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The SPME-GC-MS analysis of the aroma components of Gymnadenia conopsea subsp. conopsea, subsp. densiflora, var. alpina and Gymnadenia odoratissima var. odoratissima, var. idae were reported. The main components of in total 78 found in G. conopsea subsp. conopsea were elemicin, cis-9-hexadecenal, hexadecanal, isoelemicin and (Z)-11-hexadecen-1-ol acetate; in subsp. densiflora benzyl benzoate, eugenol and trans-isoeugenol; in var. alpina benzyl benzoate, methyleugenol and elemicin. In the scent of G. odoratissima var. odoratissima were found 2-phenylethyl acetate, eugenol and pentadecane, in var. idae mainly C15–C21 alkanes and C16, C18 carbonic acids and some isoprenoid-derivatives. As all tested Gymnadenia-taxa are allogamous, the differences in scent composition may play a role in pollinator attraction.

Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occur at a given site, often associate with different partners, possibly to avoid competition and to allow stable coexistence. However, whether differences in mycorrhizal partners directly affect seed germination and subsequent protocorm formation remains largely unknown. In this research, we used in vitro germination experiments to investigate to what extent seed germination and protocorm formation of Gymnadenia conopsea was affected by the origin and identity of fungal associates. Fungi were isolated from G. conopsea and three other co-occurring orchid species (Dactylorhiza viridis (Coeloglossum viride), Herminium monorchis, and Platanthera chlorantha). In total, eight fungal associates, belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, were successfully isolated and cultured. While all eight fungal strains were able to promote early germination of G. conopsea seeds, only fungal strain GS2, a member of the Ceratobasidiaceae isolated from G. conopsea itself, was able to promote protocorm formation and subsequent growth to a seedling. Two other fungal strains isolated from G. conopsea only supported seed germination until the protocorm formation stage. The other five fungal strains isolated from the co-occurring orchid species did not support seed germination beyond the protocorm stage. We conclude that, although G. conopsea is considered a mycorrhizal generalist that associates with a wide range of fungi during its adult life, it requires specific fungi to promote protocorm formation and growth to a seedling. ispartof: MYCORRHIZA vol:30 issue:2-3 pages:221-228 ispartof: location:Germany status: published

The paper presents data on the ecological and phytocoenotic conditions of habitats of the G. conopsea (L.) R. Br. orchid in the Northeast of European Russia (Komi Republic, Russia). The data include characteristics of the populations of this species on the northern border of its range (size and ontogenetic structure of the populations, density of specimens, phenology), as well as information demonstrating the genetic variability of the species by ISSR-markers that was not included in the main publication. The data presented here supplement our earlier published results O.E.Valuyskikh et al., 2019 and are useful for more detailed analysis of population biology and genetic variability of this rare orchid species. G. conopsea is the species of terrestrial orchids widespread in Europe and Asia and characterized by the widest ecological-cenotic amplitude and occurrence in different types of ecotopes. The size of G. conopsea populations in different parts of its range is usually small, 25–100 ind. but sometimes increases to 200–1000 ind. Hansen et al., 1999 to Travnichek et al., 2012. In the many regions of the Russian Federation, the G. conopsea are subject to protection due to the small number of habitats, long stages of ontogenesis, low population sizes and anthropogenic impact. The complex of G. conopsea s.l. included in the Red Data Book of the Komi Republic Taskaev, 2009.

Co-occurring orchid species tend to occupy different areas and associate with different mycorrhizal fungi, suggesting that orchid mycorrhizal (OrM) fungi may be unevenly distributed within the soil and, therefore, impact the aboveground spatial distribution of orchids. To test this hypothesis, we investigated spatial variations in the community of potential OrM associates within the roots of three co-habitating orchid species (Anacamptis morio, Gymnadenia conopsea, and Orchis mascula) and the surrounding soil in an orchid-rich calcareous grassland in Southern Belgium using 454 amplicon pyrosequencing. Putative OrM fungi were broadly distributed in the soil, although variations in community composition were strongly related to the proximal host plant. The diversity and frequency of sequences corresponding to OrM fungi in the soil declined with increasing distance from orchid plants, suggesting that the clustered distribution of orchid species may to some extent be explained by the localised distribution of species-specific mycorrhizal associates.

Bcud len (prounounced chu len; essence extraction) refers to the preparation of pills that provide sustenance for Tibetan yogins practicing in remote caves. Dactylorhiza hatagirea tubers (Tibetan dbang lag) which is valued as aphrodisiac in Himalaya are included in some bcud len preparations. However sex is anathema for the Yellow Sect: perhaps dbang bag’s power could be sublimated to impart vigour to spiritual practice. Some examples of bcud len materials are presented. In Dharamsala, so-called bcud len pills are marketed as health supplements claiming to prevent aging and promoted youthfulness. North of Himalaya and up to Mongolia, wangla (Dactylorhiza viridis or Gymnadenia conopsea) is tonic, aphrodisiac, almost a panacea. Chinese scientists are busy studying these orchids.

Floral scent is a crucial trait for pollinator attraction. Yet only a handful of studies have estimated selection on scent in natural populations and no study has quantified the relative importance of pollinators and other agents of selection. In the fragrant orchid Gymnadenia conopsea, we used electroantennographic data to identify floral scent compounds detected by local pollinators and quantified pollinator-mediated selection on emission rates of 10 target compounds as well as on flowering start, visual display and spur length. Nocturnal pollinators contributed more to reproductive success than diurnal pollinators, but there was significant pollinator-mediated selection on both diurnal and nocturnal scent emission. Pollinators selected for increased emission of two compounds and reduced emission of two other compounds, none of which were major constituents of the total bouquet. In three cases, pollinator-mediated selection was opposed by nonpollinator-mediated selection, leading to weaker or no detectable net selection. Our study demonstrates that minor scent compounds can be targets of selection, that pollinators do not necessarily favour stronger scent signalling, and that some scent compounds are subject to conflicting selection from pollinators and other agents of selection. Hence, including floral scent traits into selection analysis is important for understanding the mechanisms behind floral evolution.

