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Table of contents Welcome ……………………………………………………………………………………………………… 3 Organization ………………………………………………………………………………………………… 4 Sponsors ……………………………………………………………………………………………………… 4 Scientific Programme ……………………………………………………………………..…………… 5 Abstracts: Session: WORLD-WIDE DISTRIBUTION, DIVERSITY ………………………………… 14 Session: CONSERVATION GENETICS ……………………………………….………………… 20 Session: MYCORRHIZA ……………………………………..………………………………………… 24 Session: POLLINATION ………………………………………………………………….…………… 29 Session: PHYLOGENETIC RELATIONSHIPS ………………………………………………… 36 Session: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY ………………. 40 Session: POPULATION DYNAMICS ……………………………………………………………… 46 Session: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION … 50 Session: TAXONOMY …………………………………………………………………………………… 56 Session: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION 61 Session: MODELLING AND DATA ANALYSIS ……………………………………………… 69 POSTERS ……………………………………………………………………………………………………… 74 2 Welcome The 4th International Congress on Orchid Conservation follows on from the successful congresses held in Perth (Western Australia) in 2001, Sarasota (Florida) in 2004 and San José (Costa Rica) in 2007. We are now experiencing dramatic changes in most aspects of our life. Modern technologies are being devised and deployed with increasing frequency; although they make our lives easier, their consequences, such as pollution of air, soil and water, greatly increase the pressure on our already stressed environment. To keep up with the rapidly growing human population, agricultural practices tend to be more and more intensive, which has a negative effect on biodiversity in agricultural landscape. Due to human activities, global temperature is increasing, with unpredictable consequences on ecosystems. Therefore, we have decided to dedicate this congress to „Orchids in a Changing World“. We hope that this theme will offer much scope for discussion. 3 Organization Scientific Board • • • • • • • • • • • • • • • • • • • • • • • James D. Ackerman, University of Puerto Rico, Rio Piedras, USA Richard M. Bateman, Royal Botanical Gardens, Kew, UK Mark W. Chase, Royal Botanical Gardens, Kew, UK Salvatore Cozzolino, Università degli Studi di Napoli Federico II, Naples, Italy Kingsley W. Dixon, Botanic Gardens and Parks Authority, Perth, Australia Michael F. Fay, Royal Botanical Gardens, Kew, UK Michael J. Hutchings, University of Sussex, UK Hans Jacquemyn, Katholieke Universiteit, Leuven, Belgium Pavel Kindlmann (Head), CzechGlobe & Charles University, Prague, Czech Republic Tiiu Kull, Estonian University of Life Sciences, Tartu, Estonia Marilyn H.S. Light, University of Ottawa, Ottawa,Canada Hong Liu, Florida International University, Miami, USA Giuseppe Pellegrino, University of Calabria, Rende, Italy Robert W. Pemberton, United States Department of Agriculture, Fort Lauderdale, USA Franco Pupulin, Jardín Botánico Lankester, Univ. Costa Rica, Cartago, Costa Rica Hanne N. Rasmussen, University of Copenhagen, Copenhagen, Denmark David L. Roberts, University of Kent, Kent, UK Raymond L. Tremblay, University of Puerto Rico, Humacao, Puerto Rico, USA Philip T. Seaton, Royal Botanical Gardens, Kew, UK Marc-Andre Selosse, Université de Montpellier, Montpellier, France Richard P. Shefferson, University of Georgia, Athens, USA Dennis Whigham, Smithsonian Environmental Research Center, Edgewater, USA Lawrence W. Zettler, Illinois College, Jacksonville, USA Local Organizing Committee • • • • • • • • • • Pavel Kindlmann (Head), CzechGlobe & Charles University, Prague Olga Ameixa, CzechGlobe, České Budějovice Bishnu Bhattarai, CzechGlobe, České Budějovice Karolína Černá, CzechGlobe, České Budějovice Prakash Kumar