中濱 直之

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.

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.

In recent years, the increase in sika deer populations has had a significant impact on forest ecosystems. Many studies have reported that this increase has led to the dominance of plants unpalatable to the deer; however, few studies have investigated the effects of this dominance on the specialist sawfly species that feed on the unpalatable plants. We observed the mass emergence of Aglaostigma amoorensis (Hymenoptera: Tenthredinidae), a rare specialist sawfly of the false helleborine Veratrum plants unpalatable to sika deer, in areas under severe feeding pressure by sika deer. An exponential increase in the number of emergent A.amoorensis adults was observed to occur with an increase in Veratrum album patch sizes. In addition, the increase in the A.amoorensis density had a negative effect on reproductive success of V.album. Our findings suggest that the relative abundance of specialist sawfly to the host-plant abundance has changed for successive years after sika deer irruption.

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. (C) 2016 Elsevier B.V. All rights reserved.

The reintroduction of ex situ conserved individuals is an important approach for conserving threatened plants and reducing extinction risk. In this study, we elucidated the effects on the genetic diversity of wild populations of Vincetoxicum pycnostelma Kitag. [=Cynanchum paniculatum (Bunge) Kitag.] by modelling the genetic consequences of reintroducing plants using the germinated seeds of herbarium specimens. This semi-natural grassland herb is threatened in Japan. First, we tested the germinability of seeds from herbarium specimens collected from nine sites in Kinki and Tokai districts, Japan (one specimen per site, total 206 seeds). Next, we analysed the genetic diversity and structure of germinated seedlings and the current wild individuals using nine polymorphic microsatellite markers. Germination was observed for 38 seeds (18.4 %) from four specimens collected 3-18 years prior to the study. Although the genetic diversity of the specimens' seeds was lower than that of the wild population because of the small sample size, the seedlings from specimens taken from three sites had unique alleles that did not exist in the wild populations. Consequently, viable herbarium specimens' seeds with unique alleles could be useful resources for recovering the genetic diversity in threatened wild plant populations.