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This study is part of a project devoted to determining the scent of all the orchid species present in Basilicata. All the analyses were performed by using the solid-phase microextraction technique coupled with gas chromatography-mass spectrometry. The scent of eight species belonging to the Orchis genus was investigated. In the case of O. anthropophora, caryophyllene, tetradecanal and hexadecanal were the main components of the aroma, in O. purpurea, 3,5-dimethoxytoluene and elemicin were found, in O. italica, caryophyllene and 4-(3-hydroxy-2-methoxyphenyl)butan-2-one were found, in O. pauciflora, linalool and 1,4-dimethoxybenzene were found, in O. mascula, linalool was found, in O. quadripunctata, penta- and heptadecane were found, in O. provincialis, β-farnesene and farnesal were found, and in O. pallens, curcumene was the main product.

Çalışmada 9 farklı Orchis taksonununa [Neotinea tridentata (Scop.) R.M.Bateman, Pridgeon & M.W.Chase (Syn. O. tridentata Scop.), O. punctulata Stev. ex Lindl., O. purpurea Huds., O. simia Lam., Anacamptis morio (L.) R. M. Bateman, Pridgeon & M. W. Chase (Syn. O. morio L. subsp. morio), Orchis picta Raf. (Syn: O. morio L. subsp picta (Lois.) K.Richt., Anacamptis papilionacea (L.) R. M. Bateman, Pridgeon & M. W. Chase (Syn: O. papilionacea L. var. papilionacea), Anacamptis laxiflora (Lam.) R. M. Bateman, Pridgeon & M. W. Chase (Syn: O. laxiflora Lam.), Himantoglossum robertianum (Loisel.) P. Delforge (Syn: O. fragrans Pollini)] ait örneklerde labellum mikromorfolojisi Taramalı Elektron mikroskobu (SEM) yöntemi ile incelenmiş ve sonuçlar teşhis anahtarı halinde sunulmuştur. Örnekler, araziden toplandığı sırada %70 alkole alınmış, bir kısmı da herbaryum kurallarına göre kurutularak teşhisleri yapılmıştır. Alkolden çıkarılan labellum örnekleri, bisturi ve iğnelerle stereomikroskop altında kesip ayrılarak doğrudan, üzeri çift taraflı yapışkan bantlar bulunan ve SEM cihazına ait çelik aparatlara yerleştirilmiştir. Bir gece kadar bekletilerek alkolü tamamıyla uçurulan bu örnekler, doğrudan Taramalı Elektron Mikroskobu (SEM) ile incelenmiş ve labellum mikrofotoğrafları çekilmiştir. Bulgularımıza göre; O. fragrans, O. papilionacea, O. purpurea taksonlarının diğer tüm incelenen taksonlardan oldukça farklı olduğu, diğerlerinde genelde kendilerine özgü değişik türevlerde konikal papilla oluşumlarının bulunduğu, bazı türlerde (O. punctulata, O. purpurea, O. simia, O. laxiflora, O. fragrans) uç ve/veya mahmuz bölgelerinin dikkat çekici farklılıklara sahip olduğu görülmüştür. Söz konusu papillaların özellikleri ve labellum yüzeyindeki bölgesel örtüşü, bazı taksonlar için ayırt edici olmuştur. Bilhassa uç bölgesindeki epidermal hücrelerin papilla içerip içermemesi, hücrelerin düz veya farklı yüzey morfoloji (skülptür) özellikleri, hücrelerin düzenli ya da düzensiz olması, yanal çeperlerinin özellikleri taksonlar arasında önemli farklılıkları oluşturmuştur.

The names Epilobium lanceolatum, Lotus requienii (currently accepted as Lotus conjugatus subsp. requienii), Orchis romana (currently accepted as Dactylorhiza romana subsp. romana), and Romulea columnae are lectotypified on specimens kept at RO and BOLO, and on a Sebastiani’s illustration published in his Romanarum plantarum fasciculus primus. An epitype (at RO) for the name Epilobium lanceolatum is also designated.

A lectotype of Orchis × pseudoparviflora (O. coriophora × O. elegans, Orchidaceae) described from Ukraine is designated. The species was described by Ugrinski (1913, 1917, 1922) based on collections (syntypes) from the north-eastern part of Ukraine (currently Sumy and Kharkiv regions) and Crimea. Its taxonomic status and circumscription, as well as those of one of its parent species (O. elegans), have been interpreted differently. Taxonomic and nomenclatural issues of Orchis × pseudoparviflora are discussed. This hybrid is currently accepted under the name Anacamptis × timbali nothosubsp. reinhardii. The lectotypification of O. × pseudoparviflora is based on a recently discovered specimen from the Herbarium of the Nikita Botanical Garden – the National Scientific Center (YALT, s. n.). A scanned image of the designated lectotype is provided.

As orchids rely on their mycorrhizal fungi for nutrient supply, their spatial range is dependent on the distribution of orchid mycorrhizal (OM) fungi. We addressed possible correlations between mycorrhizal specificity and the geographic distribution of orchids and OM fungi in three populations of the rare sister species Orchis patens and O. canariensis. Metabarcoding of the fungal ITS2 region indicated that, although adult plants of either species were colonized by several ceratobasidioid, tulasnelloid, sebacinoid and serendipitoid fungi, the mycobiont spectra were dominated by Tulasnella helicospora (which occurred in 100% of examined plants with high read numbers), which is a globally distributed fungus. In vitro assays with a T. helicospora isolate obtained from O. patens indicated the effectiveness of this OM fungus at germinating seeds of its native host. At a local scale, higher read numbers for T. helicospora were found in soil samples collected underneath O. patens roots than at locations unoccupied by the orchid. Although these findings suggest that the geographical pattern of the main fungal symbiont does not limit the distribution of O. patens and O. canariensis at this scale, the actual causal link between orchid and OM fungal occurrence/abundance still needs to be better understood.