This article contains data on the reproduction biology of Gymnadenia conopsea (L.) R.Br. (Orchidaceae) in the Komi Republic on the northern border of the habitat of the species. Morphometric characteristics of flowers, fruits, and seeds in different coenopopulations of this species in the region have been studied. Fruit set, seed productivity, and seed quality are also defined.

Studies of plant breeding systems are particularly important when they involve facultative processes, which can be overlooked in natural conditions. This especially applies to species that are considered allogamous, due to their pollination syndrome and floral architecture. We examined the potential level and factors enabling or limiting spontaneous autogamy in three populations of the lepidopteran orchid Gymnadenia conopsea. Using a bagging experiment, we noted the stages of the anther thecae and the positions of the pollinaria at the five phenological stages of the flower, as well as the quantity of autogamously set fruits and the number of properly formed seeds. In the studied populations, autogamy represents an accidental character, with a maximum of 3.3% of fruits set spontaneously per analysed sample in a given population, and with seed numbers ranging from 29 to 354. This process is an environmentally dependent co-product of the mechanisms that enable a position appropriate for touching the stigma (bending of the caudicle) and increase male fitness (disintegration of the massulae), preceded by the gradual opening of the anther chambers. Autopollination of G. conopsea may occur in the flowers at various flowering stages (excluding the beginning of anthesis) at each position on the inflorescence.

A new ureido-substituted amino acid, conopsamide A (1), has been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Its structure was elucidated by extensive spectroscopic analysis, and the absolute configuration was assigned by Marfey's method. The new compound was evaluated for in vitro assay for HDAC1 (Histone Deacetylase 1) inhibitory activity.

BACKGROUND AND AIMS: Floral scent is considered an integral component of pollination syndromes, and its composition and timing of emission are thus expected to match the main pollinator type and time of activity. While floral scent differences among plant species with different pollination systems can be striking, studies on intraspecific variation are sparse, which limits our understanding of the role of pollinators in driving scent divergence. METHODS: Here, we used dynamic headspace sampling to quantify floral scent emission and composition during the day and at night in the natural habitat of six Scandinavian populations of the fragrant orchid Gymnadenia conopsea. We tested whether diel scent emission and composition match pollinator type by comparing four populations in southern Sweden, where nocturnal pollinators are more important for plant reproductive success than are diurnal pollinators, with two populations in central Norway, where the opposite is true. To determine to what extent scent patterns quantified in the field reflected plasticity, we also measured scent emission in a common growth chamber environment. KEY RESULTS: Both scent composition and emission rates differed markedly between day and night, but only the latter varied significantly among populations. The increase in scent emission rate at night was considerably stronger in the Swedish populations compared with the Norwegian populations. These patterns persisted when plants were transferred to a common environment, suggesting a genetic underpinning of the scent variation. CONCLUSIONS: The results are consistent with a scenario where spatial variation in relative importance of nocturnal and diurnal pollinators has resulted in selection for different scent emission rhythms. Our study highlights the importance of adding a characterization of diel variation of scent emission rates to comparative studies of floral scent, which so far have often focused on scent composition only.

Orchids are known for their species richness, intriguing ecology, rarity and the fact that they grow in almost all terrestrial ecosystems. Although numerous studies about their ecology have been carried out concerning calcareous areas, little is known about orchids that occur in serpentine habitats. The aim of this study was to investigate the ecological preferences of orchids in serpentine and non-serpentine areas on the model of the Valjevo Mountain Range (W Serbia). Niche analysis of orchids was performed using outlying mean index analysis. Data concerning geographical coordinates, altitude, habitat type, inclination, bedrock type, light regime, soil moisture, acidity, nitrogen and temperature were used as explanatory variables. Data of 33 orchid taxa from 407 localities were analysed. The most important gradients that govern orchid distribution were geological bedrock, light regime and temperature. The results have shown that only Anacamptis morio and Gymnadenia conopsea have statistically significant...

© 2014 The Authors. Plant-pollinator interactions are potential drivers of evolution in floral traits. Because nectar chemical composition is known to mediate both plant-pollinator interactions and plant reproductive success, it can be expected that chemical composition of nectar is subjected to strong pollinator-mediated selective forces. However, the extent of natural selection on different nectar components has not been studied so far. Using the Lepidoptera pollinated fragrant orchid Gymnadenia conopsea as a model species, we used high-performance anion-exchange chromatography (HPAEC) to characterize the sugar and amino acid composition of floral nectar in three calcareous grassland populations of G. conopsea. We then measured phenotypic selection on nectar composition and on other plant and floral traits through applying both linear regression and structural equation modelling. We demonstrate phenotypic selection on plant height, inflorescence height and on specific nectar amino acids, whereas spur length, total sugar and amino acid concentration were not direct targets of selection. Chemical nectar composition is thus indeed under selective pressure but nectar amino acids are much more important to fitness of G. conopsea, as compared to nectar sugars. Furthermore, as we found no evidence of selection on the total amino acid concentration, it is unlikely that amino acids increase pollinator attraction because they are a pollinator nitrogen source. To further unravel the evolutionary ecology of floral nectar, behavioural experiments with pollinators exposed to different nectar components and studies experimentally identifying the selective agents are recommended. ispartof: Oikos vol:124 issue:4 pages:421-427 status: published

Osmophores of Gymnadenia conopsea are located on the adaxial surface of labellum and on distal parts of two lateral sepals. Osmophore cells are characterised with a large nucleus, and dense, granular cytoplasm, which contains numerous membranes of ER and large lipid droplets. Plastids are probably involved in the synthesis of fragrant substances and, contrary to the most of investigated orchid species, they do not contain starch. Numerous secretory vesicles take part in the secretion. Secreted fragrant substance migrates across the cell wall and through the pores in the cuticle. It is not accumulated on the osmophore cell surface.

