Macranthos Var Research Articles

Consistent pollination services to Cypripedium macranthos var. rebunense (Orchidaceae) by Bombus pseudobaicalensis

AbstractBombus pseudobaicalensis queens are known pollinators of the threatened orchid Cypripedium macranthos var. rebunense. However, whether other bumblebee species also pollinate this orchid has not yet been ascertained. A 10‐year observation provided convincing evidence that B. pseudobaicalensis is the only consistent pollinator. B. beaticola moshkarareppus queens were newly found to rarely carry pollen smears of the orchid, whereas B. hypocrita sapporoensis and B. diversus queens were practically mere flower visitors. Why these three species do not equal B. pseudobaicalensis in pollination effectiveness remains unclear but differences in body size and individual abundance between B. pseudobaicalensis and the other species are considered as possible primary causes. Given the almost complete dependence of C. macranthos var. rebunense on B. pseudobaicalensis for pollination and its potential risk, conservation managers should take care not to reduce the bumblebee colonies.

岩手県内に栽培されている絶滅危惧植物アツモリソウCypripedium macranthos var. speciosumの遺伝的多様性および外来近縁種の混入

岩手県内に栽培されている絶滅危惧植物アツモリソウCypripedium macranthos var. speciosumの遺伝的多様性および外来近縁種の混入

Applying social network analysis to stakeholder analysis in Japan’s natural resource governance: two endangered species conservation activity cases

ABSTRACTStakeholders with diverse interests, concerns, and needs regarding the use and management of natural resources are involved in their governance process. Because of their diversity, policy practitioners need to manage their conflicting interests and establish a conservation strategy that reflects those needs. Therefore, it is necessary to identify the relevant stakeholders who should be involved in decision making. This study sought to ascertain the relevant stakeholders in Japan’s natural resource governance by using a social network analysis. The conservation activities involved in protecting two endangered species, Cypripedium guttatum (Spotted Lady’s-Slipper) and Cypripedium macranthos var. rebunense (Rebun Lady’s-Slipper), were chosen as case studies. With the data obtained from surveys, social networks were analyzed using centrality, core-periphery, and factions. Then, the stakeholders were categorized into three classes (key, primary, and secondary stakeholders) according to their location in the networks. Because the Spotted Lady’s-Slipper case included stakeholders from a wide range of activity fields and all the key stakeholders in the decision-making arena, it enjoyed a preferable condition in terms of representativeness and inclusiveness of diverse stakeholders. However, in the Rebun Lady’s-Slipper case, the guards and several key stakeholders working on on-site activities were excluded. Consequently, the analysis identified key stakeholders, who should be included in the decision-making arena. The study demonstrated the usefulness of the methodology, which would assist policy practitioners by identifying relevant stakeholders who should be involved in decision making when organizing the arena in Japan’s natural resource governance.

Post‐anthesis changes in the labellum of Cypripedium macranthos var. rebunense (Orchidaceae) and a speculation on functional design of the labellum

AbstractIn bumblebee‐pollinated Cypripedium macranthos var. rebunense, some plants have small labella that do not allow pollinating queens to enter, but they occasionally produce capsules for reasons that remain unclear. We found that after anthesis, the labellum gradually droops, and its width and length (but not height) distinctly increase, eventually producing an elongate and dorsoventrally compressed shape. The rate of increase in labellum size was significantly greater in flowers that initially had a smaller labellum, so that the post‐anthesis changes actually enabled large‐bodied bumblebees to enter those labella. Therefore, we conclude that C. macranthos var. rebunense relied on only bumblebee queens for pollination, irrespective of labellum size at the time of anthesis. Observations of in‐labellum bumblebee behavior suggested that the elongate and dorsoventrally compressed shape and the inclination seemed to result in more predictable and desirable bumblebee movements into the labellum.

Network governance of endangered species conservation: A case study of Rebun Lady's-Slipper

Since networks among stakeholders significantly affect the performance of natural resource governance, including endangered species conservation, effective co-management by diverse stakeholders is a crucial challenge. Using social network analysis, this study analyzes how stakeholders’ networks influence the performance of collaborative endangered species conservation. Conservation activities regarding the Rebun Lady's-Slipper (Cypripedium macranthos var. rebunense) are used as a case study, in which conservation activities are implemented under two formal institutions: the Rebun Lady's-Slipper Program and the Alpine Flowers Council. The former is responsible for overall policy-making, and the latter conducts monitoring and educational activities. I asked 38 actors involved in Rebun Lady's-Slipper conservation to assess the network activities. Most respondents evaluated “monitoring and patrol” and “educational activities” as good, while less than half of the respondents evaluated “policy-making arena” as good. The social network of the Alpine Flowers Council that conducts “monitoring and patrol” and “educational activities” was highly closed and heterogeneous; the network is dense, having a high proportion of ties between actors in different subgroups. This indicates that bonding and bridging social capital are developed, thus fostering the collaborative relationship necessary for monitoring and educational activities. Conversely, the network of the Rebun Lady's-Slipper Program is less closed, but is also heterogeneous. Furthermore, the network is separated into subnetworks, indicating the lower performance of the policy-making arena. Finally, these results indicate that the performance of Rebun Lady's-Slipper conservation activities is closely related to the social network structures.