For the first time, perennial materials on the Orchidacaeae Juss. family of Chechnya are summarizedб consisting of 15 families and 29 species. The largest genus in the family – Orchis L. is represented by 8 species. The article presents information that all members of the family are protected plants listed in the Red Book of Chechnya, Ingushetia, Dagestan, and the Stavropol Territory. Some species are not included in the list of plants to be protected, including those species that have underground tuberoids and can serve as a source of salep (for instance, Platanthera bifolia, P. chlorantha, Herminium monorchis, Traunsteinera sphaerica, Dactylorhisa incarnata, D. salina, D. urvilleana). In this connection, data on the resources of family members and the potential possibilities of their use as a medicinal raw material are of interest. We have carried out resource studies of the most common species of Orchis tridentata Scop, within the North Caucasus. The obtained data allow us to expand our understanding of the species role of Orchidaceae Juss family. Пoлyчeнныe дaнныe пoзBoляют pacшиpить пpeдcтaBлeниe o poли BидoB ceмeйcтBa Orchidaceae Juss. of the North Caucasus flora in the composition of vegetation cover.

espanolA partir de los resultados obtenidos de la extensa e intensa prospeccion del territorio, sepresenta informacion corologica sobre 9 taxones de la familia Orchidaceaeen la provinciade Segovia, Coeloglossum viride(L.) Hartm., Epipactis helleborine(L.) Crantz subsp. helle-borine, Epipactis microphylla(Ehrh.) Swartz., Epipactis tremolsii Pau, Ophrys fusca Link.subsp. fusca, Ophrys insectifera L. subsp. insectifera, Orchis papilionaceaL., Orchis ustulataL. y Spiranthes spiralis (L.) Chevall, asi como del hibrido Orchis x gennarii Reichenb. fil.(O. moriox O. papilionacea), los cuales ademas de constituir primeras citas provinciales,amplian notablemente el conocimiento de la orquidoflora silvestre de Segovia. Se aportandatos sobre su distribucion en la CCAA de Castilla y Leon, ecologia, estado de las pobla-ciones y conservacion. Asi mismo, se incluyen mapas de distribucion en cuadriculas UTMde 10x10 Km y material fotografico. EnglishBased on the results obtained from intense and extensive prospecting of the territory choro-logical information is hereafter put forward for 9 taxa from the Orchidaceaefamily in theprovince of Segovia, Coeloglossum viride(L.) Hartm., Epipactis helleborine(L.) Crantz subsp. helleborine, Epipactis microphylla(Ehrh.) Swartz., Epipactis tremolsii Pau, Ophrys fusca Link. subsp. fusca, Ophrys insectifera L. subsp. insectifera, Orchis papi-lionaceaL., Orchis ustulata L. and Spiranthes spiralis (L.) Chevall, as well as for the hybridOrchis x gennarii Reichenb. fil. (O. moriox O. papilionacea), which, besides representingthe first populations found in the province, noticeably increase the knowledge about thewild orchid flora in the province of Segovia. Data concerning their distribution, ecology,state of the populations and conservation in the Castilla y Leon region is brought forward.Also included is photographic material and distribution maps in 10x10 Km UTM grid cells. euskaraHango lurraldean egindako azterketa zabal eta sakonaren ondoren, Segoviako Orchidaceaefamiliako 9 taxoiren informazio korologikoa aurkezten da lan honetan. Coeloglossum viri-de(L.) Hartm., Epipactis helleborine(L.) Crantz subsp. helleborine, Epipactis microphylla(Ehrh.) Swartz., Epipactis tremolsii Pau, Ophrys fusca Link. subsp. fusca, Ophrys insectiferaL. subsp. insectifera, Orchis papilionaceaL., Orchis ustulata L. y Spiranthes spiralis (L.) Chevall, eta bai Orchis x gennarii Reichenb. fil. (O. moriox O. papilionacea) hibridoarenaere. Probintzian lehen zitak izateaz gain, Segoviako basa orkidofloraren ezagutza zabaltzeko urrats handia dira. Gaztela eta Leongo Autonomia Erkidegoan espezieek dutenbanaketaz, ekologiaz, populazioen egoeraz eta kontserbazioaz ere datuak aurkezten diralan honetan, baita 10 x 10 Km UTM kuadrikuletan banaketa mapak eta argazki-materialaere.

Muşilinde Hasköy ilçesi Altınova Beldesi civarı ve Bulanık ilçesi civarında salepelde edilişi, elde edilişte kullanılan türler, yöntemleri araştırma amacımızıteşkil etmiştir. Yapılan araştırmalar sonucunda Muş Hasköy ilçesi AltınovaBeldesi civarı ve Bulanık ilçesi civarı olmak üzere iki merkezde salep eldeedilmektedir. Ayrıca Diyarbakır’da da salep toplandığı araştırmalarımız sonucuortaya çıkmıştır. Bu merkezlerde salep elde etme yöntemleri ve salep eldeetmede kullanılan orkide türleri tespit edilmiştir. Muş Hasköy ilçesi AltınovaBeldesi civarı ve Bulanık ilçesi civarında salep karışımındaki parçalı yumrularDactylorhiza umbrosa, Dactylorhiza iberica‘ya aittir. Yuvarlakolanlar ise Anacamptis palustris, Orchis pseudolaxiflora, Anacamptiscoriophora olup, ikisi rizomlu olmak üzere 4 cinse ait 7 orkide türü tespitedilmiştir. Salep elde etmek amacıyla her yıl milyonlarca orkide tahripedilmektedir. Bu tahribat çayır salebi elde edilen orkide türlerini tehlikeyesokmaktadır., Our aim is to clarify the species used for production of salep,production methods of orchids in Altınova region of Haskoy and Bulanıkdistricts of Muş province. The orchids species were collected from Muşprovince, Hasköy district around Altınova town and around Bulanık district.Moreover, our research showed that salep is collected from Diyarbakır as well.The methods and the species used to obtain salep in these areas weredetermined. Segmentary tubers collected from Muş province, Hasköy districtaround Altınova town and around Bulanık district belong to Dactylorhizaumbrosa, Dactylorhiza iberica. As for The round tubers, they are Anacamptispalustris, Orchis pseudolaxiflora, Anacamptis coriophora. Inthe area, 7 orchids species were determined. The species belong to 4 orchidsgenera. Two of the species are rhizomatous. Every year millions of orchids arebeing destructed to obtain salep. This destruction especially endangers thesalep species growing in meadows.