Individual, intra- and interpopulation, ecological, and geographic variation has been studied in G. conopsea populations on limestones of the Timan Range. The results show that the response of plants to deterioration of growing conditions manifests itself in a decrease in the values of individual morphometric characters and the strength of correlations between them. In the northeast of European Russia, this species is represented by two forms, G. conopsea (L.) R.Br. s. str. and G. conopsea var. alpina Rchb. f. ex Beck (?), with the latter being regarded as an extreme form of its ecological variation.

Data are presented on the abundance, structure, and dynamics of hemipopulations of G. conopsea protocorms and autotrophic individuals at the northern boundary of the species range. The dynamics of onto� genetic structure of G. conopsea cenopopulations have a fluctuating pattern. Their response to adverse weather conditions is manifested in shortterm reduct ion of density and increase in cenopopulation ageness in subsequent years. Deterioration of growing conditions in a series of ecotopes is accompanied by an increase in the proportion of young individuals, which allows the species to level off the impact of adverse weather fac� tors on population size.

The life form of G. conopsea (short-lived perennial with annual vegetative renewal) makes it possible to combine the long-term, complete ontogeny and polycarpy of the genet with the short life span and monocarpy of individual daughter ramets. Annual changes in the structural organization of daughter individuals allow the plants to adapt to changing environmental conditions and retain the territory they have occupied. Organismal mechanisms providing for the sustainable development of G. conopsea plants in marginal populations are considered in the context of the concept of multivariant plant ontogeny.

Gymnadenia conopsea (L.) R. Br. (Orchidaceae) is a perennial herbaceous orchid plant that grows widely throughout Europe and in temperate and subtropical zones of Asia. In China, its tuber has been used in traditional Chinese medicines, Tibetan medicines, Mongolian medicines and other ethnic medicines, and taken to treat numerous health conditions. The present paper provides a review of the traditional uses, phytochemistry, biological activities, and toxicology to highlight the future prospects of the plant. More than 120 chemical compounds have been isolated, and the primary components are glucosides, dihydrostilbenes, phenanthrenes, aromatic compounds, and other compounds. G. conopsea and its active constituents possess broad pharmacological properties, such as the tonifying effect, anti-oxidative activity, anti-viral activity, immunoregulatory, antianaphylaxis, antigastric ulcer, sedative, and hypnotic activities, etc. However, overexploitation combined with the habitat destruction has resulted in the rapid decrease of the resources of this plant, and the sustainable use of G. conopsea is necessary to study. Meanwhile, the toxicity of this plant had not been comprehensively studied, and the active constituents and the mechanisms of action of the tuber were still unclear. Further, studies on G. conopsea should lead to the development of scientific quality control and new drugs and therapies for various diseases; thus, its use and development require additional investigation.

The subject of this diploma thesis was the research of vegetation at localities in the Protected Landscape Area Bohemian Paradise. There were choosen areas: Natural Monument Podloučky and Protected Area Fialník, where grows an orchid Gymnadenia conopsea subsp. montana. There were made 60 phytocenological samples, and described: biotop, altitude, slope, huminidy and exposure. There were found four orchid species in the localities. Phytocenological samples were compared with each other. It has been shown that differences between localities are greater than the differences between orchid and non-orchid samples. Significant differences were also found in comparing the coverage in the floor between locations and abundace of species in each floor.

This study is to offer the raw data for conservation and restoration of national park by surveying and analysing the vascular plants in the Gayasan national park. The flora were summarized as 102 families, 328 genera, 469 species, 4 subspecies, 59 varieties and 9 forms. The endangered plants designated by Ministry of Environment were 2 taxa; Pedicularis hallaisanensis Hurus. and Gymnadenia conopsea for. albiflora Y.N.Lee. The rare plants designated by Korea Forest Service were 13 taxa; Crypsinus hastatus (Thunb.) Copel., Ligusticum tachiroei (Franch. & Sav.) M. Hiroe & Constance, Primula modesta var. fauriae (Franch.) Takeda, Lilium distichum Nakai ex Kamibay and so forth. The korean endemic plants were 15 taxa; Betula ermanii Cham., Carpinus laxiflora (Siebold & Zucc.) Blume, Stewartia pseudocamellia Maxim., Galium koreanum (Nakai) Nakai, Heloniopsis koreana Fuse, N.S. Lee & M.N. Tamura and so forth. The specific plants by floristic region were 66 taxa; Betula chinensis Maxim., Spiraea fritschiana Schneid., Vaccinium hirtum var. koreanum (Nakai) Kitam., Cimicifuga heracleifolia Kom., Adoxa moschatellina L. and so forth. The plants with approval for delivering oversea were 22 taxa; Salix hallaisanensis H.Lv., Sanguisorba argutidens Nakai, Viola albida Palib., Weigela subsessilis (Nakai) L.H.Bailey and so forth. The naturalized plants were 30 taxa; Chenopodium album L., Lepidium apetalum Willd., Trifolium pratense L., Bidens frondosa L., Helianthus tuberosus L. and so forth.

