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Assisted gene flow can restore genetic diversity when genetic drift has driven deleterious alleles to high frequencies in small, isolated populations. Previous crosses among 20 populations of Gymnadenia conopsea documented the strongest heterosis and the weakest inbreeding depression in sparse and small populations, consistent with fixation of mildly deleterious alleles by genetic drift. We genotyped the populations used for crosses, and used 1200–1728 SNPs to test the following predictions: (1) heterosis increases with genetic differentiation (FST) to donor populations and decreases with genetic diversity in the recipient population, (2) inbreeding depression increases with genetic diversity, and (3) genetic diversity increases, and mean FST to other populations decreases, with population size and density. Pairwise FST ranged from very low to moderate (0.005–0.20) and genetic diversity varied moderately among populations (proportion of polymorphic loci = 0.52–0.75). However, neither FST between populations, nor genetic diversity in the recipient population, were related to the strength of heterosis. There was also no association between genetic diversity and the strength of inbreeding depression. Genetic diversity increased and mean FST decreased with population size, consistent with reduced diversity and increased differentiation of small populations by genetic drift. The results indicate that the loci conferring heterosis are not mirrored by overall population differentiation, and limited additional information on potential source populations for genetic rescue is gained by the genetic data. Instead, the use of controlled crosses can directly reveal positive effects of introducing new genetic material, and is a simple method with high potential in conservation. [ABSTRACT FROM AUTHOR]

A bioactivity-guided separation strategy was used to identify novel antistroke compounds from Gymnadenia conopsea (L.) R. Br., a medicinal plant. As a result, 4 undescribed compounds (1–2, 13, and 17) and 13 known compounds, including 1 new natural product (3), were isolated from G. conopsea. The structures of these compounds were elucidated through comprehensive spectroscopic techniques, such as 1D/2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and quantum chemical calculations. An oxygen–glucose deprivation/reoxygenation (OGD/R)-injured rat pheochromocytoma (PC12) cell model was used to evaluate the antistroke effects of the isolates. Compounds 1–2, 10–11, 13–15, and 17 provided varying degrees of protection against OGD/R injury in the PC12 cells at concentrations of 12.5, 25, and 50 µM. Among the tested compounds, compound 17 demonstrated the most potent neuroprotective effect, which was equivalent to that of the positive control drug (edaravone). Then, transcriptomic and bioinformatics analyses were conducted to reveal the regulatory effect of compound 17 on gene expression. In addition, quantitative real-time PCR (qPCR) was performed to verify the results of the transcriptomic and bioinformatics analyses. These results suggest that the in vitro antistroke effect of compound 17 may be associated with the regulation of the Col27a1 gene. Thus, compound 17 is a promising candidate for the development of novel antistroke drugs derived from natural products, and this topic should be further studied. [ABSTRACT FROM AUTHOR]

ABSTRACT Fungi and bacteria often occupy very similar niches; they interact closely with each other, and bacteria can provide direct or indirect benefits to plants that form mutualistic interactions with fungi. In orchids, successful seed germination largely depends on compatible mycorrhizal fungi, but whether and how bacteria contribute to seed germination and protocorm development remains largely unknown. Here, we performed field and laboratory experiments to assess the potential role of bacteria in mediating seed germination and protocorm development in the terrestrial orchid Gymnadenia conopsea. Our results suggested that bacterial and fungal communities differ between developmental stages in the germination process. The diversity of bacterial and fungal communities and their interaction network in germinating seeds (Stage 1) differed significantly from those in later developmental stages (Stages 2–5). Pseudomonas gradually became the dominant bacterial group as the protocorms matured and showed a positive association with Ceratobasidiaceae fungi. Seed germination tests in vitro demonstrated that co-inoculation of Ceratobasidium sp. GS2 with Pseudomonas isolates significantly improved protocorm growth and development, suggesting that the observed increase in Pseudomonas abundance during protocorm development directly or indirectly improves the growth of germinating seeds. Overall, our findings indicate that bacteria may exert non-negligible effects on seed germination of orchids and, therefore, offer valuable perspectives for future strategies for conservation and cultivating orchid species.IMPORTANCEIt is well known that orchid seeds depend on mycorrhizal fungi to supply the necessary nutrients that support germination in natural environments. Apart from fungi, bacteria may also be involved in the germination process of orchid seeds, but so far, their role has not been intensively studied. This research provides evidence that bacterial community composition changes during seed germination of the terrestrial orchid Gymnadenia conopsea. Interestingly, in vitro experiments showed that Pseudomonas spp., which were the most dominant bacteria in the later germination stages, improved protocorm growth. These results suggest that bacteria contribute to the germination of orchid seeds, which may open new perspectives to apply bacteria as a biofertilizer in the introduction and restoration of G. conopsea populations.

