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.     

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.     

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.     

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.     

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.     

Variable selection patterns on the labellum shape of Geoblasta pennicillata,a sexually deceptive orchid

By mimicking shape and female mating pheromones, flowers of sexually deceptive orchids attract sexually excited males which pollinate them while trying to copulate. Although many studies have demonstrated the crucial importance of odour signals in these systems, most flowers pollinated by pseudocopulation resemble, at least superficially, an insect body and these visual cues may be important to cheat pollinators. In this 2‐year study, we show that the shape of the labellum of Geoblasta pennicillata is a target of pollinator‐mediated natural selection. Contrary to our expectations, plants with a labellum shape more similar to female wasps were not favoured. The strength and pattern of phenotypic selection varied between study years and sexual functions. Although selection through female success was probably associated to the fine‐tuning of the mechanical fit between flower form and male wasp, shape was the target of natural selection through male success in both study years indicating that male wasps use this trait when choosing flowers. The imperfect mimicry and patterns of selection observed indicated that an exact imitation is not needed to attract and deceive the pollinators and they suggested a receiver perceptual bias towards uncommon phenotypes.     

A biochemical approach to the electrogenic potassium pump of insect sensilla: Potassium sensitive ATPases in the labellum of the fly

Summary A membrane bound, potassium sensitive ATPase which occurs in the proboscis ofProtophormia terraenovae (Diptera, Calliphoridae) is described. This ATPase activity is found only in the sensilla-rich labella but not in the haustella which contain few sensilla. Density gradient centrifugation shows that the enzyme is not of mitochondrial origin. It is insensitive to sodium azide, a specific inhibitor of mitochondrial ATPases, and has a relatively low affinity to potassium: half maximal activation is reached at approximately 70 mmol/l potassium.It is suggested that the potassium activated ATPase in the labellum of the fly is an integral constituent of the electrogenic potassium pump, which may be important for the generation of receptor currents.Abbreviation TEV transepithelial voltage Supported by the Deutsche Forschungsgemeinschaft SFB 4/C4     

Size‐dependent selection against small queens of the invasive bumblebee Bombus terrestris in Japan

Bombus terrestris (L.) (Hymenoptera: Apidae, Apinae, Bombini) has been introduced to Japan and became the dominant bumblebee in some areas, replacing native species. To implement an effective eradication program, the attributes responsible for establishment of the species must be better understood. Because body size has frequently been related to reproductive success in female insects, I examined the effect of B. terrestris queen size during hibernation, nest founding, and reproduction stages. Queens exhibited clear variation in size. Foundresses were significantly larger than gynes, indicating that larger queens may have an advantage over smaller ones in surviving hibernation. The over‐representation of queens with pollen at larger sizes suggested that larger queens may also have an advantage over smaller ones in nest founding. On the other hand, foundress size was not significantly associated with reproductive output. The number of collected foundresses in this study was not sufficient to draw a clear conclusion but factors other than foundress size may also play an important role in determining reproduction. In addition, gyne size increased significantly during the 4‐year survey. Because large queens have an advantage during hibernation, increased body size may increase the number of hibernating queens that survive, allowing them to outcompete native bumblebees.     

Comparative labellum micromorphology of the sexually deceptive temperate orchid genus Ophrys: diverse epidermal cell types and multiple origins of structural colour