Summary 1. This account presents information on all aspects of the biology of Gymnadenia conopsea (L.) R. Brown s.l. (Fragrant Orchid) that are relevant to understanding its ecological characteristics and behaviour. The main topics are presented within the framework of the Biological Flora of the British Isles: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, floral and seed characteristics, herbivores and disease, history and conservation. 2. Most available information was published before recent revisions in taxonomy split what had been known as Gymnadenia conopsea into three separate species, now designated G. borealis (Druce) R.M. Bateman, Pridgeon & M.W. Chase, G. conopsea (L.) R. Brown and G. densiflora (Wahlenb.) Dietrich. Although the three newly accepted species have at least partial reproductive isolation, they have numerous morphological and ecological similarities. Some recent work still refers to G. conopsea s.l. because of difficulties in distinguishing between the species, and inertia in adoption of the new taxonomy. Wherever possible, differences between G. borealis, G. conopsea s.s. and G. densiflora are highlighted. 3. Gymnadenia conopsea s.l. is a perennial, terrestrial orchid that is widely distributed across Eurasia. The three segregate species are all considered native. G. conopsea s.s. (Chalk Fragrant Orchid) occurs mainly in the southern half of Britain, where it is most commonly observed in dry calcareous grasslands and pastures. G. densiflora (Marsh Fragrant Orchid) occurs in damp calcareous marshes, fens and dune slacks in England and Wales; it is rare in Scotland but frequent in Ireland. G. borealis (Heath Fragrant Orchid) is mainly found on hill pastures in Wales, northern England and Scotland. The three species all avoid shade, being most abundant in open habitats. 4. Although the three segregate species cannot always be reliably distinguished on the basis of morphological characteristics, diagnostic features include plant height, leaf width and disposition, density of flowers in the inflorescence, shape of the labellum, spur width, and odour and colour of the flowers. Additionally, several cytotypes within G. conopsea s.s. have been observed in Central Europe. These often grow in mixed-ploidy populations; in contrast, the species appears to be predominantly diploid in Britain. 5. Gymnadenia conopsea s.l. is a non-bulbous geophyte. Leaves emerge in spring, and flowering takes place between the end of June and the beginning of August. Vegetative adult dormancy – the failure of above-ground parts to appear in a growing season, followed by reappearance of full-sized photosynthetic plants in subsequent years – has been observed. Such dormancy rarely lasts longer than 1 year.

Most plants attract multiple flower visitors that may vary widely in their effectiveness as pollinators. Floral evolution is expected to reflect interactions with the most important pollinators, but few studies have quantified the contribution of different pollinators to current selection on floral traits. To compare selection mediated by diurnal and nocturnal pollinators on floral display and spur length in the rewarding orchid Gymnadenia conopsea, we manipulated the environment by conducting supplemental hand-pollinations and selective pollinator exclusions in two populations in central Norway. In both populations, the exclusion of diurnal pollinators significantly reduced seed production compared to open pollination, whereas the exclusion of nocturnal pollinators did not. There was significant selection on traits expected to influence pollinator attraction and pollination efficiency in both the diurnal and nocturnal pollination treatment. The relative strength of selection among plants exposed to diurnal and nocturnal visitors varied among traits and populations, but the direction of selection was consistent. The results suggest that diurnal pollinators are more important than nocturnal pollinators for seed production in the study populations, but that both categories contribute to selection on floral morphology. The study illustrates how experimental manipulations can link specific categories of pollinators to observed selection on floral traits, and thus improve our understanding of how species interactions shape patterns of selection.

Summary 1. Understanding the many factors that affect community composition and coexistence of species in natural environments is one of the central goals in ecology. As germination and establishment of seedlings in orchids are crucially dependent on mycorrhizal availability, the diversity and spatial distribution of orchid mycorrhizal fungi are likely factors that contribute to orchid coexistence. 2. In this study, we combined molecular identification techniques with seed germination experiments and spatial point pattern analyses to investigate to what extent differences in mycorrhizal association patterns affected spatial variation in seed germination and the above-ground distribution of three co-occurring terrestrial orchid species (Anacamptis morio, Gymnadenia conopsea and Orchis mascula). 3. The three species associated with a different set of mycorrhizal fungi, except A. morio and G. conopsea, which shared one fungal associate. The number of fungal lineages detected associating with each species also differed between species, being highest in G. conopsea, which associated with five lineages, and restricted to a single lineage in O. mascula. 4. Seed germination experiments showed that below-ground seed germination was restricted to locations where orchid individuals were most abundant, and quickly declined with increasing distance from the nearest above-ground congener. Spatial point pattern analyses indicated significant fine-scale spatial clustering that was highest in O. mascula and lowest in G. conopsea. Moreover, the spatial distributions of the three species were independent from each other, except for A. morio and G. conopsea. 5. Synthesis. Our results support previous findings that co-occurring orchid species tend to use different mycorrhizal partners. Although the possibility that variation in local environmental conditions affected seed germination could not be completely ruled out, our results suggest that the presence of specific mycorrhizal fungi contributed, at least partly, to the spatial distribution and coexistence of the investigated orchid species.