[Objective] The present paper aimed to explore the effects of environmental factors on bacterial community structure in rhizosphere soil of wild Gymnadenia conopsea in Tibet. (Method) Therefore, four sampling sites were selected to collect soil samples, including the abandoned timber factory (FM), Linzhi viewing station (LZ), Linhai viewing station (LH), and the radar station (LD), and the high-throughput sequencing technology was used to analyze the diversity and structure of bacterial community in rhizosphere soil of G. conopsea at different altitudes, and the effects of different environmental factors on the diversity and structure of bacterial community were analyzed. [Result] The total of 32 phyla, 90 classes, 224 orders, 378 families, and 693 genera were detected in all samples. Proteobacteria, Actinobacteria and Acidobacteria were the dominant phyla, Bradyrhizobium and Chryseobacterium were the dominant genera. With the increase of altitude, the diversity and richness of rhizosphere soil bacterial community increased, and the difference between groups was greater than the difference within groups. [Conclusion] Altitude, TK, pH and EC significantly affected the bacterial community composition in rhizosphere soil at phylum level. Three environmental factors, namely TK, TP and altitude, had more obvious effects on bacterial community composition at genus level. Key words: Gymnadenia conopsea; Environmental factor; Rhizosphere soil bacteria; Community diversity; Community structure [ABSTRACT FROM AUTHOR]

ABSTRACTAlmost all orchids rely on mycorrhizal fungus to support their seed germination. To date, the effect of active components in mycorrhizal fungus on orchid seed germination largely remains unknown. In this study, we aimed to investigate the impact of active components found in mycorrhizal fungus on orchid seed germination. Specifically, we focused on a terrestrial orchid Gymnadenia conopsea and its host-specific seed germination supporting fungus Ceratobasidium GS2. In total, several steroids (1–7) were isolated from this fungus. Notably, compounds 1, 2, 4, and 5 exhibited significant enhancements in protocorm volume. Moreover, compounds 1–6 demonstrated strong promotion of protocorm differentiation. These findings suggest that steroids may play a crucial role in the symbiotic germination of G. conopsea seeds. Future studies should continue to explore the specific mechanisms through which these steroids exert their effects, contributing to our understanding of orchid biology and mycorrhizal interaction.

