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Aim Interactions with mycorrhizal fungi are increasingly recognized as an important factor underlying the distribution and abundance of orchid species. However, the geographical distribution of orchid mycorrhizal fungi (OMF) and how their communities vary over large geographical areas are less well understood. Because climatic and environmental similarity may decrease with geographical distance or because some OMF have limited dispersal capabilities, similarities in orchid mycorrhizal communities can be expected to decrease with increasing distances separating orchid populations. However, up till now empirical evidence is largely lacking. Location Eurasia. Taxa Gymnadenia conopsea (L.) R. Brown and Epipactis helleborine (L.) Crantz. Methods High‐throughput sequencing was used to perform a cross‐continental comparison of OMF that associate with two widespread Eurasian terrestrial orchids, Epipactis helleborine and Gymnadenia conopsea. Both phylogenetic and nonphylogenetic measures of community dissimilarity and their components were calculated and related to geographical distances using Mantel tests. Results Our results showed that in both orchid species similarity in mycorrhizal communities decreased significantly with geographical distance. Decomposing the contribution of spatial turnover and nestedness to overall dissimilarity showed that the observed dissimilarity was mainly the result of species replacement between regions, and not of species loss. Similarly, a strong relationship was observed between phylogenetic community dissimilarity and geographical distance. Decomposing PCD values into a nonphylogenetic and phylogenetic component showed that orchid populations located closely next to each other were likely to contain the same operational taxonomic units (OTUs), but that the non‐shared taxa came from different phylogenetic clades. Species indicator analyses showed that the majority of OMF OTUs were restricted to particular geographical areas. However, some OTUs occurred in both continents, indicating that some fungi have very wide distributions. Main conclusions Overall, these results demonstrate that orchid mycorrhizal communities differ substantially across large geographical areas, but that the distribution of orchids is not necessarily restricted by the distribution of particular OMF. Hence, widespread orchid species can be considered mycorrhizal generalists that are flexible in the OMF with which they associate across large geographical areas.

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.

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. 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. 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. 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.

Polyploidy is widely recognized as a major mechanism of sympatric speciation in plants, yet little is known about its effects on interactions with other organisms. Mycorrhizal fungi are among the most common plant symbionts and play an important role in plant nutrient supply. It remains to be understood whether mycorrhizal associations of ploidy—variable plants can be ploidy-specific. We examined mycorrhizal associations in three cytotypes (2x, 3x, 4x) of the Gymnadenia conopsea group (Orchidaceae), involving G. conopsea s.s. and G. densiflora, at different spatial scales and during different ontogenetic stages. We analysed: adults from mixed- and single-ploidy populations at a regional scale; closely spaced adults within a mixed—ploidy site; and mycorrhizal seedlings. All Gymnadenia cytotypes associated mainly with saprotrophic Tulasnellaceae (Basidiomycota). Nonetheless, both adults and seedlings of diploids and their autotetraploid derivatives significantly differed in the identity of their mycorrhizal symbionts. Interploidy segregation of mycorrhizal symbionts was most pronounced within a site with closely spaced adults. This study provides the first evidence that polyploidization of a plant species can be associated with a shift in mycorrhizal symbionts. This divergence may contribute to niche partitioning and facilitate establishment and co-existence of different cytotypes.

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.

Geographically structured phenotypic selection can lead to adaptive divergence. However, in flowering plants, such divergent selection has rarely been shown, and selection on floral signals is generally little understood. In this study, we measured phenotypic selection on display size, floral color, and floral scent in four lowland and four mountain populations of the nectar-rewarding terrestrial orchid Gymnadenia odoratissima in two years. We also quantified population differences in these traits and pollinator community composition. Our results show positive selection on display size and positive, negative, or absence of selection on different scent compounds and floral color. Selection on the main scent compounds was consistently stronger in the lowlands than in the mountains, and lowland plants emitted higher amounts of most of these compounds. Pollinator community composition also differed between regions, suggesting different pollinators select for differences in floral volatiles. Overall, our study is the first to document consistent regional differences in selection on floral scent, suggesting this pattern of selection is one of the evolutionary forces contributing to regional divergence in floral chemical signaling.

