中濱 直之

The impact of biological invasions is unpredictable, and hence it is important to provide information at the earliest stage of invasion. This is the first report of the web spinner Haploembia solieri (Rambur, 1842) (Insecta: Embioptera) in Japan. We found this species in the Port of Kobe, on an artificial island in Hyogo Prefecture. The locality is clearly distant from its known distribution H. solieri is native in the Mediterranean region and introduced into the United States. In our surveys, 90 individuals were collected, but no males. This is also the first report of H. solieri in East Asia. Because we observed the H. solieri population in the fall of 2016 and early summer of 2017, this species may have been able to overwinter in Japan.

Calanthe izuinsularis is a rare, beautiful and fragrant orchid endemic to the Izu archipelago. Although it is known that mainland populations of closely related Calanthe species are pollinated by medium-to large-sized bees, it is likely that C. izuinsularis has been forced to alter its floral biology to attract alternative pollinators, as large-sized bees are rarely found on the Izu Islands. Indeed, the unusual floral characteristics of C. izuinsularis, which produces pale flowers that emit a strong and fragrant scent during the night, are considered the adaptation to a moth-pollinator syndrome. As expected, our time-lapse photography using a digital camera revealed that the inflorescences of C. izuinsularis were most frequently visited by nocturnal moths such as Noctuidae, Geometridae and Crambidae, which accounted for 50% of all insect visits ( in terms of both the number of frames with captured visitors and the number of times visited). However, our study could not provide evidence of a pollinator shift towards moths because none of the moth species was observed with pollinaria. On the other hand, our study found that the pollinaria of C. izuinsularis could become attached to the mesothorax of the small-sized sweat bee Lasioglossum occidens. Therefore, our study suggests that C. izuinsularis is still somewhat dependent on bee pollination, while the orchid has changed its floral colouration and scent. The occurrence of bee-pollination in C. izuinsularis would provide an explanation for how the natural hybridisation between C. aristulifera, C. discolor and C. izuinsularis can occur.

Knowledge of the temporal changes in genetic diversity and structure is important for identifying factors causing a decline in threatened insect species, and for establishing conservation programs for these species. Thus, there is recently an increasing interest in the restoration of genetic diversity in conservation programs using DNA data from historical museum specimens. For butterfly specimens, we measured the yields and fragment sizes of the extracted DNA and investigated the genotyping success probability of nine short microsatellite markers (allele size 73-191 bp). We used leg samples of specimens of a medium-sized butterfly species, Melitaea ambigua (Lepidoptera; Nymphalidae), collected from the 1960s to the 2010s. The yields of specimen-extracted DNA longer than 150 bp decreased with increasing specimen age. There were negative correlations between the genotyping success probability and specimen age for each of all microsatellite markers. A negative correlation was also observed between the genotyping success probability and allele size of each microsatellite marker. We conclude that short microsatellite markers and analysis of recently obtained specimens are particularly suitable for microsatellite analysis of butterfly specimens.

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.

Celes akitanus was rediscovered in semi-natural grasslands in Japan. This species is one of the most endangered insects in Japan; most local populations were thought to be extinct. Reliable records of this rare species had been obtained only from Yamagata, Niigata, Tokyo and Nagano Prefectures. We compared the morphology of the holotype of Oedipoda akitana (= C. akitanus) and newly collected specimens and found that they matched with each other. The species was redescribed based on the holotype and new specimens. A molecular phylogenetic analysis involving other acridid species indicated that our samples were practically identical to C. akitanus from China. We conclude that the specimens we collected are C. akitanus, which has been rediscovered in Japan after about 30years. Because the discovered populations were restricted to narrow and scattered areas, conservation of these populations should be a high priority. Population monitoring and habitat assessment are required. Because C. akitanus and many other endangered species are found in the grassland areas, these habitats should be protected and maintained.

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.

Premise of the study: Veronicastrum japonicum (Plantaginaceae) grows in grasslands on Honshu Island, Japan, and is threatened by habitat loss because of rapid land development over recent decades. For the genetic characterization of the remaining populations, microsatellite markers were developed.Methods and Results: Twelve polymorphic microsatellite loci were developed using next-generation sequencing. The number of alleles per locus ranged from two to 24 (mean 7.7), and the expected heterozygosity per locus ranged from 0.35 to 0.94 (mean 0.68).Conclusions: These markers can be used for genetic studies in conservation, such as the evaluation of genetic diversity and genetic structure.

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.

Melitaea ambigua and M. protomedia (Nymphalidae) are widely distributed in the grasslands of Northeast Asia. In Japan, these species have been threatened by habitat loss because of the rapid expansion of land development over recent decades. We isolated and characterised 18 polymorphic microsatellite loci (9 loci for each species) using a next-generation sequencer. The number of alleles and the expected heterozygosity per locus was 2-12 and 2-7, 0.10-0.86 and 0.13-0.76 in M. ambigua and M. protomedia, respectively. The markers can be useful for genetic studies for conservation such as evaluation of genetic diversity and genetic structure.

Premise of the study: Microsatellite markers were developed for the coastal shrub species Scaevola taccada to estimate the population genetic structure, which may reflect different seed dispersal patterns.Methods and Results: Thirteen microsatellite primer sets were developed for S. taccada using 454 pyrosequencing. The primer sets were tested on 64 individuals sampled from two populations in Japan. Fragments were amplified using the primers, with one to 10 alleles per locus, and the expected heterozygosity ranged from 0.00 to 0.85.Conclusions: These results indicate the utility of markers in S. taccada for broad estimations of the population genetic structure of this species.

pVincetoxicum pycnostelma Kitag. (Apocynaceae) is a perennial herb that grows in grassland habitats in East Asia and the western part of Amur. Urbanization, land development, abandonment of traditional management of grasslands, or any combination of these factors are rapidly reducing grassland habitats, and V pycnostelma is now ranked as a "Near Threatened" species on the Red List of Japan. Identification and characterization,of pollinators of this endangered species are important in its conservation. We observed and collected insect visitors to the flowers at night and in the morning in the populations of V pycnostelma on the banks of the Kidzu River, Kyoto, Japan from June to August in 2011. Pollinators were characterized as those insects on which flower pollinaria were attached. Twenty-seven species (9 families; 3 orders) were observed visiting the flowers; individuals of 14 lepidopteran species (6 families) had pollinaria attached to their bodies and most visited the flowers at night. Adult lepidopterans with a proboscis length of 0.9 - 4.5 mm were the primary carriers of pollinaria. Examination of stigmatic chambers of randomly selected flowers showed that the percentage of flowers in which pollinaria was inserted was much higher than the percentage of fruit set observed. Reasons for the observed low fruit set may be self-incompatibility or resource limitation. These preliminary observations were conducted at only 1 site and were restricted to a short period of time; yet, these data could be useful for elucidation of effective pollinators of this species and, thus, its conservation.

Vincetoxicum pycnostelma (Apocynaceae) is distributed widely in East Asia; however, over the last several decades, the number of populations of the species in Japan has been rapidly decreasing because of the loss of its grassland habitat. Existing population sizes are extremely small. Here, we isolated and characterized 9 microsatellite loci of V. pycnostelma by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 5-22 (mean, 8.7) and 0.41-0.92 (mean, 0.74), respectively. The markers described in this study will be useful for parentage analysis of V. pycnostelma (combined first-parent non-exclusion probability < 0.005) and conservation genetic studies such as evaluating the genetic diversity within and between the remaining populations.