[Objective] The study aimed to analyze the diversity of fungi community in the rhizosphere soil of Gymnadenia conopsea and its correlation with soil environmental factors, so as to provide reference for exploring the work of resource protection and sustainable utilization such as artificial cultivation and ecological planting of G. conopsea. [Method] High-flux sequencing and comparative analysis were carried out on the diversity of rhizosphere soil fungal communities of G. conopsea in Aruhorqin Banner, Dongwuzhumuqin Banner and Ongniud Banner of Inner Mongolia by using Illumina Miseq high-throughput sequencing technology, and the physical and chemical properties and correlation of plant rhizosphere soil were analyzed. [Result] The rhizosphere soil of G. conopsea was acidic, rich in organic matter and available potassium, but low in nitrogen and phosphorus, and the soil physical and chemical properties in different distribution areas were significantly different. A total of 5125 OTUs were obtained from soil samples from three distribution areas, belonging to 11 phyla, 31 classes, 65 orders, 161 families and 158 genera. At the phylum level, the dominant phyla of fungi in the rhizosphere soil of the three distribution areas were Ascomycota and Basidiomycota. At the genus level, Trichoglossum, Hygrocybe and Mortierella were the dominant groups of G. conopsea rhizosphere soil fungi. Alpha diversity index showed that the general trend of diversity and richness of rhizosphere soil fungal community in G. conopsea in the three distribution areas was Al > D3 > W2; Beta diversity index showed that there were some differences in the composition of true communities in rhizosphere soil of G. conopsea in the three distribution areas. The contents of total potassium, total phosphorus, total nitrogen and water in the soil may be the key factors affecting the composition of the fungal community in the rhizosphere soil of G. conopsea distributed in Inner Mongolia. Among them, Hygrocybe and Clavulinosis had significant positive correlation with the key factors of soil environment; However, there was a significantly negative correlation between the key factors of soil environment and the pathogenic species of Hyphomycea and Fusarium. [Conclusion] Through the results of the study, we have a preliminary understanding of the growth soil conditions and characteristics of the rhizosphere soil fungal community of G. conopsea distributed in Inner Mongolia. Among the three different distribution areas, the diversity and richness of the rhizosphere soil fungal community of G. conopsea in Aruhorqin Banner are the highest. Soil physical and chemical properties have a great impact on the diversity of fungi in the rhizosphere soil of G. conopsea, and other influencing factors need to be further studied. [ABSTRACT FROM AUTHOR]

Almost all orchids rely on mycorrhizal fungus to support their seed germination. To date, the effect of active components in mycorrhizal fungus on orchid seed germination largely remains unknown. In this study, we aimed to investigate the impact of active components found in mycorrhizal fungus on orchid seed germination. Specifically, we focused on a terrestrial orchid Gymnadenia conopsea and its host-specific seed germination supporting fungus Ceratobasidium GS2. In total, several steroids (1–7) were isolated from this fungus. Notably, compounds 1, 2, 4, and 5 exhibited significant enhancements in protocorm volume. Moreover, compounds 1–6 demonstrated strong promotion of protocorm differentiation. These findings suggest that steroids may play a crucial role in the symbiotic germination of G. conopsea seeds. Future studies should continue to explore the specific mechanisms through which these steroids exert their effects, contributing to our understanding of orchid biology and mycorrhizal interaction. [ABSTRACT FROM AUTHOR]

Searching for PD-1/PD-L1 inhibitor from medicinal plants has become a potential method to discover small molecular cancer immunotherapy drugs. Using PD-1/PD-L1 inhibitory activity assay in vitro, a bioactive fraction was obtained from the ethanol extract of Gymnadenia conopsea. A sensitive UPLC-HRMS/MS method was established for the rapid screening and identification of compositions from bioactive fraction. Based on the characteristic fragmentation patterns of standards analysis and extracted ion chromatogram (EIC) method, 46 compounds were rapidly screened and identified (including 35 succinic acid ester glycosides and 11 other compounds), among which 17 compounds were tentatively identified as new compounds. [ABSTRACT FROM AUTHOR]