Vegetation factors influencing the establishment and growth of the endangered Japanese orchid, Cypripedium macranthos var. rebunense

AbstractCypripedium macranthos var. rebunense is a well‐known endangered terrestrial orchid that is endemic to the cool‐temperate Rebun Island off the coast of northwestern Hokkaido, Japan. The present study aimed to identify suitable methods for conservation of this orchid through management of the natural vegetation. We analyzed the vegetation types and plants growing sympatrically with this orchid using two‐way indicator species analysis (TWINSPAN) and detrended correspondence analysis (DCA) ordination. Reproductive orchids can survive in various broad‐leaved and short to medium‐height herbaceous meadows with high plant species richness at a small scale in coastal regions. In order to maintain C. macranthos var. rebunense populations, succession to dense and tall large‐leaved herbs, Sasa scrub, shrubs, and trees, should be suppressed. Favorable vegetation for the establishment of seedlings of this orchid includes narrow‐leaved, medium‐sized grasses, sedges, herbs, mosses, and prostrate mat‐forming shrubs. The ground cover offered by these plants and their litter may favor stability of moisture, temperature, and suitable light conditions at the ground surface. In situ seed sowing at such ‘safe sites’ is a potential low‐impact technique to enhance seedling recruitment of this orchid.

Comparative pollination biology in two sympatric varieties of Cypripedium macranthos (Orchidaceae) on Rebun Island, Hokkaido, Japan

AbstractCypripedium macranthos sensu lato typically has purple‐pink flowers with no nectar and harvestable pollen. On Rebun Island, Hokkaido, Japan, purple‐pink‐flowered C. macranthos var. macranthos individuals rarely grow among numerous pale‐cream‐flowered C. macranthos var. rebunense plants. In both varieties, flower size is similar, their flowering periods completely overlap, and they share the same pollinator (Bombus pseudobaicalensis). However, in only one of 12 years from 2001 to 2012 did var. macranthos attain an annual fruit‐set ratio (an estimate of pollination success) higher than that of sympatric var. rebunense plants. These findings strongly suggest that in C. macranthos on Rebun Island, flower color results in the differential pollination success, because the pollinator prefers pale‐cream Cypripedium flowers and/or avoids purple‐pink ones, thereby producing pollinator‐mediated selection favoring pale‐cream flowers.

In vitro proliferation and genetic diversity of Cypripedium macranthos var. rebunense

In vitro culture from seeds to full maturity of Cypripedium macranthos var. rebunense, one of the most endangered species in Japan, was studied. Immature seeds from wild plants germinated efficiently in Tsutsumi-Tomita medium and 1/3 strength Murashige-Skoog medium both supplemented with 1% sucrose. Formed protocorms were transferred onto the same Murashige-Skoog medium developed into seedlings, which were subjected to a cold-treatment. The seedlings were further successively cultured in artificial soils (Florialite and then Cryptomoss) under artificially controlled photoperiod and temperature conditions. After 7 years, fully matured plants successfully flowered. The genetic diversity of the wild colony of this orchid in Rebun Island was examined. Leaf samples randomly collected were subjected to random amplified polymorphic DNA analysis. Initially examined 31 samples were classified into independent 5 groups, indicating that the colony consists of genetically different individuals. This suggested that plants in the habitat are heterogenous as to the genetic structure. Introduction of artificially propagated plants with a similar genetic background to this habitat would match the genetic diversity of the existing population. The results of this study may be helpful in planning the rescue of other endangered plant species.

An antifungal compound involved in symbiotic germination of Cypripedium macranthos var. rebunense (Orchidaceae)

Germination of orchid seeds fully depends on a symbiotic association with soil-borne fungi, usually Rhizoctonia spp. In contrast to the peaceful symbiotic associations between many other terrestrial plants and mycorrhizal fungi, this association is a life-and-death struggle. The fungi always try to invade the cytoplasm of orchid cells to obtain nutritional compounds. On the other hand, the orchid cells restrict the growth of the infecting hyphae and obtain nutrition by digesting them. It is likely that antifungal compounds are involved in the restriction of fungal growth. Two antifungal compounds, lusianthrin and chrysin, were isolated from the seedlings of Cypripedium macranthos var. rebunense that had developed shoots. The former had a slightly stronger antifungal activity than the latter, and the antifungal spectra of these compounds were relatively specific to the nonpathogenic Rhizoctonia spp. The level of lusianthrin, which was very low in aseptic protocorm-like bodies, dramatically increased following infection with the symbiotic fungus. In contrast, chrysin was not detected in infected protocorm-like bodies. These results suggest that orchid plants equip multiple antifungal compounds and use them at specific developmental stages; lusianthrin maintains the perilous symbiotic association for germination and chrysin helps to protect adult plants.