In this study, the goal was the germination and propagation of Orchis sancta L. which is endangered due to uncontrolled collection and used in obtaining salep, in vitro conditions. By the addition of 0 (control group), 20, 40, 60, 80, and 100 g/l concentrations of sucrose, glucose, maltose, galactose, and fructose of carbohydrate forms into Van WaesDebergh culture medium, sugars of effects were determined on germination and development of Orchis sancta L. seeds. In the study, germination, formation of protocorm and shoot periods and ratios were examined. The highest average germination ratio was obtained as 77.85% in Maltose40, the lowest average as 44.36% in Galaktose100 mediums. In protocorm formation, when the highest average ratio was detected as 68.53% in Sucrose100, the lowest average as 25.33% in Fruktose100 mediums. Germination period as 12.50-21.33 days, protocorm formation period as 22.50-50.83 days and shoot formation period as 50.66-105.33 days’ intervals were determined., Siirt University Coordinator of Scientific Research and Projects-SİÜ-FEB-25

Germination and propagation of terrestrial orchids is highly challenging due to their small size seeds and absence of endosperms. Some orchid species are extremely dependent on fungi for germination of their seeds. Although germination occurs symbiotically between the seed and the fungus, it is not coincidental that fungus species are specific to orchid species. Aim of this study is to show the impact of the fungi, isolated from the tubers of ten naturally grown species of orchids, collected from mountainous area of Van province, Turkey, on in vitro symbiotic germination of the seeds. The collected orchids species were Dactylorhiza romana (Seb.) Soo subsp. georgica (Klınge) Soo ex Renz & Taub., Orchis pinetorum Boissier & Kotschy, Orchis spitzelii Saut. ex W.D.J. Koch, Orchis coriophora L., Orchis collina Banks & Solander, Orchis anatolica Boiss., Orchis simia Lamarck, Ophrys straussii H. Fleischmann & Bornmüller, Dactylorhiza umbrosa (Kar. & Kir.) Nevski, Anacamptis pyramidalis (L.) L. C. Rich. In the study from orchid tubers, Rhizoctonia sp., Aspergillus sp., Alternaria sp., Penicillium sp., Trichoderma sp. and Fusarium sp. fungi species are isolated. Firstly, orchid seeds and fungal isolates placed in oat culture medium were transferred to Van Waes & Debergh culture medium in subculture. The highest germination rates obtained in the seeds are 71.19% in Dactylorhiza romana subsp. georgica species with Rhizoctonia sp.; 78.26% in Orchis pinetorum species with Penicillium sp.; 27.77% in Orchis spitzelii species with Fusarium sp.; 83.07% in Orchis coriophora species with Rhizoctonia sp.; 75.00% in Orchis collina species with Fusarium sp.; 38.88% in Orchis anatolica species with Alternaria sp.; 73.91% in Orchis simia species with Fusarium sp.; 91.60% in Ophrys straussii species with Fusarium sp.; 93.75% in Dactylorhiza umbrosa species with Fusarium sp. and 56.00% in Anacamptis pyramidalis species with Rhizoctonia sp. At the end of the study, it was understood that the fungi isolated from their own or other tubers had different effects on the germination of each orchid species., Siirt University Coordinator of Scientific Research and Projects-2013-SİÜZİR-Z2

The ultrastructure of proplastids in the leaves of five species of orchids was investigated: Paphiopedilum, Stanhopea, Cymbidium, Coelogyne and Orchis. The proplastids of the different species differ in the density of their stroma. The most electron dense is the stroma of Paphiopedilum proplastids and the lowest electron-density is shown by Orchis proplastids. A characteristic feature of most of the investigated proplastids is the presence of prolamellar bodies. The frequency of their occurrence and their structure differ from species to species. The remaining membrane system is limited to single peripheral vesicles. Dividing forms of the proplastids have been observed. The number of dividing proplastids averages 5 per cent. The proplastids have been observed to divide by constriction, invagination of the inner membrane or arrangement of vesicles in the division plane.

In the stroma of leaves proplastids of all investigated species of orchids: Paphiopedilum, Stanhopea, Cymbium, Coleogyne and Orchis occur starch grains of different shape, size and electron density. The number of starch grains vary from one to several. The proplasitids from cells of the vascular bundle sheath are completely filled with numerous starch grains and the stroma is strongly reduced. The formation of protein crystaloids was observed only in the proplastids of Paphiopedilum. In the stroma of proplastids in investigated species the DNA-containing areas are present. A tendency of their peripheral situation and of the formation of contacts between DNA fibirils and the inner surrounding membrane or with rare thylakoid's membranes was observed. In these areas in priopastids of Stanhopea, Cymbidium, Coelogyne and Orchis starch grains of different size, shape and electron density occur.