Effective population size should be positively related to census size and density, and it is expected to influence the strength of genetic drift, inbreeding and response to selection, and thus the distribution of the genetic load across populations.We examined whether census population size and density predict the strength of inbreeding depression, heterosis and population mean fitness at the seed stage in the terrestrial orchid Gymnadenia conopsea by conducting controlled crosses (self, outcross within and between populations) in 20 populations of varying size (7–30,000 individuals) and density (1–12.8 individuals/m2). In the largest population, we also examined how local density affects the occurrence of self‐pollination with a pollen staining experiment.The majority of populations expressed strong inbreeding depression at the seed stage (mean δID: min–max = 0.26: −0.53 to 0.51), consistent with a mainly outcrossing mating system and substantial genetic load. The effect of between‐population crosses varied from strong outbreeding depression to heterosis (mean δOD: min–max = 0.05: −0.22 to 0.92), indicating varying influence of drift and selection among populations.Census population size did not significantly predict the strength of inbreeding depression, heterosis or population mean fitness. However, inbreeding depression was positively and heterosis negatively correlated with population density. The proportion of self‐massulae deposition was three times higher in sparse patches compared to dense ones (41% vs. 14%).Combined effects of density‐dependent pollinator behaviour and limited seed dispersal may cause stronger genetic sub‐structuring in sparse populations and reduce the strength of the correlation between census and effective population size. The results point to the importance of considering population density in addition to size when evaluating the distribution of recessive deleterious alleles across populations.Synthesis and applications. Management plans for threatened species often involve crosses between populations to restore genetic variation, a process termed genetic rescue. This study indicates that such conservation efforts should be more successful if designed on the basis of population density in addition to population size, because we found population density predicted both the strength of heterosis and inbreeding depression across populations of Gymnadenia conopsea. [ABSTRACT FROM AUTHOR]

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There are 32 species from the family Orchidaceae in Latvia, and 26 are rare and endangered, and their preservation in an in vitro bank is vital. The death of in vitro grown wild terrestrial orchids is mainly caused by the release of phenolic compounds from root tissues. As mineral nutrients form a significant component of culture media, a hypothesis was advanced that increased doses of both Ca and Fe could prevent phenol oxidation and improve micropropagation. Liparis loeselii (L.) Rich. and Gymnadenia conopsea (L.) R. Br. calcicole plants, and Dactylorhiza russowii (Klinge) Holub, a non-calcicole plant, were used as model species. Modification of the culture media with increased Ca gluconate monohydrate concentrations significantly improved the quality of L. loeselii and D. russowii plants, especially in the presence of elevated levels of ferric citrate. There was no benefit of Ca for G. conopsea, except in combination with the highest level of ferric citrate tested. The results revealed species-specific stimulatory or inhibitory impacts of changes in the pH of culture media on orchid plantlet quality. These findings demonstrate the crucial role of iron to prevent necrosis. The results indicated that for species with relatively higher adaptation potential to growth in habitats with different pH levels (L. loeselii and D. russowii), the shoot quality in vitro was better if the Ca to Fe ratio in the culture medium was 2:1. Despite the fact that G. conopsea is a calcicole species, the optimal Ca to Fe ratio in the culture medium was 1:1. [ABSTRACT FROM AUTHOR]

Using ISSR-PCR approach, the genetic structure of G. conopsea populations have been studied in the Komi Republic (Russia) at the northern boundary of its range, where the populations have a high level of phenotypic variation. It was established that the reaction of G. conopsea specimens to the lack of heat was expressed as a decrease in the most plastic phenotypic traits (shoot height, inflorescence length, number of flowers, leaf size) and determined a clear morphological differentiation in karst landscapes of the Timan Ridge with different temperature conditions. Two ISSR primers made it possible to reveal 839 loci, 37.79% of which were polymorphic. UPGMA analysis divided the pool of G. conopsea populations into two clusters: populations from the limestones of Timan (180 plants) and population from the Mezen-Vychegda Plain (20 plants). The assessment of genetic variability revealed the decreased parameters in the G. conopsea population from the Mezen-Vychegda Plain (P = 26.94%, He = 0.050, Is = 0.084). At limestones of South Timan, the species had both highly variable populations (P = 55.5–60.6%, He = 0.084–0.089) and populations with decreased parameters of genetic variability (P = 29.3–29.6%, He = 0.053–0.059). Despite the high diversity of karst landscapes and territorial fragmentation of the populations, we found a low level of interpopulation differentiation (F = 0.017–0.036) and strong genetic flows between the populations (D = 0.007–0.020, I = 0.980–0.993). A Mantel test did not show a correlation between the genetic and geographic distances among populations (r = –0.047; p = 0.04). A majority of the revealed genetic variability was realized inside the populations. The share of interpopulation variability was only 14%. An analysis of genetic relationships based on the Structure v2.3 software program allowed us to divide the samples according to two geographical areas and to suggest that in South Timan there are at least four separate groups differing in ISSR markers. No clear dependence between the type of karst landscape and genetic variability of the populations was detected. [ABSTRACT FROM AUTHOR]