Paudel, CzechGlobe, České Budějovice Iva Schodelbauerová-Traxmandlová, CzechGlobe, České Budějovice Lenka Atexingerová, CzechGlobe, České Budějovice Michal Heřman, CzechGlobe, České Budějovice Luboš Matějíček, Charles University, Prague Věra Klestilová, CzechGlobe, České Budějovice Sponsors • CzechGlobe, Global Change Research Centre, Brno, Czech Republic • Biodiversity Research Centre, České Budějovice, Czech Republic • Institute for Environmental Studies, Faculty of Science, Charles University, Prague, Czech Republic • Orchid Specialist Group, IUCN 4 Monday, May 30 Session: WORLD-WIDE DISTRIBUTION, DIVERSITY Room: Orangery Moderators: James D. Ackerman and David L. Roberts 8:30-9:00 9:00-9:30 9:30-10:00 10:00-10:30 Opening session The natives fight back: endemic florivorous weevils attack flowers of an alien orchid and reduce plant reproductive success James D. Ackerman, Carlos Vega, Isamalish Espino, Ana A. Cuevas What determines worldwide orchid species richness? Pavel Kindlmann, James D. Ackerman, Raymond L. Tremblay, Dave Roberts, Iva Traxmandlová, Luboš Matějíček Flora Malesiana. Orchids of New Guinea Ed De Vogel, André Schuiteman, Jaap Vermeulen 10:30-11:00 Coffee break 11:00-11:30 Revealing the decline and expansion of orchids of NW European Russia Peter G. Efimov Session: MYCORRHIZA Room: Orangery Moderator: Richard P. Shefferson and Mélanie Roy 11:30-12:00 12:00-12:30 12:30-14:00 North American Orchid Conservation Center - a concept for a national focus on orchid conservation and restoration Dennis Whigham, Barbara Faust, Holly Shimizu, Gary Krupnick, Frank Clements, Melissa Mccormick, John Kress A biological comparison between non-photosynthetic (albino) and photosynthetic individuals in Cephalanthera damasonium suggests why green orchids rarely shift to mycoheterotrophy M. Roy, C. Gonneau, A. Rocheteau, D. Berveiller, B. Schatz, J.-C. Thomas, C. Damesin, M.-A. Selosse Lunch Session: CONSERVATION GENETICS Room: Regent's Hall Moderators: Mike Fay and Ovidiu Paun 14:00-14:30 14:30-15:00 Genetic variability in widespread Cephalanthera species with different reproductive systems Mike Fay Adaptation in Dactylorhiza allopolyploids - a story from beyond genetics Ovidiu Paun, Richard M. Bateman, Michael F. Fay, Mark W. Chase 5 15:00-15:30 Coffee break 15:30-16:00 Pollination in Ophrys: insights from population genetics and genomics 16:00-16:30 16:30-17:00 17:00-17:30 Helena C. Cotrim, Filipa A. Monteiro, Maria S. Pais Genetic differentiation among Gymnadenia conopsea and G. densiflora, and their conservation genetics in anthropogenic vs. seminatural habitats Walter Durka, Christiane Stark A comparative study of two leafless Vanilla species V. roscheri Rchb. and V. humblotii Rchb. f. from the South West Indian Ocean, using reproduction biology and genetic analyses Rodolphe Gigant, Laurence Humeau, Thierry Pailler, Alexandre De Bryun, Guillaume Viscardi, Brigitte Church, Valérie Guiot, Michel Grisoni, Pascale Besse Addressing the flow cytometric and morphological puzzle in the fragrant orchid (Gymnadenia conopsea agg.) Vladislav Čurn, Barbora Kubátová, Eva Krajníková, Kristina Kotlanová, Jana Rauchová, Pavel Trávníček, Jana Jersáková, Jan Suda Session: MYCORRHIZA Room: Orangery Moderator: Richard P. Shefferson and Mélanie Roy 15:00-15:30 Specialised symbioses and their role in orchid rarity 15:30-16:00 16:00-16:30 Ryan D. Phillips, Rod Peakall, Matthew D. Barrett, Kingsley W. Dixon And Stephen D. Hopper Coffee break Specificity of mycorrhizal associations in photosynthetic terrestrial orchids: an open question Lorenzo Pecoraro, Tiiu Kull 16:30-17:00 Stable isotopes as a tool to study nutrient exchange between orchids and fungi Gerhard Gebauer WELCOME PARTY 19:00-22:00 Restaurant „Knights of Rose“ 6 