The pollination success of two nonrewarding orchids in two coarse habitats was examined. The comparative results of Orchis punctulata and O. purpurea showed a significant differentiation. The fruit set of O. purpurea was significantly greater compared to O. punctulata , whereas both species also had significantly higher fruit set in the grassland compared to the forest. Although the pollination success of O. purpurea was not significantly correlated with plant height, inflorescence size, or nearest neighbor distance, these factors were found to be significantly correlated to the pollination success of O. punctulata . Among these factors, the nearest neighbor distance had the highest impact on the pollination success of O. punctulata , whereas the respective effects to the individuals of O. purpurea were found to be nonsignificant. The results demonstrate that the distribution of O. punctulata at the edges of its range is not delimited by factors related to its pollination effectiveness and that other factors, mostly related to the microsite conditions, might play a more significant role in the colonization of more western areas. Moreover, suitable management treatments that would increase the light levels within the forest sites studied may also increase the pollination success and the fruit production of the studied orchids.

Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and their life cycle. The level of specificity of the association between orchid species and fungi can be related to the number of co-occurring orchid species. To investigate orchid mycorrhizal associations in adult-photosynthetic orchids, 16 Mediterranean orchid species belonging to 4 genera (Anacamptis, Ophrys, Orchis, and Serapias) at 11 different sites were subjected to DNA-based analysis. Eighteen operational taxonomic units representing two fungal families, Tulasnellaceae and Ceratobasidiaceae, were identified. All examined orchid species associated with different mycorrhizal fungi. Interestingly, there was a positive correlation between number of orchid species and number of mycorrhizal. Monospecific populations showed a lower number of fungi, while sympatric populations had a higher number of mycorrhizal fungi. Our results showed that Mediterranean orchid...

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post-pollination pre-zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.

For the first time, changes of the growth and accumulation of phenolic compounds (PHCs) in different parts of underground organs of the tuberoid orchid Dactylorhiza maculata during vegetation and the winter dormancy period were studied. It was shown that the replacement of old tuberoids and adventitious roots by new ones is accompanied by active growth, assimilates reallocation, and PHCs accumulation. It was found that the tuberoid performing the function of a storage organ possessed a low PHCs content, which increased at the end of vegetation and was stably high during the winter dormancy period; in spring the content decreased, mosr likely as a result of PHCs catabolism and utilization for growth processes. It was proposed that the higher PHCs content in adventitious roots and tuberoid terminal parts is determined by mycotrophy of the organs.

WOS: 000394538200002 Orchis forms a big group in terms of the number of species it includes, and studies conducted in recent years reveal results which are different from each other in terms of the systematics and phylogeny of the genus. In order to reveal both qualitative and quantitative seed properties of the species belongs to phylogenetically related genera in detail and to investigate which properties are diagnostic among these taxon, the seed micromorphology and morphometry of 10 taxa from Orchis, Anacamptis and Neotinea genera were examined using a light and electron microscope. The seed micromorphological and morphometric characteristics were examined with the help of principle component analysis, canonical discriminant analysis and hierarchical clustering analyses by taking into consideration the taxonomic status of closely-related Anacamptis, Neotinea and Orchis genera. In this study, significant differences were detected at the genus level in terms of morphometric and micromorphological characteristics such as seed shape, seed length, periclinal wall ornamentation, chalazal region cell shape, embryo length, width and volume. Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [114Z702] We acknowledge the Karadeniz Research and Application Center of Advanced Technology (KITAM) for providing the equipment and technical support for experiments involving electron microscopy. This work was supported through The Scientific and Technological Research Council of Turkey (TUBITAK, 2014, grant number 114Z702).

Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle-class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL-F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL-F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree-based methods for nrITS in 67% of the tested tubers, 58% for trnL-F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross-border trade of Salep tubers. This is the peer reviewed version of the article, which has been published in final form at doi: 10.1111/1755-0998.12615. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

In this study, the seed micromorphologies of eight taxa of Anacamptis, Neotinea and Orchis growing around Edirne province (Turkey) were investigated using light microscopy and scanning electron microscopy (SEM). Slides prepared with glycerin jelly were used for measurements under the light microscope and fine details of seed testae characteristics were observed with SEM. Seeds of the investigated orchid taxa are fusiform shaped and of different shades of brown. Their lengths and widths are different among the taxa and range between 0.263-0.640 mm and 0.118-0.208 mm, respectively. Testa surfaces of Orchis mascula subsp. mascula, Orchis purpurea subsp. purpurea and Orchis simia subsp. simia, are smooth while those of Anacamptis coriophora, Anacamptis laxiflora subsp. laxiflora, Anacamptis morio subsp. morio, Anacamptis papilionacea and Neotinea tridentata subsp. tridentata are reticulate. An identification key based on seed morphologies and sizes is suggested for the first time, including testae structures of orchids growing in Edirne province. The overall results of the study showed that morphological structures of orchid's seeds could be used as diagnostic characters in identification.