Genetic diversity of an endangered plant, Cypripedium macranthos var. rebunense (Orchidaceae): background genetic research for future conservation

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, na and ne were 1.85 and 1.28 respectively, and Ho and He were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.

Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment

Cypripedium macranthos var. rebunense is a well‐known wild orchid in Japan, and is considered to be a symbol for rare plant conservation. A fungus isolated from roots of C. macranthos var. rebunense induced symbiotic germination of the species in vitro. Cold treatment of the seeds at 4°C prior to fungal inoculation was required for the symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency. Maximum germination was attained after seeds were inoculated just after the cold treatment for 12 weeks, and approximately 20% of the seeds developed into protocorms more than 1 mm long. These results suggest that fungal inoculation takes place at the beginning of spring in nature, and the tough impervious seed coat may preserve the seed from the infection during autumn and winter seasons. The lengthy culture period of more than 16 weeks at 20°C on the same medium with the fungus caused gradual browning and rot of the protocorms. By elimination of the fungus with a fungicide and by transfer to a nutrient rich medium, approximately 20% of the protocorms developed into healthy plantlets. The methods obtained here appear to be applicable to symbiotic germination of many other threatened Cypripedium spp.

Genetic Analyses of Genus Cypripedium Found in Northern Japanese Islands and Related Species Endemic to Northeast China

Several species and varieties of genus Cypripedium grown in Japanese Islands and northeast China were investigated using genetic analysis. C. macranthos var. hotei-atsmorianum and C. macranthos var. rebunense were collected in northern Japan. They are regarded as varieties or forms of C. macranthos of Eurasia. Although the large subunit of ribulose-1, 5-diphosphate decarboxylase/oxydase (rbcL) of chloroplast did not show any difference among the species, a difference between C. macranthos var. hotei-atsmorianum and the other two was found in the sequence of nuclear DNA encoding the internal transcribed spacer (ITS) region of ribosomal RNA. Random amplified polymorphic DNA method (RAPD) revealed a difference between C. macranthos var. rebunense from others. That C. calceolus and C. macranthos which were both collected in the Eurasia differed was demonstrated by their rbcL analyses and ITS sequences. The genetic analyses confirmed that C. × ventricosum was a hybrid of C. calceolus × C. macranthos. Thus, analyses using rbcL and ITS region are useful to distinguish species and varieties of Cypripedium.

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.

Bumblebee pollination of Cypripedium macranthos var. rebunense (Orchidaceae); a possible case of floral mimicry of Pedicularis schistostegia (Orobanchaceae)

Nectarless flowers of Cypripedium macranthos var. rebunense are pollinated by only queen Bombus pseudobaicalensis, which also pollinates nectar-producing flowers of Pedicularis schistostegia. Our previous study (Sugiura et al. 2001) suggested that they form a floral mimicry system: (1) Flowering phenology of both plants overlapped greatly. (2) Cypripedium occurred with lower frequency relative to Pedicularis. And (3) in a mixed stand of both plants, foraging bumblebees were sometimes confused between the mimic and model flowers. The present study clarified the system with new evidence. (4) Flower colour of Cypripedium and Pedicularis would be similar within the range of a bumblebee's visual spectrum. And (5) both species largely overlapped in spatial distribution. Based on these findings, we discuss how C. macranthos var. rebunense differs in pollination mechanism from other congeneric species, especially C. calceolus.

Notes on the Pollination of Cypripedium macranthos var. speciosum and Dactylorhiza aristata (Orchidaceae)

Anthecological observations on two Japanese orchids, Cyperipedium macranthos var. speciosum and Dactylorhiza aristata, were preliminarily carried out. The results suggested that nectarless flowers of

A Preliminary Report on the Symbiotic Germination of Nine Japanese Terrestrial Orchids.

To develop an effective propagation method, a symbiotic culture was attempted in nine species of Japanese terrestrial orchids. Seeds of three species of Goodyera (Goodyera biflora var. macrantha, G. foliosa var. laevis, and G. hachijonesis var. matsumurana), when inoculated non-symbiotically or with fungi from the binucleate Rhizoctonia fungus group among the 20 fungus strains tested germinated with virtually the same TTC activity level as embryos. Those on a symbiotic culture with binucleate Rhizoctonia grew markedly well compared with seeds on a non-symbiotic culture. The low germination rates in six of the species in the TTC test results are attributed to the unsuitability of the test culture conditions. There were numerous effective fungal strains with 5 species other than Cypripedium macranthos var. speciosum, which developed a symbiotic relationship with only one of the fungal isolates tested. Among the 3 species (Aorchis cyclochila, Dactylorhiza aristata, and Gymnadenia camtschatica) the binucleate Rhizoctonia fungal group would be suitable for symbiotic germination and subsequent growth, whereas the 2 species Amitostigma kinoshitae and Ponerorchis graminifolia var. graminifolia. responded better to R. repens