Conservation of species threatened by habitat fragmentation is a major global challenge, and determining the genetic and demographic processes associated with isolation and reductions in population size will be critical for an increasing number of species. We conducted controlled crosses and field germination experiments to quantify the effects of inbreeding and outbreeding in the declining orchid Gymnadenia conopsea in two Norwegian populations that differ in size. We further compared our results with published estimates of inbreeding depression in orchids. There was severe inbreeding depression for seed production (δ = 0.41–0.67) and germination (δ = 0.46–0.66) in both populations, with stronger inbreeding depression in the large population. Compared to outcrossing, selfing reduced female fitness (number of seeds per fruit × proportion of seeds germinating) by 76 and 54 % in the large and small population, respectively. The magnitude of inbreeding depression for seed production was higher than the average reported for orchids, while for germination it was similar to earlier estimates. The large population also experienced considerable outbreeding depression for seed production (δ = 0.23–0.27), germination (δ = 0.33) and female fitness (δ = 0.47) following crosses with a population 1.6 km away. The strong inbreeding depression indicates that both populations harbour a substantial genetic load, and suggests that fragmentation may reinforce population decline in G. conopsea via increased inbreeding. Moreover, the local outbreeding depression indicates substantial genetic differentiation at a moderate spatial scale. This has important implications for the use of crosses between populations or plant translocations as conservation approaches.

Morphological trait variation in populations of rhizomatous (Cypripedium calceolus, Listera ovata) and root-tuber orchid species (Platanthera bifolia, Gymnadenia conopsea) has been studied along a latitudinal gradient in Europe. It has been shown that the structure of shoots changes with transition from the center to the northern or southern periphery of the species range. The reduction in the number of metameres at the range periphery is more distinct in orchids with multimetameric shoots, compared to those with oligometameric shoots. The plasticity of shoot structure observed along the geographic gradient is contingent on climatic factors and can be manifested regionally in cases of long-term temperature deviations. Other morphological parameters, such as shoot height and the sizes of leaves and flowers, can serve as indicators of ecological conditions in habitats.

A new approach to reveal the dynamics of taxa on incompletely investigated territories was developed. The decline/expansion rate of orchids in the North Western part of European Russia (Leningrad, Novgorod and Pskov Regions, an area approx. 195 000 km 2 .) was estimated using this method. The method is based on comparison of numbers of grid cells where a certain species was recorded in various time intervals using specially designed software. More than 9000 records were used, however the territory remained insufficiently and unevenly studied both spatially and over time. The study revealed a statistically significant (p

Habitat fragmentation decreases plant population sizes and increases spatial isolation, which hampers the exchange of seeds and pollen between fragmented populations. This may result in decreased population viability. We compared the effects of population size and isolation on the reproductive success of two orchid species, Gymnadenia conopsea (nectar-producing) and Orchis mascula (nectarless) growing in highly fragmented calcareous grassland in southern Belgium. We expected that the nectar-producing species would be more susceptible to the negative reproductive consequences of habitat fragmentation compared to the nectarless species. Nectar production has been associated with increased geitonogamous pollination and, therefore, with lower seed viability. Our results show that seed viability increased with increasing population size in O. mascula, whereas it was always low in G. conopsea, even in large populations. In contrast, percentage fruit set was positively related to population size in the nectar-producing G. conopsea, but no such effect was observed in the nectarless O. mascula, where fruit set was low even in large populations. Population isolation was not related to reproductive success for either species. Our results suggest that even in large populations, where pollinators are expected to be more abundant, increased geitonogamous pollination reduces seed viability in the nectar-producing G. conopsea. In contrast, seed viability in O. mascula seems to benefit from increased pollinator availability in larger populations. For the latter species, however, fruit set remains low, even in large populations, compared to G. conopsea. This may be explained by the relatively low attractiveness of nectarless orchid species for pollinators. Our results indicate that small population size may negatively influence reproductive success in both nectarless and nectar-producing orchids by reducing seed viability and fruit set, respectively.

†Background and Aims One of the prerequisites for polyploid research in natural systems is knowledge of thegeographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in theGymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequencesof the observed spatial patterns investigated.†Methods DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determineploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cyto-type distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions.†Key Results Gymnadeniatissues undergo a progressively partial endoreplication, which accounts for about 60 %and 75 % of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are there-fore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x,8x)and three minority (6x,10x,12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to allfive different ploidy levels). The scarcity of the minority cytotypes (about 2.7 %) suggests the existence of strongpre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cyto-type variation, including clumping of individuals of the same ploidy and negative association between tetra- andoctoploids.†Conclusions The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception ofintrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studyingprocesses governing the formation and establishment of polyploids and assessing the evolutionary significance ofthe various pre- and postzygotic mating barriers that maintain this ploidy mixture.Key words: Coexistence, contact zone, cytotype mixture, flow cytometry, Gymnadenia conopsea, hybridization,mating barriers, polyploidy, progressively partial endoreplication, spatial distribution, sympatry.

Floral diversification and specialization are thought to be driven largely by interactions with pollinators, but the extent to which current selection on floral traits is mediated by pollinators has rarely been determined experimentally. We documented selection through female function on floral traits in two populations of the rewarding orchid Gymnadenia conopsea in two years and quantified pollinator-mediated selection (Δβpoll) by subtracting estimates of selection gradients for plants receiving supplemental hand pollination from estimates obtained for open-pollinated control plants. There was directional selection for taller plants, more flowers, larger corollas, and longer spurs in the study populations. Pollinator-mediated selection ranged from weak to moderately strong (Δβpoll, range −0.01–0.21, median 0.08). All observed selection on spur length could be attributed to interactions with pollinators, while the proportion of observed selection on plant height (0%–77%), number of flowers (13%–42%), and ...