Gymnadenia conopsea R. Br. is a traditional Tibetan medicinal plant that grows at altitudes above 3000 m, which is used to treat neurasthenia, asthma, coughs, and chronic hepatitis. However, a comprehensive configuration of the chemical profile of this plant has not been reported because of the complexity of its chemical constituents. In this study, a rapid and precise method based on ultra-high performance liquid chromatography (UPLC) combined with an Orbitrap mass spectrometer (UPLC−Orbitrap−MS/MS) was established in both positive- and negative-ion modes to rapidly identify various chemical components in the tubers of G. conopsea for the first time. Finally, a total of 91 compounds, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others, were identified in the tubers of G. conopsea based on the accurate mass within 3 ppm error. Furthermore, many alkaloids, phenolic acid derivates, and terpenes were reported from G. conopsea for the first time. This rapid method provides an important scientific basis for further study on the cultivation, clinical application, and functional food of G. conopsea.

The anatomy and the ultrastructure of spur nectaries of Cymnadenia conopsea at different developmental stages were investigated. The secretory epidermis surrounded the inside of spur and formed many unicellular papillae, which significantly enlarged the secretory surface. At the activity stage the epidermal cells contained characteristic plastids with well developed intraplastidal membrane system and numerous osmiophillic globules. The contact of plastids and endoplasmic reticulum indicates a possibility of the involvement of these structures in the secretory processes. The cell wall and the cuticle did not form a barrier for the secreted nectar and no pores or cracks were visible in the cuticle covering secretory papillae.

The link between biotic interaction intensity and strength of selection is of fundamental interest for understanding biotically driven diversification and predicting the consequences of environmental change. The strength of selection resulting from biotic interactions is determined by the strength of the interaction and by the covariance between fitness and the trait under selection. When the relationship between trait and absolute fitness is constant, selection strength should be a direct function of mean population interaction intensity. To test this prediction, we excluded pollinators for intervals of different length to induce five levels of pollination intensity within a single plant population. Pollen limitation ( PL) increased from 0 to 0.77 across treatments, accompanied by a fivefold increase in the opportunity for selection. Trait-fitness covariance declined with PL for number of flowers, but varied little for other traits. Pollinator-mediated selection on plant height, corolla size, and spur length increased by 91%, 34%, and 330%, respectively, in the most severely pollen-limited treatment compared to open-pollinated plants. The results indicate that realized biotic selection can be predicted from mean population interaction intensity when variation in trait-fitness covariance is limited, and that declines in pollination intensity will strongly increase selection on traits involved in the interaction. [ABSTRACT FROM AUTHOR]

Costs of reproduction are expected to vary with environmental conditions thus influencing selection on life-history traits. Yet, the effects of habitat conditions and climate on trade-offs among fitness components remain poorly understood. For 2-5 years, we quantified costs of experimentally increased reproduction in two populations (coastal long-season vs. inland short-season) of two long-lived orchids that differ in natural reproductive effort ( RE; 30 vs. 75% fruit set). In both species, survival costs were found only at the short-season site, whereas growth and fecundity costs were evident at both sites, and both survival and fecundity costs declined with increasing growing season length and/or summer temperature. The results suggest that the expression of costs of reproduction depend on the local climate, and that climate warming could result in selection favouring increased RE in both study species. [ABSTRACT FROM AUTHOR]

