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.     

Genetic diversity of an endangered plant, <Emphasis Type="Italic">Cypripedium macranthos</Emphasis> var. <Emphasis Type="Italic">rebunense</Emphasis> (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, n _a and n _e were 1.85 and 1.28 respectively, and H _o and H _e 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.     

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.     

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.     

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.     

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.     

气候变化情景下大花杓兰在中国的适生区预测

大花杓兰(Cypripedium macranthos)隶属兰科杓兰属,是国家二级重点保护野生植物,与大多数杓兰属植物分布在我国西南山区不同,主要分布于我国的华北、东北和台湾等地区。多年来,过渡采挖等导致了大花杓兰种群数量和个体数目急剧下降。鉴于大花杓兰特殊的分布格局和濒危现状,选择过去、当前和未来8个气候情景,利用MaxEnt物种分布模型结合38个环境变量及来源于数据库和最新实地调查的80个分布位点进行建模,分析了影响大花杓兰分布的关键环境变量,预测了其在当前、过去和未来气候情景下的适生区及其分布中心和迁移趋势。结果表明：当前情景下,大花杓兰适生区主要分布在我国东北和华北地区。影响其分布的5个关键环境变量分别是：UV-B最强月份均值(UV-B3,贡献率：54.0%)、森林覆盖率(FOR,贡献率：14.3%)、降水量季节性变化(BIO15,贡献率：7.4%)、温度季节性变动系数(BIO4,贡献率：6.8%)和草/灌木/林地(GRS,贡献率：4.6%)。其中,紫外辐射相关变量是首次被运用在杓兰属植物的适生区分布预测中,并被证实对大花杓兰的分布具有重要影响。过去3个气候情景下大花杓兰总适生...     

Deceptive pollination of the Lady's Slipper Cypripedium tibeticum (Orchidaceae)

To test whether the nectarless flowers of Cypripedium tibeticum attract pollinators through mimicry like the allied species C. macranthos var. rebunense, pollination biology of C. tibeticum was investigated in western China. Although C. tibeticum was also pollinated by bumble bee queens, i.e. Bombus lepidus , B. lucorum and B. hypnorum , no special, rewarding model plants were found in the habitat. Field experimentation confirmed that the flowers were self-compatible but insects were required to transfer orchid pollen to the stigma. Both Bombus queens and workers were visitors, but queens were much more frequent than workers and only queens were effective pollinators. Floral functional morphology analysis showed that it was large queens rather than small workers that fitted well with the flowers of C. tibeticum. With the faint sweet-fruity scent, the minor floral fragrance compound, ethyl acetate, probably plays a role in attracting bumble bees by food deception. The dark flowers with the inflated, trap-like labellum are hypothesized to mimic the nest site of queens. Therefore, bumble bee queens tend to be attracted by C. tibeticum through nest site mimic combined with food deception. Considering that the co-blooming flowers of C. flavum are pollinated by the Bombus workers, and C. smithii pollinated by a queen, we suggest that using the same bumblebees with different body sizes as the pollinators is the main reproductive isolation between interfertile C. tibeticum and C. flavum, while C. tibeticum and C. smithii tend to hybridize naturally.     

Variation in stigma morphology – How does it contribute¶to pollination adaptation in Pedicularis (Orobanchaceae)?

 Stigma morphology in Pedicularis and its role in pollination have not been evaluated. This paper reports for the first time the relationship between stigma morphology and other aspects of floral design. 17 species of Pedicularis representing all the different types of corolla structure in the genus were studied in the field and in the laboratory. Results indicate that the stigmatic surface morphology of Pedicularis species is related to the type of corolla structure occurring in the species. Pollination studies in eight Pedicularis species demonstrated adaptation between stigmatic surface morphology and other aspects of floral biology. Species with larger, more corrugated stigmatic surfaces always possess a subsidiary construction (“tooth” or “beak”) on the galea. In addition, they have relatively longer flower tubes, higher stigmatic pollen loads, bigger pollen size and a shorter blooming period for individual flowers. Finally, the authors discuss the evolutionary significance of floral design in genus Pedicularis. Received April 8, 2002; accepted June 11, 2002 Published online: December 11, 2002     

Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

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     

Diversity of ericoid mycorrhizal fungi isolated from hair roots of Rhododendron obtusum var. kaempferi in a Japanese red pine forest

 The diversity of ericoid mycorrhizal fungi of Rhododendron obtusum var. kaempferi was examined in a stand of Pinus densiflora at Tsukuba, Japan. In total, 153 slow-growing fungal isolates were obtained from roots of R. obtusum var. kaempferi, in which 113 isolates formed an ericoid mycorrhizal structure in vitro. Among them, 53 isolates were morphologically identified as Oidiodendron maius, but the others were not identified due to their sterilities. PCR-RFLP analysis in the rDNA-ITS region divided them into four different RFLP types. Phylogenetic analysis from sequence data of the region suggested that the four RFLP types belonging to distinct taxa and one sterile type are considered to be Hymenoscyphus ericae. This study is the first report of ericoid mycorrhizal fungi in a natural habitat in Japan. Received: August 23, 2002 / Accepted: December 11, 2002 Acknowledgments We thank Dr. K. Narisawa, Plant Biotechnology Institute, Ibaraki Agricultural Center, and Dr. R.S. Currah, Department of Biological Science, University of Alberta, for their helpful advice. Contribution no. 176, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan Correspondence to:M. Kakishima     

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.     