Members of the orchid genus Nigritella are widespread in European mountains, but species circumscriptions and evolutionary patterns in the genus are subjects to conflicting opinions. We analyzed a representative material of Nigritella for differentiation at nuclear and plastid marker loci. In agreement with predictions from embryological studies, diploid members of Nigritella are sexual and mostly out-crossing, whereas triploid, tetraploid and pentaploid members are apomicts. The diploid taxa were poorly differentiated in the investigated molecular markers, except for the western N. gabasiana, which was separated in plastid haplotypes. Polyploid Nigritella aggregate into three groups and within each of these groups apomictic polyploids have given rise to new species. Within the N. nigra group, the tetraploid N. nigra subsp. austriaca is somewhat differentiated from the triploid subsp. nigra at nuclear as well as plastid loci. Fusion of an unreduced egg cell from subsp. nigra with a haploid microgamete from Gymnadenia conopsea gave rise to Gymnigritella runei. Within the N. widderi group, N. archiducis-joannis is poorly separated from N. widderi in molecular markers, and the pentaploid N. buschmanniae has evolved by fusion of an unreduced egg cell from N. widderi with a haploid microgamete from a diploid Nigritella. Within the N. miniata group, N. stiriaca is somewhat differentiated from N. miniata at nuclear loci, but no other segregates of N. miniata are supported at species level. Polyploid Nigritella species accumulate genetic diversity by somatic mutations. In the widespread N. nigra subsp. austriaca and N. miniata this diversity is correlated to geography. Although some polyploids may be of recent origins, each polyploid contain genetic markers no longer encountered in diploid members of the genus. According to plastid marker data, Nigritella and Gymnadenia may constitute monophyletic sister genera. (Less)

Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixed-ploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygotically isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora. We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths’ responses to the olfactory display of the plants, and by following the pollinator’s behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to physiologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of pre-mating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes’ coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype’s origin.

Glucosyloxybenzyl 2-isobutylmalates are one group of important active constituents in the tubers of Gymnadenia conopsea R. Br. and Coeloglossum viride (L.) Hartm. var. bracteatum (Willd.). For the purpose of quality evaluation of these two Chinese herbal medicines, it is necessary to use a rapid and reliable assay that is suitable for the determination of their active constituents. A high-performance liquid chromatography method is firstly developed for the simultaneous quantification of five glucosyloxybenzyl 2-isobutylmalates in the tubers. The analytes including dactylorhin B, dactylorhin E, loroglossin, dactylorhin A, and militarine are isolated from the tubers of G. conopsea. The compounds are separated on an Agilent Hydrosphere C(18) (150 x 4.6 mm i.d., 5 microm) column using a mobile phase of acetonitrile-water including 0.3% acetic acid (adjusted with 36% acetic acid) with gradient elution at a flow rate of 1.0 mL/min. Detection is set at a UV wavelength of 221.5 nm. The recovery of the method is 97.7-101.0%, and linearity (r0.9998) is obtained for all the analytes. The assay is successfully applied to determine the contents of the analytes in the tubers of G. conopsea and C. viride var. bracteatum collected from different regions of China.

The expected presence/absence of a target species outside the area of actual observations is commonly estimated using statistical models or decision criteria. This investigation demonstrates a similarity-based solution for predictive mapping as an alternative to generalizing models. The maps of the expected distribution of 12 orchids were created using find sites from field observations and absence sites generated onto the observation track. The expected presence/absence of a species in a location was calculated according to the similarity between the location and selected examples of presence and absence sites. A machine learning system selected the best predictive sets for each species out of 161 cartographic and remote sensing features. The usefulness of the predictive distribution maps was expressed as the ratio of the density of find sites per track in the predicted presence area relative to the density per track in the predicted absence area. The predictive mapping was more efficient for Dactylorhiza incarnata, D. russowii, Gymnadenia conopsea, and Goodyera repens. Soil properties and the proportion of find sites for the other species in the vicinity were the most indicative site characteristics. The rarer species were found to be better indicators of the occurrence of the other species than were the more common orchids. The proposed approach—to direct subsequent field observations to sites where the occurrence of the target species was predicted but has not yet been recorded—helped discover new populations of orchids and enhance the representativeness of absence sites.

Ten minor new glycosidic constituents (1- 10), together with 10 known compounds, have been isolated from a neuroprotective fraction of an ethanolic extract of the tubers of Gymnadenia conopsea. The structures of 1-10 were determined using spectroscopic and chemical methods. The compounds isolated were evaluated for activity in in vitro assays for acetylcholine esterase and monoamine oxidase inhibitory activities.

Previous research has shown that the presence of a reward on average doubles reproductive success in orchids by correspondingly increasing the frequency of pollinator visitations. We examined whether such reward-induced increases have a downstream effect on the behaviour of ambush predators concealed in orchid inflorescences, extending observations begun at Downe by Charles Darwin. Specifically, we studied three orchid-rich sites in southern England, in order to compare the occurrence of crab spiders on three coexisting species of terrestrial orchids: the nectariferous Gymnadenia conopsea versus the nectar-less Dactylorhiza fuchsii and Anacamptis pyramidalis. No significant difference was observed between rewarding and non-rewarding inflorescences at Risborough, whereas at Aston Clinton the nectar-less species supported significantly more crab spiders, albeit mainly in relatively short grass. Comparison of the two non-rewarding species present at Downe approximated a significant preference by the spiders for Anacamptis, which more closely resembles the rewarding Gymnadenia. The presence of a floral reward does not result in a higher frequency of crab spiders. We speculate that concealment quality of the inflorescence, the nature of the dominant pollinator(s), and/or ease of movement of spiders between inflorescences may be more important than frequency of visits by potential prey, particularly when no other form of concealment is readily available. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92, 763‐771. ADDITIONAL KEYWORDS: Anacamptis ‐ crab spider ‐ Dactylorhiza ‐ floral predator ‐ Gymnadenia ‐ nectar ‐ Orchidaceae ‐ tritrophic interaction.