Orchidaceae are one of the most diversified Angiosperm families, with more than 27,000 accepted names (The Plant List 2013). The majority of orchid species rely on biotic pollen vectors to reproduce (Nilsson 1992), so do 88% of flowering plant species (Ollerton et al. 2011). Usually, plants attract pollinators with floral signals that advertise rewards (Schiestl and Johnson 2013). However, some plant species also display attractive floral signals but don’t provide any reward (or low or wrong reward) (Jersakova et al. 2009); they are called deceptive species (Renner 2006). Deceptive pollination occurs in about 30% of orchid species (Jersakova et al. 2006, Claessens and Kleynen 2011), and the commonest systems involve the exploitation of the food-seeking behavior of the pollinators (food deception—Jersakova et al. 2006). Under natural conditions, insect visits on rewardless flowers are rare events difficult to record (Cozzolino et al. 2005, Claessens and Kleynen 2011), making direct observation time consuming (Widmer et al. 2000). Indirect approaches have been developed to study more easily orchid-pollinator relationships: pollinaria carried by insects (caught on rewarding plants growing in the vicinity of studied orchid populations) allow the identification, through molecular techniques, of the corresponding orchid species (Widmer et al. 2000); pollinaria found on insects can also be identified through specific morphological features (Singer et al. 2008, Micheneau et al. 2009); scales and hairs found on pollinated stigmas can allow the identification of the corresponding insect species (Micheneau et al. 2009). Videotape observations are another alternative to direct observations (Micheneau et al. 2009, Lortie et al. 2012, Nakase and Suetsugu 2016), but observation-based techniques don’t allow accurate identification of numerous insects (especially dipterans and hymenopterans). The latter have to be collected and examined carefully in order to draw up accurately visitor and potential pollinator lists. In the genus Orchis Tourn. ex L. (Orchidaceae), none of the species produce nectar and the majority depends on food deception to attract pollinators (Scopece et al. 2014). Visitor and pollinator guilds have been studied more or less extensively according to the species (e.g. van der Cingel 1995, Berger 2003, 2004; Claessens and Kleynen 2011). In O. militaris (L.), most data were collected in Austria by Voth (1987) and in the United Kingdom by Farrell (1985). Other contributions come from Berger (2003, 2004), Ebert and Rennwald (1993), Petit (1998), Voth (1999), etc. Among (altitudinal) regions, a plant species can be pollinated by different insect species. This spatial variation in pollinator identity is well known in various species, including orchids (e.g. Schatz et al. 2005, Gomez and Zamora 2006, Gonzalez et al. 2014, Sun et al. 2014, Gross et al. 2016). The aim of this study was thus to determine the guilds of floral visitors and potential pollinators to O. militaris in a region that was not widely studied so far: Southern Belgium (Wallonia).

Despite highly specialized pollination strategies, hybridization is a common phenomenon among Mediterranean deceptive orchids. Food-deceptive species sire a progeny of F 1 unfertile plants, which work as a late post-zygotic barrier. Conversely, when pre-zygotic barriers of sexually deceptive ( Ophrys ) species are absent, the hybrids are fertile and an extensive introgression may occur. Here, we have performed molecular analysis and hand pollination treatments to characterize a hybrid zone of two food-deceptive species, O. mascula and O pauciflora . Hybrids (called O. × colemanii ) have shown different amounts of parental nrDNA, strongly supporting that they are F 2 and/or successive hybrid generations. Comparable high levels of reproductive success have been detected in natural conditions and in experimental crosses suggesting the absence of effective reproductive barriers either between hybrids, either between hybrids and parental species. In light of ecological and distributional features of O. × colemanii across its distribution range, we hypothesize that these populations have originated by secondary contact in the periglacial belt of Apennines. Moreover, the rare and localized O. pauciflora could benefit a genetic enrichment by hybridizing with a widespread related species. O. × colemanii is not a dead end population, but may have a role as potential reserve of adaptive variability and is an unusual stage along the speciation process.

Seed micromorphology of 24 taxa of the genera Anacamptis and Orchis was examined under light and scanning electron microscopy. Seed qualitative characters appear very useful at the supraspecific level in the subtribe Orchidinae. Based on our observations, the sculpturing of the periclinal walls of the medial testa cells, the seed shape and several features of the anticlinal walls of the apical cells showed variability between the studied taxa, with special relevance of the first character. According to this factor, we found one type of seed for Anacamptis and two for Orchis. The taxonomic value of seed coat characters is compared with the last taxonomic proposals for the genera Anacamptis and Orchis.

Summary • Nonrandom species–species associations may arise from a range of factors, including localized dispersal, intra- and interspecific interactions and heterogeneous environmental conditions. Because seed germination and establishment in orchids are critically dependent upon the availability of suitable mycorrhizal fungi, species–species associations in orchids may reflect associations with mycorrhizal fungi. • To test this hypothesis, we examined spatial association patterns, mycorrhizal associations and germination success in a hybrid zone containing three species of the genus Orchis (Orchis anthropophora, Orchis militaris and Orchis purpurea). • Hybridization occurred predominantly between O. purpurea and O. militaris. The spatial distribution patterns of most pure species and hybrids were independent from each other, except that of O. purpurea and its hybrids. The fungal community composition of established individuals differed significantly between pure species, but not between hybrids and O. purpurea. Seed germination experiments using pure seeds showed that the highest number of protocorms were found in regions where adult individuals were most abundant. In the case of hybrid seeds, germination was restricted to areas where the mother plant was most abundant. • Overall, these results suggest that the observed nonrandom spatial distribution of both pure and hybrid plants is dependent on the contingencies of the spatial distribution of suitable mycorrhizal fungi.

Summary 1. This account presents information on all aspects of the biology of Orchis anthropophora (L.) All. (Man Orchid) that are relevant to understanding its ecological characteristics and behaviour. The following 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. Orchis anthropophora is a native species in Britain, where it occurs predominantly in chalk and limestone grassland and dune grassland. It can also be found along field margins and in old abandoned chalk pits and limestone quarries, where it is usually found at the foot of slopes. It also grows on road verges and sometimes in quite dense scrub. Orchis anthropophora tolerates some shade, but it flowers most profusely in open conditions. It is a common species in the Mediterranean region. 3. Orchis anthropophora is wintergreen and has leaves throughout the winter. Flowering takes place between mid-May and mid-June. Vegetative adult dormancy – the failure of above-ground parts to appear in a growing season, followed by the reappearance of full-sized photosynthetic plants in subsequent seasons – has been observed. In most cases, dormancy does not last longer than 1 year. Orchis anthropophora is long-lived (half-life of cohorts ranges from 4.0 to 7.8 years). 4. Orchis anthropophora is not autogamous, and pollinators are required for successful pollination and fruit-set. Flowers are predominantly pollinated by beetles and sawflies. Terpenes are the major compounds of the floral scent profile of O. anthropophora. Natural levels of fruit-set are generally low. Recorded values are mostly 80% in a population in southern France. 5. Orchis anthropophora hybridizes with several other species in the genus Orchis, including O. italica, O. simia, O. purpurea and O. militaris. Intergeneric hybrids have been reported with Barlia, Dactylorhiza and Neotinea. In Britain, hybrids with O. simia and O. purpurea have been reported. 6. Orchis anthropophora has declined markedly in Britain during the last century, and at present, it is rare and classified as endangered. Very few new populations are becoming established, and many populations have become extinct, mainly because of agricultural intensification (ploughing and fertilization of old fields). Conservation of the remaining populations requires moderate grazing by sheep to maintain a short turf and to promote seedling recruitment.