Background and Aims Polyploidization, the doubling of chromosome sets, is common in angiosperms and has a range of evolutionary consequences. Newly formed polyploid lineages are reproductively isolated from their diploid progenitors due to triploid sterility, but also prone to extinction because compatible mating partners are rare. Models have suggested that assortative mating and increased reproductive fitness play a key role in the successful establishment and persistence of polyploids. However, little is known about these factors in natural mixed-ploidy populations. This study investigated floral traits that can affect pollinator attraction and efficiency, as well as reproductive success in diploid and tetraploid Gymnadenia conopsea (Orchidaceae) plants in two natural, mixed-ploidy populations.Methods Ploidy levels were determined using flow cytometry, and flowering phenology and herbivory were also assessed. Reproductive success was determined by counting fruits and viable seeds of marked plants. Pollinator-mediated floral isolation was measured using experimental arrays, with pollen flow tracked by means of staining pollinia with histological dye.Key Results Tetraploids had larger floral displays and different floral scent bouquets than diploids, but cytotypes differed only slightly in floral colour. Significant floral isolation was found between the two cytotypes. Flowering phenology of the two cytotypes greatly overlapped, and herbivory did not differ between cytotypes or was lower in tetraploids. In addition, tetraploids had higher reproductive success compared with diploids.Conclusions The results suggest that floral isolation and increased reproductive success of polyploids may help to explain their successful persistence in mixed-ploidy populations. These factors might even initiate transformation of populations from pure diploid to pure tetraploid. [ABSTRACT FROM AUTHOR]

Water soluble polysaccharides from seven batches of Gymnadenia conopsea were firstly investigated and compared using high performance size exclusion chromatography coupled with multi-angle laser light scattering/refractive index detector (HPSEC-MALLS/RID) and saccharide mapping based on polysaccharide analysis by carbohydrate gel electrophoresis (PACE), respectively. The results showed that the weight-average molecular weight ( M w ) and the radius of gyration (< S 2 > z 1/2 ) of polysaccharides were ranging from 4.46 × 10 5 to 7.41 × 10 5 Da and 73.3–94.2 nm, respectively. By applying the polymer solution theory, the exponent ( ν ) values of < S 2 > z 1/2 = k M w ν were ranging from 0.36 to 0.42, which indicated that polysaccharides from G. conopsea existed as globular in the aqueous solution. Furthermore, the results showed that α-1,4- and β-1,3(4)-glucosidic, α-1,5-arabinosidic, β-1,4-mannosidic and α-1,4-D-galactosiduronic linkages existed in polysaccharides from G. conopsea . The similarity of the hydrolysates of polysaccharides in G. conopsea collected from different regions was high. Moreover, the nitric oxide released from RAW 264.7 cells induced by polysaccharides were significantly affected by their α-1,5-arabinosidic and β-1,3(4)-glucosidic, especially α-1,4-D-galactosiduronic and β-1,4-mannosidic linkages. These results are beneficial for better understanding of the structures–bioactivity relationship of polysaccharides from G. conopsea , and helpful to improve their pharmacological activity-based quality control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spatial variation in pollinator-mediated selection (Δ βpoll) is a major driver of floral diversification, but we lack a quantitative understanding of its link to pollen limitation (PL) and net selection on floral traits. For 2-5 years, we quantified Δ βpoll on floral traits in two populations each of two orchid species differing in PL. In both species, spatiotemporal variation in Δ βpoll explained much of the variation in net selection. Selection was consistently stronger and the proportion that was pollinator-mediated was higher in the severely pollen-limited deceptive species than in the rewarding species. Within species, variation in PL could not explain variation in Δ βpoll for any trait, indicating that factors influencing the functional relationship between trait variation and pollination success govern a major part of the observed variation in Δ βpoll. Separating the effects of variation in mean interaction intensity and in the functional significance of traits will be necessary to understand spatiotemporal variation in selection exerted by the biotic environment. [ABSTRACT FROM AUTHOR]

