Naoyuki Nakahama

Development of microsatellite markers for two endangered grassland butterflies, Melitaea ambigua and M. protomedia (Nymphalidae), using Ion Torrent next-generation sequencing

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.

Availability of short microsatellite markers from butterfly museums and private specimens

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.

Mass emergence of a specialist sawfly species on unpalatable herbs under severe feeding pressure by sika deer

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.

The Sweat Bee Lasioglossum occidens is a Potential Pollinator of Calanthe aristulifera in the Oceanic Izu Islands

The genus calanthe comprises approximately 200 species of primarily terrestrial or lithophilic orchids distributed throughout Africa, Madagascar, China, Japan, South East Asia, and Australia (Karasawa and Ishida 1998). Although the pollination biology of most calanthe species remains unknown, at least eight species have been confirmed to be autogamous (Catling 1990; Jacquemyn et al., 2005). It has been suggested that the calanthe are most likely pollinated by Lepi doptera given the similarity of their floral characteristics to the genus Epi den drum (Dressler 1993), which is known to be pollinated by this group of insects. Several reports have provided observational evidence for this hypothesis; for instance, it has been noted that milkweed butterfly ideopsis similis has the ability to transfer the pollinaria of calanthe triplicata on its proboscis (Sugiura and Miyanaga 1996), while the cabbage white butterfly Pieris rapae has been confirmed as a pollinator for transplanted calanthe argenteostriata in Southeast China (Zhang et al., 2010). Taken together, these findings indicate that some long-spurred calanthe species can indeed be pollinated by butterflies or moths. However, there is also substantial evidence that other calanthe species are pollinated by bees. For example, intensive observation of the short-spurred species calanthe striata revealed it was pollinated exclusively by the carpenter bee, Xylocopa appendiculata circumvolans (Sugiura 2013), while the spur-less spec ies calanthe reflexa appears to be pollinated by two bumblebee species, Bombus diversus diversus and B. hypocrita (Sakata et al., 2013). Similarly, there is evidence that the short-spurred species calanthe yaoshanensis is pollinated by Bom bus patagiatus as well as by large hoverflies belonging to the genera Erista lis and criorhina (Ren et al., 2014). Furthermore, Suetsugu and Fuku shi ma (2014) have recently reported that medium to large bees, such as Eucera nipponensis, are the main pollinators of calanthe discolor, which has relatively long spurs, while Suhara (1993) has suggested that the long-spurred species calanthe aristulifera is exclusively pollinated by E. nipponensis. Such findings indicate that, even in long-spurred species, the pollination of the calanthe by larger bees could be more widespread than previously thought. It is well-established that the pollinator assemblage of small islands can have reduced diversity relative to mainland habitats (Carlquist 1974; Inoue and Amano 1986). For example, the insect fauna of many oceanic islands tend to lack large bees, which are considered important pollinators on the mainland (Kato et 368 EntomoloGical nEws

Recent transitions in genetic diversity and structure in the endangered semi-natural grassland butterfly, Melitaea protomedia, in Japan

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 1374 bp 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.

Time-lapse photography reveals the occurrence of unexpected bee-pollination in Calanthe izuinsularis, an endangered orchid endemic to the Izu archipelago

ABSTRACT 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.

Rediscovery of Celes akitanus (Orthoptera: Acrididae) from semi-natural grasslands in Japan

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 30 years. 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.

Preliminary Observations of Insect Visitation to Flowers of Vincetoxicum pycnostelma (Apocynaceae: Asclepiadoideae), an Endangered Species in Japan

Abstract Vincetoxicum 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.

Historical changes in grassland area determined the demography of semi-natural grassland butterflies in Japan

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.

Development of microsatellite markers for the endangered orchid Calanthe izu-insularis (Orchidaceae)

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.