae) in Japan. First, we examined changes in genetic diversity and structure from the 1980s to the 2010s from archival and contemporary DNA samples using eight microsatellite markers. Second, we estimated the genetic structure based on 1374bp of mitochondrial COI gene from contemporary samples. We also defined the conservation units of M. protomedia based on the above analysis.The genetic diversity of M. protomedia has significantly declined from the 1980s to the 2010s. Although genetic differentiation was very strong among populations in c. 2010, there was only weak genetic structure in c. 1990. In addition, the number of haplotypes based on mitochondrial DNA is now very low due to recent declines.These findings suggest that effective conservation units for critically endangered species should be determined based on historic (i.e. prior to population declines) as well as contemporary genetic diversity and differentiation, because genetic structure may have changed over time. Genetic analysis of archival DNA is useful to obtain historic genetic information.