Genetic variation was examined in five microsatellite loci to seek evidence of genetic differentiation and restricted gene flow that would support the taxonomic division of Gymnadenia into three species (G. borealis, G. conopsea, and G. densiflora). A total of 107 alleles was detected in 17 populations from England, Scotland, and Ireland. The mean expected heterozygosities ranged from 0.48 to 0.81. The differentiation in allele frequencies amongst populations that had been assigned to each taxon on the basis of morphology was sufficiently large to support the taxa as distinct species. Phylogenetic trees based on microsatellite allele frequencies, as well as assignment tests, supported the existence of three distinct groups with at least partial restriction of gene flow between them. There was substantial homozygote excess, leading to high FIS estimates, for most loci in most populations. This is unlikely to have been a result of widespread null alleles, and more probably reflects a high level of inbreeding in G. conopsea. This inference requires further investigation. The implications of the results of this and other taxonomic studies for the conservation of Gymnadenia in Britain are discussed.

Mutualists and antagonists are known to respond to similar floral cues, and may thus cause opposing selection on floral traits. However, we lack a quantitative understanding of their independent and interactive effects. In a population of the orchid Gymnadenia conopsea, we manipulated the intensity of pollination and herbivory in a factorial design to examine whether both interactions influence selection on flowering phenology, floral display, and morphology. Supplemental hand-pollination increased female fitness by 31% and one-quarter of all plants were damaged by herbivores. Both interactions contributed to selection. Pollinators mediated selection for later flowering and herbivores for earlier flowering, while both selected for longer spurs. The strength of selection was similar for both agents, and their effects were additive. As a consequence, there was no. net selection on phenology, whereas selection on spur length was strong. The experimental results demonstrate that both pollinators and herbivores can markedly influence the strength of selection on flowering phenology and floral morphology, and cause both conflicting and reinforcing selection. They also indicate that the direction of selection on phenology will vary with the relative intensity of the mutualistic and antagonistic interaction, potentially resulting in both temporal and among-population variation in optimal flowering time.

Floral traits have evolved to maximize reproductive success by attracting pollinators and facilitating pollination. Highly attractive floral traits may, however, also increase the degree of self-pollination, which could become detrimental for plant fitness through inbreeding depression. Floral nectar is a trait that is known to strongly mediate pollinator attraction and plant reproductive success, but the particular role of the nectar amino acid (AA) composition is poorly understood. Therefore, we experimentally manipulated the nectar AA composition and abundance of the Lepidoptera-pollinated orchid Gymnadenia conopsea through soil fertilization, and we quantified AA content and AA composition through high performance anion exchange chromatography with pulsed amperometric detection. Mixed models were then used to evaluate differences in pollinia removal, fruit set, seed set and degree of selfing between fertilized and control individuals. Selfing rates were estimated using microsatellite markers. We found that fertilized individuals had a significantly higher nectar AA content and an altered AA composition, whereas plant height, number of flowers, nectar volume and sugar concentration remained unchanged. Fertilized individuals also had significantly more pollinia removed and a higher fruit set, whereas control plants that did not receive the fertilization treatment had significantly fewer selfed seeds, and more viable seeds. Although we cannot exclude a role of changes in floral scent following the fertilization treatment, our results strongly suggest a relation among nectar AA composition, fruiting success and selfing rates. Our results also indicate potential consequences of nutrient pollution for plant reproductive success, through the induced changes in nectar AA composition.

We have recently found that the morphologically determined subspecies Gymnadenia conopsea ssp conopsea in Sweden includes early and late flowering individuals. We were interested in the interactions between the flowering time groups; if there were gene flow between them and if so this was detrimental or advantageous. A spatially mixed population of early and late flowering individuals was studied using three microsatellite loci. We measured patterns in genetic differentiation and inferred occurrence of hybridisation and introgression. Variation in flowering time, fertility and relative and absolute fruit set was measured. The pattern of introgression between flowering-time groups differed between loci. In two of the three investigated loci, allele separation was distinct between early and late flowering plants and one genetically obvious hybrid was infertile. In the third locus, several alleles were shared between the two flowering time variants. The degree of introgression was associated to fruit set failure, which was higher in the late flowering plants and lower in early flowering plants. A small group of early flowering individuals with somewhat delayed flowering compared to the main group was genetically distinct and had lower relative and absolute fruit set. This group was not genetically intermediate, but rather constituting an independent group, with lower fruit set possibly caused by absence of pollinators. There seem to be a strong barrier against introgression into the late flowering group which is kept genetically distinct and less diverse. The early flowering group is diverse, includes two subgroups and seems to benefit from gene flow.

Seven new glucosyloxybenzyl 2-isobutylmalates, gymnosides I-VII (1-7), were isolated from the tubers of Gymnadenia conopsea. The structures of 1-7 were determined on the basis of chemical and physicochemical evidence.

The methanol-eluted fraction of the methanolic extract from the tubers of Gymnadenia conopsea was found to show radical scavenging activities for DPPH and super oxide anion (.O2-) radicals. Three new glucosyloxybenzyl 2-isobutylmalates, gymnosides VIII, IX, and X, were isolated from this natural medicine together with 58 known constituents. The stereostructures of gymnosides were elucidated on the basis of chemical and physicochemical evidence. In addition, the phenanthrene and dihydrostilbene constituents showed radical scavenging activities and suggested the following structural requirements on radical scavenging activities; a) phenanthrenes: 1) dihydrogenation at the 9,10-positions enhances the activities, 2) the 1 or 3-p-hydroxybenzyl group enhances the activities; b) dihydrostilbenes: 1) methylation of the 3'-position reduces the activities, 2) the 2- and/or 6-p-hydroxybenzyl groups enhance the activities.