We investigated scent composition and pollinator attraction in two closely related orchids, Gymnadenia conopsea (L.) R.Br. s.l. and Gymnadenia odoratissima (L.) Rich. in four populations during the day and night. We collected pollinators of both species using hand nets and sampled floral odour by headspace sorption. We analysed the samples by gas chromatography with mass spectrometry to identify compounds and with electroantennographic detection to identify compounds with physiological activity in pollinators. In order to evaluate the attractiveness of the physiologically active compounds, we carried out trapping experiments in the field with single active odour substances and mixtures thereof. By collecting insects from flowers, we caught eight pollinators of G. conopsea, which were members of four Lepidoptera families, and 37 pollinators of G. odoratissima, from five Lepidopteran families. There was no overlap in pollinator species caught from the two orchids using nets. In the scent analyses, we identified 45 volatiles in G. conopsea of which three (benzyl acetate, eugenol, benzyl benzoate) were physiologically active. In G. odoratissima, 44 volatiles were identified, of which seven were physiologically active (benzaldehyde, phenylacetaldehyde, benzyl acetate, 1-phenyl-2,3-butandione, phenylethyl acetate, eugenol, and one unknown compound). In field bioassays using a mixture of the active G. odoratissima compounds and phenylacetaldehyde alone we caught a total of 25 moths, some of which carried Gymnadenia pollinia. A blend of the active G. conopsea volatiles placed in the G. odoratissima population did not attract any pollinators. The two orchids emitted different odour bouquets during the day and night, but G. odoratissima showed greater temporal differences in odour composition, with phenylacetaldehyde showing a significant increase during the night. The species differed considerably in floral odour emission and this differentiation was stronger in the active than non-active compounds. This differentiation of the two species, especially in the emission of active compounds, appears to have evolved under selection for attraction of different suites of Lepidopteran pollinators.

The unique geographical environment at high altitudes may cause a series of diseases, such as acute altitude reaction, cerebral edema, and pulmonary edema. (L.) R. Br. has been reported to have an effect on high-altitude hypoxia. However, the molecular mechanism, especially the expression of long noncoding RNAs (lncRNAs), is not yet clear. The expression profiles of lncRNAs in high-altitude hypoxia-induced brain injury mice treated with (L.) R. Br. by using a microarray method. A total of 226 differentially expressed lncRNAs, 126 significantly dysregulated mRNAs and 23 differentially expressed circRNAs were detected (>2.0-fold, p<0.05). The expression of selected lncRNAs, mRNAs and circRNAs was validated by qRT-PCR. KEGG analysis showed that the mRNAs coexpressed with lncRNAs were involved in inflammation and hypoxia pathways, including the HIF-1, PI3K-Akt, and NF-kappa B signaling pathways. The lncRNA-TF network analysis results indicated that the lncRNAs were regulated mostly by HMGA2, SRY, GATA4, SOX5, and ZBTB16. This study is the first to report the expression profiles of lncRNAs, mRNAs and circRNAs in mice with high-altitude hypoxia-induced brain injury treated with (L.) R. Br. and may improve the understanding of the molecular mechanism of (L.) R. Br. in treating high altitude hypoxia-induced brain injury.

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.

A novel species, Gymnadenia winkeliana, has been identified in the Bucegi Natural Park ROSCI0013, located in the Southern Carpathians of Central Romania. Two moderately sized populations of Gymnadenia winkeliana, totalling 120–140 individuals, were discovered inhabiting the alpine grasslands of the park, situated 2.000 m above sea level. To describe this newly found population as comprehensively as possible, 44 vegetative and floral organs/organ parts were directly studied and measured from living plants. Special attention was focused on the characteristics that proved to have taxonomic significance, particularly those involving distinctive details in the morphology of the leaves, perianth, labellum and gynostemium. A total of 223 characteristics were analysed encompassing the morphology of every organ of the plant, cytology and breeding system. Furthermore, comprehensive taxonomic treatment and description, accompanied by colour photographs illustrating the holotype, are provided. Voucher specimens were deposited at the Herbarium of the University of Agriculture and Veterinary Medicine, Bucharest (USAMVB Herbarium barcode: 40102, NEA); Gymnadenia winkeliana, a (micro)endemic species, is characterized as a putative allogamous, facultatively apomict that significantly differs from other Gymnadenia R.Br. species found in Romania. Notably, it distinguishes itself through its smaller habitus (reaching heights of up to 8–10 cm), its two-coloured, rounded/hemispherical inflorescence displaying a gradient of pink hues in an acropetal fashion (ranging from whitish-pink at the base to vivid-pink at the topmost flowers), and its limited distribution in high-altitude areas, encompassing approximately 8–10 km2 in the central area of the Bucegi Natural Park. This species has been under observation since 2005, with observed population numbers showing a significant increase over time, from ca. 50–55 (counted at the time of its discovery) to 120–140 individuals (counted in June 2023). Additionally, comprehensive information regarding the habitat, ecology, phenology and IUCN conservation assessments of Gymnadenia winkeliana are provided, including maps illustrating its distribution.