Understanding landscape scale variation in reproductive and pollination success is a major aim of plant population biology. A potential determinant of reproductive success that has received surprisingly little attention so far is variation in nectar chemical composition across plant individuals and populations. Using the Lepidoptera pollinated fragrant orchid ( Gymnadenia conopsea) as a model species, we aimed at providing a first account of landscape scale variation in nectar amino acid and sugar composition, its environmental drivers, and how it may affect plant fruit set., Nectar was sampled from 986 flowers from 296 individuals across eleven discrete populations of the G. conopsea. The proportions of sugars and amino acids were determined using high-performance anion-exchange chromatography. Nectar composition was related to soil characteristics of the populations, and to fruit set, using linear mixed models., Approximately, 20% of the variance in nectar traits was situated between populations, whereas the highest proportion of variance ( c. 45%) was found among flowers within individuals. Soil carbon and nitrogen content affected both nectar concentration and composition. Furthermore, fruit set of G. conopsea individuals was found to be significantly related to nectar amino acid and sugar composition., Synthesis. These results show that landscape scale variation in nectar amino acid and sugar composition should be taken into account in future studies of plant reproductive success. Furthermore, there is also high within-plant variation in both nectar amino acid and sugar composition. This variation may reduce geitonogamous pollination, but it may also limit rapid pollinator-mediated selection on nectar composition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ontogenetic structure of G. conopsea cenopopulations have a fluctuating pattern. Their response to adverse weather conditions is manifested in short-term reduction 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 factors on population size. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Floral spurs are regarded as features affecting pollinator behaviour. Anacamptis pyramidalis is regarded as a deceitful, non-rewarding orchid species. In the form fumeauxiana, additional spurs occur on the lateral sepals. In this study we analyse micromorphological and ultrastructural floral features and suggest the mechanism of deception in A. pyramidalis and A. pyramidalis f. fumeauxiana. In f. fumeauxiana, the adaxial surface of the lip, the lip calli, the tips of the lateral sepals, the abaxial and adaxial epidermises of the lip spur, and the lateral sepal spur have a secretory function. Numerous stomata were observed on the abaxial surfaces of spurs and sepals. The characteristic features of the ultrastructure of osmophore cells were noted: dense cytoplasm with numerous profiles of ER, mitochondria, plastids with plastoglobuli and tubular structures, a large nucleus, lipid droplets, and vesicles fusing with the plasmalemma. The similarity of the floral morphology and anatomy, the flowering period, and pollinators of A. pyramidalis, A. pyramidalis f. fumeauxiana and Gymnadenia conopsea suggest a possible food-deceptive mechanism-imitation of nectar presence produced in the spurs of Gymnadenia. [ABSTRACT FROM AUTHOR]

The fragrant orchid Gymnadenia conopsea s.l. is a controversial taxon with two commonly distinguished species, G. conopsea s.str. and G. densiflora. Despite morphological similarity, differentiation between the taxa has been reported for several characters; however, character variation within taxa has obviated a clear consensus. We assessed ITS sequences, microsatellite variation and chromosome numbers on the European scale (1,420 samples) and conducted morphological analyses for 626 samples from Germany. ITS analysis revealed a 2% nucleotide divergence between the taxa, similar to the divergence between other Gymnadenia species. The ITS sequences of G. densiflora form a well-supported monophyletic group sharing a most recent common ancestor with G. nigra and G. austriaca. Thus, G. conopsea and G. densiflora are not sister species, and a species rank is supported for G. densiflora (Wahlenb.) Dietrich and G. conopsea (L.) R.B r. s.str. This was confirmed by the microsatellite analysis, which revealed a strong genetic differentiation between the taxa because of largely non-overlapping sets of alleles. Chromosome numbers showed that G. conopsea was either diploid or tetraploid, whereas G. densiflora was diploid throughout. Morphologically, the taxa differed significantly in the mean value of a number of diagnostic characters. However, a discriminant analysis showed that the morphological variability is substantial, and on the individual level an unequivocal assignment is not possible as 96% of G. conopsea, but only 77% of G. densiflora could be assigned correctly. Further studies are needed on character variation within and among species and ploidy levels to allow for a better identification of the genetically differentiated but morphologically similar taxa. [ABSTRACT FROM AUTHOR]