†Background and Aims The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid‐mycorrhizal associations may be involved in acting as a post-mating barrier. †Methods We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology. †Results Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents. †Conclusions Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Premating barriers are probably the main factors determining hybridization rates between the investigated species.

WOS: 000439934400002 We aimed to describe and analyse the morphological, anatomical and micromorphological traits of 36 Turkish orchids representing 12 genera (e.g. Anacamptis, Cephalanthera, Dactylorhiza, Orchis, Serapias) in detail and analyse their usability for solving phylogenetic and taxonomic issues. We applied UPGMA cluster analysis to anatomical, morphological and micromorphological characters such as root tubers, leaf and flower structures, pit, sclerenchymatic sheath, vascular bundle shape, crystal and starch, exodermis and endodermis structure, stomata type, bulliform cells in roots, shoots and leaves, surface structures like papillae, hairs and ornamentation on flower parts, leaves, fruits and seeds. Furthermore, in a phylogenetic framework, we analysed nuclear ribosomal ITS diversity in the same orchid species belonging, and in a combined Bayesian phylogenetic analysis based on anatomical, morphological, micromorphogical and ITS data we confirmed the usefulness of multiple data sets for effectively assessing taxonomically critical orchids. In the combined analysis, all genera were resolved as monophyletic with topologies congruent with recently published more thorough molecular phylogenetic reconstructions, while the trees obtained by seprately analysing the ITS and the anatomical, morphological, micromorphogical data were less resolved and partly inconclusive. Scientific and Technological Research Council of Turkey (TUBITAK, 2014) [114Z702] This work was funded by The Scientific and Technological Research Council of Turkey (TUBITAK, 2014, grant no. 114Z702).

Salep samples obtained from 10 different Orchidaceae spp., namely Dactylorhiza osmanica var. osmanica, Ophrys mammosa, Orchis anatolica, Orchis coriophora, Orchis italica, Orchis morio, Orchis palustris, Orchis simia, Orchis tridentata and Serapias vomeracea ssp. orientalis, in Anatolia, were analyzed for moisture, glucomannan, starch, protein, ash contents, pH and viscosity values. Depending on the species, the samples showed statistically significant differences in glucomannan, starch and viscosity values. It was observed that the salep samples obtained from the tubers of O. italica, O. morio, O. anatolica and O. tridentata and S. vomeracea ssp. orientalis, respectively, had higher glucomannan contents and viscosities. To ensure a supply of high-quality salep, the uncontrolled collection of tubers from the wild, especially the species O. italica, O. morio and O. anatolica, should be prevented, and research into methods of cultivation should be carried out.

1. This account presents information on all aspects of the biology of Orchis mascula (L.) L. (early purple orchid) 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. Orchis mascula is a native herb of the British flora. It is mostly found in woodland, copses, grasslands and open pastures, mostly on neutral or base-rich soils. It can also occur in hedgerows, scrub, on roadsides and railway banks, in grikes on limestone pavement and on moist cliff ledges. It is absent from very acid or very wet sites. It tolerates a sparse to moderately dense canopy, but it does not flower in deep shade. 3. Orchis mascula is a non-bulbous geophyte with little or no capacity for vegetative spread. The main perennating organ is a tuber (strictly a rootstem tuber). In most years the tuber generates a rosette of expanded leaves, and, at the end of every year of the plant's life, the tuber is replaced by at least one new tuber. Dormancy, the failure of above-ground parts to appear in a growing season, and the subsequent reappearance of full-sized photosynthetic plants in subsequent seasons, has been observed, but does not last longer than 1 year. The species is long-lived: it takes at least 4 years from first appearance above-ground to achieve flowering for the first time. The maximum recorded lifetime after first appearance is 13 years. 4. Orchis mascula is not autogamous and pollinators are necessary for successful pollination and fruit set. Flowers are nectarless, and pollinated by deceit, mainly by bumblebees and solitary bees. Natural levels of fruit set are usually < 20%. Hand-pollination can increase fruit set to approximately 80%, indicating that seed production is strongly pollen-limited. The lowest four flowers are more likely to be pollinated than the upper flowers, probably because pollinators learn to avoid the species after discovering that it offers no reward. 5. As in most other European countries, Orchis mascula has declined in the British Isles, although it is not at threat of extinction at a national level. Most sites from which it has been lost are in central England and Scotland. Most losses have been caused by woodland clearance and coniferization, intensification of grassland management and ploughing. The cessation of traditional coppicing practises has also led to a decline in the abundance of O. mascula. Since the species is slow to colonize new forest stands or grasslands, management should focus mainly on conservation of ancient forest habitats and grasslands in which fertility is moderate to low and grazing is absent or low in intensity. Restoration of traditional coppicing practices could also lead to higher chances of the species flowering and surviving in the long-term.