Highly efficient pollination by bumblebees ensures seed production in Pedicularis lachnoglossa (Orobanchaceae), an early‐flowering Himalayan plant

Abstract Pedicularis (Orobranchaceae) is a common high altitude genus of the Himalayas that may be affected by pollination limitation. Using Pedicularis lachnoglossa from Yulong (Jade Dragon) Snow Mountain in Lijiang (Yunnan Province, southwest China), we investigated the effects of high altitude habitats on the process of pollination and seed production. Floral biology, pollinator foraging behavior, breeding system, and pollination efficiency were examined using observation and exclusionary techniques. Pedicularis lachnoglossa was found to be entomophilous and exclusively pollinated by Bombus friseanus and B. yunnanicola. Our results indicated that pollination limitation in P. lachnoglossa was not significant. Under open pollination, approximately 80% of flowers were successfully pollinated and developed to fruits, and about 38% of ovules developed to mature seeds. Bumblebee pollination is highly precise and efficient in P. lachnoglossa, because its flowering phenology and floral characters enhance the foraging of bumblebees on flowers. This study supports that animal pollination plays a crucial role in the outbreeding of the early flowering Pedicularis. The evolution of floral specification in Pedicularis has the advantages of adaptation to bumblebee pollination in adverse high altitude habitats.     

First successful isolation of Entoloma clypeatum species complex from basidiospores

The Entoloma clypeatum species complex, known as “Harushimeji” in Japan, associates with Rosaceae and Ulmaceae plant species. In this study, we successfully isolated cultures of this fungal group via basidiospore isolation from tentative four Harushimeji species using modified Norkrans's C (MNC) medium and MNC medium containing n-butyric acid. Colony formations were observed on 22 of 25 basidioma samples; however, most exhibited slow and unsteady growth. The isolated mycelia contained dikaryotic hyphae and were identified through molecular phylogenetic analyses of the internal transcribed spacer region of fungal ribosomal RNA. Six isolates showing steady growth were deposited in the fungal culture collection of the Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, Japan. This result indicates that basidiospore isolation is a useful method for obtaining Harushimeji strains.     

Insect pollination of Musella (Musaceae), a monotypic genus endemic to Yunnan, China

 The pollination biology of Musella lasiocarpa (Franch.) C. Y. Wu ex H. W. Li, a monotypic genus in the banana family (Musaceae) endemic to southwestern China, was investigated. Floral phenology, nectar production, and flower visitors were observed, measured, and recorded. Bagging experiments and comparative breeding system studies were carried out at different study sites and in different seasons. Our results revealed four important aspects of the reproductive biology of Musella. 1) This species blooms year round, but most flowering is concentrated from February to August. The longevity of female flowers is greater than that of male flowers and opening of both types of flowers occurs at anytime during the day, but is concentrated in the morning. 2) Although nectar is produced uniformly while flowers are open, the nectar volume and rate of female flowers are significantly higher than that of male flowers. 3) Insects, such as bumblebees (Bombus eximius and B. montivolans), honeybees (Apis cerana and A. florea), and wasps (Vespa mandarinia) are the primary floral visitors and show a preference for female flowers. 4) Both fruit and seed set are pollinator-limited at both study sites. A comparison of the floral biology and pollination characteristics among the three genera in the banana family suggests that insect pollination has played an important role in the reproductive isolation and breeding system evolution of Musella. Received February 14, 2002; accepted March 26, 2002 Published online: November 22, 2002 Addresses of authors: Aizhong Liu, W. John Kress (correspondence, e-mail: kress.john@nmnh.si.edu), Botany, MRC-166, United States National Herbarium, National Museum of Natural History, Smithsonian Institution, PO Box 37012, Washington DC 20013-7012, USA. Hong Wang, De-Zhu Li (dzl@public.km.yn.cn), Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China.     

Mixed bumblebee and blowfly pollination of Cypripedium flavum (Orchidaceae) in Sichuan,China

Most Cypripedium species are specialized orchids pollinated by, in a broad sense, bees or flies. Here we present the first evidence that a slipper orchid, Cypripedium flavum, is pollinated by both bees and flies, i.e., bumblebees and blowflies. Artificial pollination experiments demonstrated that the flowers of C. flavum are self-compatible, but need pollen vectors for successful reproduction. Field observations detected 25 insects visiting the flowers, and 14 of these insects entered into the labellum of the flowers, but only female bumblebees, Bombus hypnorum, B. remotus, and the blowfly Calliphora vomitoria exited of the labellum with pollen smears of C. flavum. The floral functional morphology of C. flavum appears to be more suited to bumblebees than to blowflies. The bumblebees are more efficient pollinators of the orchid, but blowflies are more frequent visitors, so they pollinated more flowers despite being less efficient.     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)