Labellum micromorphology was imaged via scanning electron and light microscopy in 32 microspecies and one artificial hybrid of the European terrestrial orchid genus Ophrys, together representing all ten macrospecies circumscribed in the genus via molecular phylogenetics. Imaging of homologous regions of the adaxial surface, paying particular attention to the diagnostic feature of the comparatively reflective speculum, revealed the presence of between three and seven epidermal cell types on each labellum, the less complex labella being plesiomorphic. Epidermal protuberances range from short, domed papillae to long, twisted unicellular filaments. Multiple origins are inferred for pale labellar margins, large yellow appendices (both putative osmophores exuding pseudopheromones), broad flat labella and long lateral horns. Homoplasy in the speculum is manifested in unusually complex or simple outlines and the presence or absence of a pale margin or iridescence. The reflectivity of the speculum is caused by a combination of chemical and physical colour, whereas iridescence can be caused only by physical properties. The specula of most microspecies studied bear striated trichomes, albeit maturing comparatively late in ontogeny and being sufficiently narrow to allow light to reach the flat polygonal trichome bases. Reflectivity appears to be negatively correlated with the convexity and degree of cuticular corrugation shown by these epidermal cells. Two clades (the Speculum + Tenthredinifera + Bombyliflora group and Bertolonii subgroup of the Sphegodes group) have lost specular trichomes and include the most iridescent species; their flat, polygonal, nonstriated cells resemble those observed on the paired pseudoeyes that bracket the stigmas of all Ophrys except the Fusca group. The smooth thick‐layered cuticle and dense layers of organelles and starch bodies revealed by preliminary transmission electron microscopy study provide alternative candidates for the primary reflective surface of the speculum; in contrast, the trichomes and conical cells that dominate Ophrys labella, and occur on the specula of all but the most reflective species, absorb and/or diffuse light. Multiple MYB family genes are hypothesized to control epidermal micromorphology. The relative contributions of olfactory, visual and tactile cues to the sophisticated pseudocopulatory pollination mechanism that characterizes Ophrys remain unclear, but the degree of reproductive isolation achieved, and thus the speciation rate, have certainly been greatly exaggerated by most observers. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162 , 504–540.     

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.     

A field experiment on the effect of Nosema bombi in colonies of the bumblebee Bombus terrestris

Abstract 1. Nosema bombi is a microsporidian parasite of bumblebees that is of potentially great ecological and economic importance. Nosema bombi has been shown to have detrimental fitness effects on the bumblebee Bombus terrestris under laboratory conditions. The present study investigates whether N. bombi also has negative fitness effects under natural conditions. 2. A field experiment was conducted with B. terrestris spring queens caught from the wild. In the laboratory these queens were assigned to either infected or control treatment groups. On the emergence of workers, colonies were placed in the field and a number of fitness and life‐history traits were measured. 3. The size of colonies produced by infected queens was significantly smaller than those of control queens. None of the 14 infected colonies produced sexual offspring, whereas four of the 14 control colonies reached the male production stage. Results of the field experiment are in agreement with prior laboratory‐based results, and even imply that such effects are amplified under natural conditions. 4. The evidence suggests that, on entering the colony early, N. bombi has a highly detrimental effect on the fitness of its host. It would seem that this parasite reduces its opportunities to transmit into the next host generation.     

Expressed sequence tag analysis of the diapausing queen of the bumblebee Bombus ignitus

We constructed a full‐length cDNA library from diapausing queens of the bumblebee Bombus ignitus. A total of 480 randomly selected clones was sequenced by single‐run 5′‐end sequencing. Of these, there were 437 high quality clones, 23 poor quality clones and 20 read‐fail clones. Each high quality clone sequence was searched against a public protein database. The most frequently found matching genes were ribosomal proteins (12.5%), p10 (3.58%), cytochrome P450 monooxygenase (3.13%) and sensory appendage protein (2.9%). Sequence similarity analysis between bumblebees and other insect species showed that 72 out of 437 (16.5%) bumblebee expressed sequence tags (EST) matched sequences of Apis mellifera, with matches to Drosophila melanogaster (6.6%), Caenorhabditis briggsae (6.2%), Lysiphlebus testaceipes (4.8%), Periplaneta americana (3.7%) and Anopheles gambiae (3.4%) following, suggesting that sequence similarity of bumblebee EST is closest to that of A. mellifera. Functional classification of EST based on Gene Ontology showed that most genes found by sequencing are associated with physiological processes in the bumblebee. The results of sequencing and analysis of our 437 cDNA demonstrated that high‐throughput EST sequencing and data analysis are powerful means for identifying novel genes and for expression profiling. Our bumblebee EST collection could be a useful platform for further studies of gene expression in diapausing bumblebees.     