We report the first confirmed occurrence in Britain of Orchis × angusticruris Franch. ex Rouy, a hybrid between two closely related orchid species of anthropomorphic Orchis (O. purpurea Huds. × O. simia Lam.) that hybridize frequently in Continental Europe. Seven individual hybrids, most likely F1 plants representing a single interspecific pollination event, first flowered with both parents in May 2006 at a nature reserve in the Chiltern Hills near Goring, Oxfordshire. Univariate and multivariate morphometric analyses (43 characters plus 12 indices), internal transcribed spacer sequencing, plastid microsatellites and amplified fragment length polymorphism (AFLP) analyses together readily separate the parents and confirm that O. purpurea was the ovule parent and O. simia the pollen parent, presumably reflecting the greater frequency and/or later flowering period of the latter at the site. This study reinforces a more general observation that, in most orchids, the ovule parent contributes substantially more to the hybrid phenotype than does the pollen parent, perhaps reflecting cytoplasmic inheritance. In contrast, the hybrids are placed closer to O. simia than to O. purpurea in the AFLP tree. Apparently recent arrivals, the few O. purpurea plants at Goring contrast genetically with the two other small populations of this species known in the Chilterns, but rather are consistent with relatively uncommon Continental populations. This suggests that the plants may have been deliberately introduced at Goring by man, although transport from the Continent in high-level air currents cannot be ruled out. The Goring population of O. simia is likely to have become genetically impoverished through (1) preferential removal of many relatively fit plants to herbaria in the 19th century and/or (2) a catastrophic population crash in the first half of the 20th century. However, both our re-examination of herbarium specimens and our population genetic data indicate past hybridization among anthropomorphic Orchis species occurring naturally in the Chilterns. Thus, we tentatively recommend retention of the hybrid plants at Goring, despite their likely anthropogenic origin from Continental material and the partial viability of their pollen and seeds, which offers opportunities for future introgression. Although the Goring hybrids broadly resemble morphologically O. militaris, another anthropomorphic Orchis still found at two Chiltern localities, sufficient morphological and molecular differences were observed to strongly refute our initial hypothesis that O. militaris could have originated through hybridization between ancestors that resembled O. purpurea and O. simia. The comparatively complex genetic properties evident in both O. simia and O. purpurea merit further study. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 687–711.

Northeastern Estonia is home to extensive oil shale mines. Associated with these are desolate and environmentally damaging hills of ash and semicoke tailings. Interestingly, some of the first plants to colonize these hills are rare orchids. Here, we assess the identities of the mycorrhizal fungi associated with these orchids, in particular Epipactis atrorubens, Orchis militaris, and Dactylorhiza baltica, and compare them with mycorrhizal fungi from orchids from pristine habitat. Epipactis atrorubens associated with the widest breadth of fungi, including unnamed members of the basidiomycete family Tulasnellaceae and the potentially ectomycorrhizal ascomycetes Trichophaea woolhopeia and Geopora cooperi. Orchis militaris also associated with unnamed members of the Tulasnellaceae. Dactylorhiza baltica associated with Ceratobasidium albasitensis. In Epipactis and Orchis, the same fungi associated with plants in the pristine habitat as with those on ash hills. The tulasnelloid and ceratobasidioid fungi mycorrhizal with these orchids appear closely related to common orchid mycorrhizal fungi, while one of the ascomycetes mycorrhizal with E. atrorubens is closely related to a mycorrhizal fungus with E. microphylla. Our results suggest that these orchids and their fungi are not limited to pristine habitats and that environmentally polluted sites may present novel habitats that may be exploited for endangered plant conservation.

Eighteen species in six genera [Oberonia, Ophrys, Orchis, Oreorchis, Ornithochilus (=Phalaenopsis) and Otochilus ] are described in this chapter. A detailed historical account of salep which makes use of tubers of Ophrys, Orchis and numerous other orchids described in this volume is included in this chapter. Usage of Otochilus species is confined to the Himalayas, while phytochemical studies are being undertaken in China. Some species of Oberonia contain alkaloids.

The pollination of the non-rewarding Orchis pauciflora was studied. The most abundant visitors of O. pauciflora were B. terrestris queens. The fragrance of O. pauciflora inflorescence was dominated by a sesquiterpene (E)-β-farnesene. (E,E)-α-Farnesene, (E)-2,3-dihydrofarnesol, geranylcitronellol, and the monoterpenes limonene and 1,8-cineol were found among less abundant constituents. The sesqui- and diterpenes detected in O. pauciflora fragrance are frequent constituents of male marking pheromones of many bumble bee species. Enantioselective analysis of O. pauciflora scent and B. terrestris male marking pheromone revealed the presence of the (S)-isomer of (E)-2,3-dihydrofarnesol in both samples, and electrophysiological experiments showed that mainly the (S)-isomer activated the antennal receptors. In field experiments, O. pauciflora inflorescences were enriched with the main compound (E)-β-farnesene resulting in significantly increased pollinia export. We here discuss the chemical similarities between orchid and bumble bees and whether the presence of bumble bee male pheromone components in O. pauciflora fragrance increases its fitness.

Hybridization is frequent in Orchidaceae and may play an important role in their speciation. However, information about pollinators and their role in hybridization is sparse. In the Mediterranean region of France, confirmed pollinators observed on Orchis simia and O. anthropophora included only one species common to both, the beetle Cidnopus pilosus. This beetle thus appears responsible for hybridization. Cross-specific pollen transfer is bidirectional, but hand pollination showed higher fruit set after deposition of O. anthropophora pollen on the stigma of O. simia than in the opposite direction. Both locally and over a 1600-km2 study area, the presence of hybrids was shown to be strongly predicted by the microhabitat preferences of this beetle. To my knowledge, this is a novel demonstration that the spatial distribution of a natural hybrid in plants can be predicted by the local occurrence of a pollinator species identified as effecting hybridization. I suggest that similar interaction could ex...