Tuesday, May 31 Session: POLLINATION Room: Orangery Moderators: Giuseppe Pellegrino and Robert W. Pemberton 9:00-9:30 Biotic resource needs of specialist orchid pollinators Robert W. Pemberton 9:30-10:00 Differences in pollen viability in Mediterranean orchids Giuseppe Pellegrino, Francesca Bellusci Alessia Luca, Aldo Musacchio 10:00-10:30 Challenges in pollinator restoration with considerations for orchid conservation Myles H.M. Menz, Ryan D. Phillips, Kingsley W. Dixon, Rod Peakall 10:30-11:00 11:00-11:30 Coffee break Orchids of the genus Broughtonia: a small Caribbean group with contrasting reproductive strategies Ángel Vale, Danny Rojas, Julio C. Álvarez, Luis Navarro, Yosvanis Acanda Artiga 11:30-12:00 Compatible and incompatible pollination of Dendrobium flowers 12:00-12:30 Saichol Ketsa, Kanjana Luangsuwalai Effects of light quality on vegetative growth, flowering and expression of FT-like gene in Phalaenopsis Kumala Dewi, Yohana, M. T. Astuti, Yekti A. Purwestri, Lila Natasaputra And Parmi 12:30-14:00 14:00-14:30 Lunch Role of stigmatic fluid on ovary growth of pollinated Dendrobium 'Pompadour' flowers Saichol Ketsa, Apinya Wisuttiamonkul Session: PHYLOGENETIC RELATIONSHIPS Room: Orangery Moderators: Salvatore Cozzolino and Mark W. Chase 14:30-15:00 An intriguing morphological variability of Platanthera s.l. Peter G. Efimov 15:00-15:30 15:30-16:00 16:00-16:30 Where have the hexaploids vanished? The role of breeding barriers in coexistence of Gymnadenia conopsea cytotypes Jana Jersáková, Stefan Dötterl, Sílvia Castro, Pavel Trávnícek, Jana Rauchová, Barbora Kubátová, Vladislav Curn, Jan Suda Coffee break Conservation significance of the renewed assessment of the molecular phylogenetic and mycorrhizal relationships in Pterostylidinae (Cranichideae) Mark Alwin Clements, Christopher Garry Howard, Joel Tupac Otero, Joseph Miller 7 Session: CONSERVATION GENETICS Room: Orangery Moderators: Mike Fay and Ovidiu Paun 16:30-17:00 The Orchid Conservation Strategy and Research in China Jing Cai, Yong Fu Yu, Sheng Li Zhao, Si Peng Lei, Lai Qiang Huang, Yi Bo Luo, Wen Chieh Tsai, Yu Yun Hsiao, Hong Hwa Chen, Xun Xu, Jun Wang, Jian Wang, Zhong Jian Liu Session: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Room: Regent's Hall Moderators: Marilyn H.S. Light and Dennis Whigham 17:00-17:30 Orchids of the cloud forests of southwestern Colombia and opportunities for their conservation Jorge E. Orejuela-Gartner Session: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Room: Regent's Hall Moderators: Kingsley W . Dixon and Hanne N. Rasmussen 14:00-14:30 Methods of studying field germination and seedling physiology: present potential and drawbacks Hanne N. Rasmussen 14:30-15:00 Seed propagation and re-introduction of the U.S. Federally endangered Hawaiian endemic, Platanthera holochila (Hbd.) Kral. (Orchidaceae) Lawrence W. Zettler, Erin M. Wood, Lynnaun J.A.N. Johnson, Anna K. Kirk, Steve Perlman 15:00-15:30 Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers Laura L. Corey, Lillian L. Moller-Jacobs, April Y. Ross, Lawrence W. Zettler, Larry W. Richardson 15:30-16:00 Coffee break 16:00-16:30 The embryology of Gastrodia nantoensis: embryo development Yung-I Lee 16:30-17:00 Limitations to orchid distribution inferred from in situ germination experiments Tamara Malinová, Jana Jersáková, Jakub Tešitel, Marc-André Selosse 8 Wednesday, June 1 WHOLE DAY TRIP 9:00-18:00 Sumava mountains – orchids and bark beetle, historical city of Cesky Krumlov OPTIONAL DINNER 19:00-22:00 Restaurant „Hunting lodge“ 9 Thursday, June 2 Session: POPULATION DYNAMICS Room: Orangery Moderators: Michael J. Hutchings and Tiiu Kull 9:00-9:30 9:30-10:00 A lifetime of sex and indolence: reflections on a 30-year-long relationship with