Background and Aims One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in the Gymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequences of the observed spatial patterns investigated. Methods DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determine ploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cytotype distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions. Key Results Gymnadenia tissues 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 therefore 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 all five different ploidy levels). The scarcity of the minority cytotypes (about 2·7 %) suggests the existence of strong pre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cytotype variation, including clumping of individuals of the same ploidy and negative association between tetra- and octoploids. Conclusions The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception of intrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studying processes governing the formation and establishment of polyploids and assessing the evolutionary significance of the various pre- and postzygotic mating barriers that maintain this ploidy mixture. [ABSTRACT FROM AUTHOR]

Four new minor constituents including two cyclodipeptides (1 and 2) and two cyclopentene derivatives (3 and 4), together with four known cyclodipeptides, have been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Their structures including absolute configurations were determined by spectroscopic data interpretation combined with chemical methods. Among them, compound 1 contains an abnormal S-(4''-hydroxybenzyl)cysteine residue, 3 and 4 possess [(4-methylcyclopentyl)methyl]benzene and (4-hydroxymethylcyclopentyl)benzene carbon skeletons, respectively, both of which are first found from the natural source. [ABSTRACT FROM AUTHOR]

Abstract: The fungal community associated with the terrestrial photosynthetic orchid Gymnadenia conopsea was characterized through PCR-amplification directly from root extracted DNA and cloning of the PCR products. Six populations in two geographically distinct regions in Germany were investigated. New ITS-primers amplifying a wide taxonomic range including Basidiomycetes and Ascomycetes revealed a high taxonomic and ecological diversity of fungal associates, including typical orchid mycorrhizas of the Tulasnellaceae and Ceratobasidiaceae as well as several ectomycorrhizal taxa of the Pezizales. The wide spectrum of potential mycorrhizal partners may contribute to this orchid''s ability to colonize different habitat types with their characteristic microbial communities. The fungal community of G. conopsea showed a clear spatial structure. With 43 % shared taxa the species composition of the two regions showed only little overlap. Regardless of regions, populations were highly variable concerning taxon richness, varying between 5 and 14 taxa per population. The spatial structure and the continuous presence of mycorrhizal taxa on the one hand and the low specificity towards certain fungal taxa on the other hand suggest that the fungal community associated with G. conopsea is determined by multiple factors. In this context, germination as well as pronounced morphological and genetic differentiation within G. conopsea deserve attention as potential factors affecting the composition of the fungal community. [Copyright &y& Elsevier]

Abstract: A high-performance liquid chromatography–diode array detection–tandem mass spectrometry (HPLC–DAD–MS n ) method has been firstly developed for chemical fingerprint analysis of rhizomes of Gymnadenia conopsea R. Br. and rapid identification of major compounds in the fingerprints. Comparing the UV and MS spectra with those of reference compounds, seven main peaks in the fingerprints were identified as adenosine (1), 4-hydroxybenzyl alcohol (2), 4-hydroxybenzyl aldehyde (3), dactylorhin B (4), loroglossin (5), dactylorhin A (6) and militarine (7). Compounds 4–7 were succinate derivative esters and firstly discovered from this species. The Computer Aided Similarity Evaluation System (CASES) for chromatographic fingerprint of traditional Chinese medicine was employed to evaluate the similarities of 10 samples of the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provinces, Tibet autonomous region of China, and Nepal. These samples from different sources had similar chemical fingerprints. This method is specific and may serve for quality identification and comprehensive evaluation of this traditional Tibetan remedy. [Copyright &y& Elsevier]