Chromosome numbers in 29 populations belonging to 21 species of Orchidinae from the Iberian Peninsula have been counted. For four Iberian endemic species, Anacamptis coriophora subsp. carpetana, Dactylorhiza caramulensis, Orchis tenera, and Serapias perez-chiscanoi, the number of chromosomes is reported here for the first time. For another three, Anacamptis champagneuxii, Himantoglossum hircinum, and Orchis langei, this number has been established in Iberian representatives. Moreover, new ploidy levels have been obtained for Anacamptis champagneuxii and Dactylorhiza sambucina. Some taxonomic, phylogenetic, and phytogeographic comments are made for several species or groups of species from the Western Mediterranean region.

The new orchid genus Sirindhomia, named after H. R. H. Princess Maha Chakri Sirindhorn of Thailand, is established to accommodate three species from Thailand (Chiang Mai and Tak provinces), Myanmar (Shan State), and China (Yunnan province). Two new species are described and a new combination, based on Habenaria monophylla Collett & Hemsl., is made; all of the three species are illustrated, and a distribution map is provided. Sirindhomia belongs to subtribe Orchidinae and has an overall similarity with Ponerorchis, Hemipilia, and Amitostigma. However, it significantly differs in its column morphology which is more reminiscent of that found in the vegetatively distinct genus Orchis. The new species, Sirindhomia pulchella and S. mirabilis, seem to be local endemics, as they are only known from the Thai mountains Doi Chiang Dao and Khao Hua Mot, respectively. Sirindhomia monophylla, on the other hand, is known from a very large part of the range of the genus. All Sirindhomia species known are restricted to limestone mountains at 800–2200 m alt., mainly growing in rock crevices and among scrub as well as on grassy slopes. They flower from April to June.

Some species belonging to the genus Orchis Tourn. ex L. (Orchidaceae) are of great economic importance as their tubers or corms are used to produce a hot beverage called salep. Nevertheless, these plants are not cultivated but are rather collected from nature, and due to careless collection many have already been listed as endangered plants. In order to assess the possibility of in vitro propagation, an orchid, Orchis coriophora L., was selected as a model plant, and the effects of basal media and plant growth regulators on in vitro seed germination, protocorm development, and plantlet formation were studied. Mature seeds were cultured in 4 different basal media, each supplemented with various concentrations and/or combinations of auxins and cytokinins/cytokinin-like substances. The highest germination rate (44.2%) was observed in Orchimax medium including activated charcoal plus 1 mg/L indole-3-acetic acid. Protocorms developed plantlets in all the tested media. Orchimax medium including activated charcoal and supplemented with 0.25 mg/L 6-benzyladenine was found to be the most suitable medium for the formation of plantlets from protocorms.

The flower structure and development of ten species in six genera of the orchid subtribe Orchidinae are described and illustrated by scanning electron micrographs. Particular attention is given to the structure of the gynostemium, which for most species is interpreted from ontogenetic data. All the species studied here share a series of features, e.g. the sequence of tepal and anther initiation, the shape and position of the anther, the presence of auricles and basal bulges, the three-lobed condition of the median carpel apex and the lateral lobes of the median carpel embracing the basal ends of the thecae. However, the form and structure of the three carpel apices are most varied in the later development stages or in the adult flower. The genus Hemipilia shows a series of peculiar characters that are quite different from those of the other genera in Orchidinae. The peculiar structure and development of the viscidia in both Amitostigma and Neottianthe indicate that both of them are different from other genera in Orchidinae. The adult floral morphology shows that the genera Galearis and Chusua are not congeneric with Orchis. The separation of the lateral lobes of the rostellum in most genera studied here as well as in the Brachycorythis group from South Africa suggests that this is the ancestral state in the subtribe Orchidinae. In contrast, the conjoining of lateral lobes in Dactylorhiza and Orchis is suggested as a derived character.

1. Annual censuses of Orchis morio (green-winged orchid) flowering spikes have been taken over a 27-year period in a replicated factorial experiment on the effects of fertilizer application. Census data, combined by block or treatment, were used in time-series analyses to test for density dependence. 2. Partial autocorrelation functions revealed the importance of positive correlations at lag 1 and negative correlations at lag 5. Stepwise multiple regressions provided evidence of delayed density dependence, again with a delay of about 5 years, with no evidence of direct (first-order) density dependence. 3. First-order autocorrelations and delayed density dependence were considered in the light of the known stage structure and generation time of the plant and the possibility of density dependence at different points in the life history. 4. Model structure affects the detection of density dependence, increasing the propensity for type I errors.

A small population (16 individuals) of Orchis × colemanii Cortesi, a natural hybrid between O. mascula (L.) L. and O. pauciflora Ten. was sampled from a locality near Sassano (National Park of Cilento, province of Salerno, Southern Italy) in order to assess its genetic structure. Filter hybridizations of O. × colemanii, O. mascula and O. pauciflora DNAs against a PCR-amplified ribosomal fragment indicated that all the hybrid individuals in study have the ribosomal repeats of both parents, even if not always combined in equal proportions, and that no specimen morphologically identified as O. mascula or O. pauciflora possessed detectable rDNA of the other species. The majority of the hybrid specimens (12 individuals) showed a preponderance of O. mascula rDNA, three had approximately equal proportions of parental repeats and one had a preponderance of O. pauciflora rDNA. As the parental species have different amounts of rDNA, as inferred from dot blot hybridization, it has been deduced that the 12 specimens may represent F1 or successive, presumably non-recombinant progenies, while the last four specimens may represent either F2 genotypes or backcrosses with O. pauciflora.

Se neotipifica Orchis entomantha Lex. (=Habenaria entomantha (Lex.) Lindl.), se lectotipifica H. acutiflora A. Rich. & Galeotti que se considera sinónimo de H. entomantha y se describen como nuevas para la ciencia Habenaria rosulifolia Espejo & López-Ferrari y H. gonzaleztamayoi García-Cruz, R. Jiménez & L. Sánchez, ambas procedentes del estado de Morelos.