Floral vasculature in Alpinia hainanensis in relation to the nature of the labellum in gingers

Observations presented here on floral vasculature in Alpinia hainanensis indicate that the labellum incorporates elements of five androecial members rather than two or three, as suggested by previous authors for Zngiberaceae flowers. The pedicel contains an outer ring and a central region of vascular bundles. Three carpellary dorsal bundles (CDs) and three alternatively arranged parietal bundles (PBs) separate from the central region successively. The remaining bundles of the central region run upwards and become the placental bundles to supply ovules. The placental bundles terminate between the top of the locular region and the base of the prolongation. The three PBs divides into about five strands respectively. Of which the outer strand enters into the petal being its midrib and the remaining strands move into the stamen adaxially being the vasculature of the functional stamen and the labellum abaxially being the lateral strands of the labellum. The three CDs divide into about five traces, of which the outer strand becomes the midrib of each sepal and the inner strand runs into the style. The remaining traces re‐unite, re‐divide again in the course up and the two adaxial sets of carpellary dorsals finally enter into the labellum being the marginal traces of it while the abaxial single strand enters into the labellum being its midrib. The two antero‐lateral glands receive small traces without lignified tube elements from the vascular plexus, which fonn in prolongation from both PBs and CDs and a few small strands in the ovary wall. There are no subulate appendages differentiated in the flower of Alpinia hainanensis. Hereby, the median of the sepals, both the marginal portions and the median of labellum, and the style have the same origin in vasculature from the CDs and so do the stamen, the lateral portions of labellum and the median of the petals from PBs. The labellum is supposed to represent three members of the outer androecial whorl by its two marginal portions and the median and two members of the inner whorl by its two lateral parts except the median.     

Host choice in the phoretic mite Parasitellus fucorum (Mesostigmata: Parasitidae): which bumblebee caste is the best?

Host caste recognition may be important for the dispersal of phoretic mites associated with social insects. All developmental stages of the mite Parasitellus fucorum (Acari: Mesostigmata: Parasitidae) live in the nests of bumblebees (Hymenoptera: Apidae: Bombus). Dispersal occurs by specialised phoretic instars, deutonymphs, which attach to adult bumblebees. Since bumblebee colonies are annual and only young queens overwinter, deutonymphs that are able to discriminate between bumblebee castes and preferentially attach to queens should be favoured by selection. In the field, deutonymphs of P. fucorum were found to be phoretic on bumblebee workers and queens, and in behavioural experiments all castes proved to be attractive as carriers for the mites. However, they preferred queens that had hibernated as carriers when they could choose between workers and queens. In a further experiment, when given a choice, deutonymphs switched from males to young queens but never transferred from a queen to a male. These results suggest that deutonymphs preferentially attach to queens but may also use other castes for transport. Those dispersing on workers and males may try to switch to queens later. Host-switching is possible during copulation and on flowers, where bees of all castes forage. Received: 14 November 1997 / Accepted: 16 February 1998     

Mating system evolution and worker caste diversity in Pheidole ants

The efficiency of social groups is generally optimized by a division of labour, achieved through behavioural or morphological diversity of members. In social insects, colonies may increase the morphological diversity of workers by recruiting standing genetic variance for size and shape via multiply mated queens (polyandry) or multiple‐breeding queens (polygyny). However, greater worker diversity in multi‐lineage species may also have evolved due to mutual worker policing if there is worker reproduction. Such policing reduces the pressure on workers to maintain reproductive morphologies, allowing the evolution of greater developmental plasticity and the maintenance of more genetic variance for worker size and shape in populations. Pheidole ants vary greatly in the diversity of worker castes. Also, their workers lack ovaries and are thus invariably sterile regardless of the queen mating frequency and numbers of queens per colony. This allowed us to perform an across‐species study examining the genetic effects of recruiting more patrilines on the developmental diversity of workers in the absence of confounding effects from worker policing. Using highly variable microsatellite markers, we found that the effective mating frequency of the soldier‐polymorphic P. rhea (avg. me_N = 2.65) was significantly higher than that of the dimorphic P. spadonia (avg. me_N = 1.06), despite a significant paternity skew in P. rhea (avg. B = 0.10). Our findings support the idea that mating strategies of queens may co‐evolve with selection to increase the diversity of workers. We also detected patriline bias in the production of different worker sizes, which provides direct evidence for a genetic component to worker polymorphism.     

