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.     

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

Cypripedium 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.     

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.     

Flowering Phenology, Pollination, and Fruit Set of Cypripedium macranthos var. rebunense, a Threatened Lady's Slipper (Orchidaceae)

Cypripedium macranthos var. rebunense, was preliminarily studied on Rebun Island, Hokkaido, Japan, for conservation of this threatened orchid. The functional longevity of individual flowers varied from 0 to 18 days, a determining factor being the wind conditions at the growing sites. The flower was self-compatible but needed a pollen vector for fruit production. It lacked nectar and adopted a deceptive pollination system. Queens of a bumblebee, Bombus pseudo-baicalensis, were found to be pollinators but their visits were infrequent. Fruit set was 8.3% at one study site but only 1.2% at another, probably due to interference from the many visitors who came to see the flowers. Based on the results, conservation and management of lady's slipper were discussed. The need for less human interference and for preservation of the interactions between lady's slipper and its pollinator or other plant species with floral rewards is stressed. Received 1 November 2000/ Accepted in revised form 15 March 2001     

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

Bombus 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.     

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.     

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

Cypripedium 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.     

Stimulatory effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of Cypripedium macranthos seed in vitro

Cypripedium macranthos is a wild orchid that is becoming endangered. Efficient methods for its propagation from seed, which is indispensable for conservation, production and breeding, have not been reported. The effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of seeds of Cypripedium macranthos Swartz were examined. The duration of treatment with a solution of hypochlorite prior to sowing was one of the critical factors that affected germination. Approximately 70% of seeds that had been treated with either a solution of NaClO that contained 0.5% available chlorine for 60 min or with one of Ca(ClO)₂ with 3.2% available chlorine for 7 h, germinated after 3 months of culture at 20°C, subsequent to 2 months chilling at 4°C. Chilling seeds at 4°C prior to culture at 20°C was another factor that stimulated germination. Even chilling for 2 weeks had a promotive effect on germination, and chilling for 2 months enhanced it most effectively: the frequency of germination was 67% after 3 months of culture at 20°C. However, the promotive effects of chilling on germination were reduced by holding seeds at 20°C for 3 and 6 weeks prior to chilling treatment. Germination of 58‐70% was achieved by the addition of 1 µ M cytokinin to the medium, while the frequency was only 17% in cytokinin‐free medium. We report a reproducible and efficient method for enhancing seed germination of C. macranthos , which involves treatment with hypochlorite prior to sowing, and the combination of chilling at 4°C prior to germination and exposure to a cytokinin.     

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. Received April 23, 2002; accepted August 23, 2002 Published online: November 28, 2002 Addresses of the authors: Naoto Sugiura (e-mail: sugiura@aster.sci.kumamoto-u.ac.jp), Department of Environmental Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan. Masashi Goubara, United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8553, Japan. Kenji Kitamura, Division of Environmental Biology, Faculty of Life and Environmental Science, Shimane University, Mastue 690-8504, Japan. Ken Inoue, Biological Institute and Herbarium, Faculty of Science, Shinshu University, Matsumoto 390-8621, Japan.     

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

In 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.     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.     

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid

We isolated Rhizoctonia-like fungi from populations of the threatened orchid Cypripedium macranthos. In ultrastructural observations of the septa, the isolates had a flattened imperforate parenthesome consisting of two electron-dense membranes bordered by an internal electron-lucent zone, identical to the septal ultrastructure of Rhizoctonia repens (teleomorph Tulasnella), a mycorrhizal fungus of many orchid species. However, hyphae of the isolates did not fuse with those of known tester strains of R. repens and grew less than half as fast as those of R. repens. In phylogenetic analyses, sequences for rDNA and internal transcribed spacer (ITS) regions of the isolates were distinct from those of the taxonomically identified species of Tulasnella. On the basis of the ITS sequences, the isolates clustered into two groups that corresponded exactly with the clades demonstrated for other Cypripedium spp. from Eurasia and North America despite the geographical separation, suggesting high specificity in the Cypripedium–fungus association. In addition, the two phylogenetic groups corresponded to two different plant clones at different developmental stages. The fungi from one clone constituted one group and did not belong to the other fungal group isolated from the other clone. The possibility of switching to a new mycorrhizal partner during the orchid’s lifetime is discussed.