This investigation evaluates the usage of genetic markers, microsatellites, to distinguish and re-identify individual plants in a population of the orchid Gymnadenia conopsea. The study also illust ...

Spatial variation in plant-pollinator interactions may cause variation in pollinator-mediated selection on floral traits, but to establish this link conclusively experimental studies are needed. We quantified pollinator-mediated selection on flowering phenology and morphology in four populations of the fragrant orchid Gymnadenia conopsea, and compared selection mediated by diurnal and nocturnal pollinators in two of the populations. Variation in pollinator-mediated selection explained most of the among-population variation in the strength of directional and correlational selection. Pollinators mediated correlational selection on pairs of display traits, and on one display trait and spur length, a trait affecting pollination efficiency. Only nocturnal pollinators selected for longer spurs, and mediated stronger selection on the number of flowers compared with diurnal pollinators in one population. The two types of pollinators caused correlational selection on different pairs of traits and selected for different combinations of spur length and number of flowers. The results demonstrate that spatial variation in interactions with pollinators may result in differences in directional and correlational selection on floral traits in a plant with a semi-generalized pollination system, and suggest that differences in the relative importance of diurnal and nocturnal pollinators can cause variation in selection.

The crucial role of reproductive isolation in speciation has long been recognized; however, a limited number of studies quantify different isolation barriers and embed reproductive isolation in a phylogenetic context. In this study, we investigate reproductive isolation between the often sympatrically occurring orchid species, Gymnadenia conopsea and G. odoratissima. We examine the phylogenetic relationship between the two species and analyse floral isolation, fruit set and seed viability from interspecies crosses, as well as the ploidy level. Additionally, we quantify interspecies differences in floral signals and morphology. The results suggest that the two species have a sister-species relationship. In terms of reproductive isolation, we found complete floral isolation between the two species, but little to no post-pollination isolation; the species also mostly had the same ploidy level in the studied populations. We also show clear distinctions in floral signals, as well as in floral size and spur length. We propose that respective adaptation to short- vs. long-tongued pollinators was the driver of speciation in the here studied Gymnadenia species. Our study supports the key role of floral isolation in orchid speciation and shows that floral isolation is not restricted to highly specialized pollination systems, but can also occur between species with less specialized pollination.

Gymnadenia conopsea (L.) R. Br., or the fragrant orchid, is one of many plant species negatively influenced by new practices in agriculture and forestry during the last decades. This study describes the level of microsatellite variation within and among 10 Swedish populations of this species. It was not possible to detect strong effects of small population size or fragmentation. In general, the species had high genetic variation within and low genetic divergence among populations, although the correlation between population size and number of alleles was close to significance at the 95% level. Also, a significant isolation by distance effect was observed, indicating the presence of modest restrictions in gene dispersal between the investigated populations.

Differences in reproductive biology between two orchid species were examined: Eight Danish populations of the nectarless Dactylorhiza majalis ssp. majalis and six Swedish populations of the nectar-producing Gymnadenia conopsea ssp. densiflora. Population size varied from 24 to 1700 flowering individuals in D. majalis and from 100 to 1200 in G. conopsea. Dactylorhiza majalis had only half as many flowers per spike as G. conopsea. Fruit-set of D. majalis ranged from 16 to 39%, much lower than the fruit-set of G. conopsea (78–91%). Thus in the species with a few flowers and no nectar a lower fruit-set was observed than in the species with many flowers and presence of nectar. The low fruit-set of D. majalis was pollen-limited and fruit-set was positively correlated with pollinia removal and the latter was negatively correlated with population size. In both species, fruit-set and population size were uncorrelated.

Gymnadenia conopsea s.l. is a common orchid in central Europe, where early- and late-flowering populations can be distinguished. The early-flowering form is recognized as subspecies conopsea and the late-flowering form as subspecies densiflora. The two subspecies can occur in sympatry, but their flowering periods are separated. We investigated whether early- and late-flowering subspecies are genetically differentiated, whether they diverged once or repeatedly, and we tried to identify potential evolutionary forces involved in the divergence of the two subspecies. We used genetic markers to estimate genetic divergence within and among populations of early- and late-flowering G. conopsea, and to reconstruct their evolutionary history. In addition, we assessed morphological variation between subspecies. Allozyme variation indicated that subspecies conopsea was significantly more variable than ssp. densiflora and that gene flow among populations of ssp. conopsea was higher than among populations of ssp. densiflora. Gene flow between subspecies was low, indicating that the difference in flowering phenology represented an effective barrier to gene flow. A neighbor-joining tree based on allozyme frequencies indicated that early- and late- flowering populations did not diverge repeatedly in sympatry. Levels of cpDNA variation were generally low, even between G. conopsea s.l. and Gymnadenia odoratissima, chosen as an outgroup. Four cpDNA haplotypes were found, which differed only in the number of microsatellite repeats. Their distribution among subspecies of G. conopsea s.l. and G. odoratissima indicates that microsatellite haplotypes have evolved repeatedly, and their occurrence in different taxa thus represents a homoplasy. Floral characters were variable within and among populations and subspecies but did not consistently separate early- from late-flowering populations. A weak separation between subspecies was found in vegetative characters that presumably reflected habitat and competitive differences experienced by early- and late-flowering populations.

Spermosporina gymnadeniae (Hyphomycetes) on Gymnadenia sp. (Orchidaceae) collected in the Polish part of the Tatra Mts was described in 2003. The fungus was recently found at new localities in Poland and Slovakia. It was recorded on Gymnadenia conopsea s.l. and Dactylorhiza fuchsii, which are new hosts of this parasite. The fungus is described and additional illustrations are provided.