Lifetime reproductive success and longevity of queens in an annual social insect

Although central to understanding life‐history evolution, the relationship between lifetime reproductive success and longevity remains uncertain in many organisms. In social insects, no studies have reported estimates of queens’ lifetime reproductive success and longevity within populations, despite the importance of understanding how sociality and associated within‐group conflict affect life‐history traits. To address this issue, we studied two samples of colonies of the annual bumblebee, Bombus terrestris audax, reared from wild‐caught queens from a single population. In both samples, queens’ lifetime reproductive success, measured as either queens’ inclusive fitness or as total biomass of queen‐produced sexuals (new queens and males), was significantly positively associated with queen longevity, measured from the day the first worker was produced. We suggest that a positive relationship between reproductive success and longevity was inherited from nonsocial ancestors showing parental care and maintained, at least in part, because the presence of workers buffers queens against extrinsic mortality.     

Cryptically dimorphic caste differences in a Neotropical, swarm-founding paper wasp genus, Parachartergus (Hymenoptera: Vespidae)

Morphological differences in body shape between females of different reproductive conditions (in terms of insemination and ovarian development) were examined in two species of the Neotropical polistine genus Parachartergus: P. smithii and P. fraternus. The present study shows, for the first time, that non‐size‐based morphological divergence between queens and workers occurs in Parachartergus, an epiponine genus once believed to have little or no morphological caste differences. In the P. smithii colony examined, queens were significantly larger than workers in five of the eight body parts measured (head width, eye width, genal width, mesosomal length, wing length, first metasomal tergum width, and width and length of the second tergum), but the mean values of wing length and first and second tergum widths were not significantly different between them. The queen : worker size ratios tended to be greater anteriorly and smaller posteriorly, although the size ratio was greatest in second tergum length. Analysis of covariance (ancova ) with mesosomal length as covariate showed that queens had proportionally wider heads and narrower first terga than did workers. In the P. fraternus colony, size differences between queens and workers were not significant, and there was little or no difference in shape, but queens had significantly proportionally wider first terga than did workers.     

Targeted agri‐environment schemes significantly improve the population size of common farmland bumblebee species

Changes in agricultural practice across Europe and North America have been associated with range contractions and local extinction of bumblebees (Bombus spp.). A number of agri‐environment schemes have been implemented to halt and reverse these declines, predominantly revolving around the provision of additional forage plants. Although it has been demonstrated that these schemes can attract substantial numbers of foraging bumblebees, it remains unclear to what extent they actually increase bumblebee populations. We used standardized transect walks and molecular techniques to compare the size of bumblebee populations between Higher Level Stewardship (HLS) farms implementing pollinator‐friendly schemes and Entry Level Stewardship (ELS) control farms. Bumblebee abundance on the transect walks was significantly higher on HLS farms than ELS farms. Molecular analysis suggested maximum foraging ranges of 566 m for Bombus hortorum, 714 m for B. lapidarius, 363 m for B. pascuorum and 799 m for B. terrestris. Substantial differences in maximum foraging range were found within bumblebee species between farm types. Accounting for foraging range differences, B. hortorum (47 vs 13 nests/km²) and B. lapidarius (45 vs 22 nests/km²) were found to nest at significantly greater densities on HLS farms than ELS farms. There were no significant differences between farm type for B. terrestris (88 vs 38 nests/km²) and B. pascuorum (32 vs 39 nests/km²). Across all bumblebee species, HLS management had a significantly positive effect on bumblebee nest density. These results show that targeted agri‐environment schemes that increase the availability of suitable forage can significantly increase the size of wild bumblebee 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