the rare terrestrial orchid (Ophrys sphegodes) Mike Hutchings 25 years in life of Cypripedium calceolus genets Tiiu Kull Risk aversion and specificity in euglossine bee pollinated plants: do pollinator 10:00-10:30 population stabilities, bee abundances, and flowering phenologies matter? James D. Ackerman, David W. Roubik 10:30-11:00 Coffee break 11:00-11:30 Conservation of Isotria medeoloides, a tale of dormancy and fungi Melissa K. McCormick, Dennis F. Whigham, John P. O'neill 11:30-12:00 Vegetative dormancy, shrinkage, and fitness in two orchids Richard Shefferson 12:00-14:00 Lunch Session: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Room: Orangery Moderators: Philip T. Seaton and Lawrence W. Zettler 14:00-14:30 Seed quality of endangered terrestrial orchid species Coeloglossum viride (L.) Hartm in Estonia Mirjam Metsare, Kadri Tali 14:30-15:00 Mycorrhiza assisted conservation of two commercially important and endangered orchids of India: Dactylorhiza hatagirea D.Don (Soo) and Vanda coerulea Griff Ex Lindl. Simmi Aggarwal, C. Nirmala, Shanuja Beri, Sachin Rastogi, Alok Adholeya 15:00-15:30 Conservation through in vitro propagation and acclimatization of the endangered orchid Laelia speciosa Irene Ávila-Díaz, Martha Mireya Ortega-Loeza And Rafael Salgado-Garciglia 10 Session: TAXONOMY Room: Regent's Hall Moderator: Ed De Vogel 14:00-14:30 Colour-scent associations in a tropical orchid: three colours but two odours Thierry Pailler, Roxane Delle-Vedove, Nicolas Juillet, Jean-Marie Bessiere, Claude Grison, Nicolas Barthes, Laurent Dormont, Bertrand Schatz Sympodial and monopodial types of growth: why it's important in orchid biology and 14:30-15:00 ecology? Irina Tatarenko 15:00-15:30 Taxonomic uncertainty in species identifications and its potential consequences David L. Roberts 15:30-16:00 Coffee break 16:00-16:30 Scientific Committee meeting - Proceeedings 18:00-20:00 Poster setup in Parkhotel 20:00-21:00 Orchid Specialist Group meeting Room: Orangery Mike Fay 11 Friday, June 3 Session: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Room: Orangery Moderators: Marilyn H.S. Light and Dennis Whigham 9:00-9:30 Potential impact of insect herbivores on orchid conservation Marilyn H. S. Light, Michael MacConaill 9:30-10:00 Conservation and reintroduction of the native orchids of Singapore Tim Wing Yam, Peter Ang, Felicia Tay, Weijing Soh 10:00-10:30 Conservation recommendations from a large survey of French orchids Daniel Prat 10:30-11:00 Coffee break 11:00-11:30 Use and conservation of Vanilla planifolia J. in México Victor Manuel Salazar-Rojas, Braulio Edgar Herrera-Cabrera, Adriana Delgado-Alvarado, Marcos Soto-Hernández, Fernando Castillo González, Jorge Campos-Contreras, Juana Cervantes-Vargas Session: MODELLING AND DATA ANALYSIS Room: Orangery Moderator: Pavel Kindlmann 11:30-12:00 Darwin's "uniform green carpet": modelling the theoretical limits to the dispersal of orchid seeds David L. Roberts, Gil Bohrer 12:00-12:30 Population viability in Cypripedium calceolus in sites with different light regimes Anne Jäkäläniemi, Juha Tuomi 12:30-14:00 Lunch 14:00-14:30 Ex situ conservation of tropical African orchids Tariq Stévart, Bonaventure Sonké, Murielle Simo, Jean Philippe Biteau, Faustino De Oliveira, Steven Dessein, Claire Micheneau, Vincent Droissart 14:30-15:00 Closing session – future of IOCC, next venue POSTER SESSION and refreshments 16:00-17:30 Parkhotel 17:30-18:00 Poster removal - Parkhotel FAREWELL BANQUET 19:00-22:00 Restaurant „Knights of Rose“ 12 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 The natives fight back: endemic florivorous weevils attack flowers of an alien orchid and reduce plant reproductive success James D. ACKERMAN1,2, Carlos VEGA1, Isamalish ESPINO1, Ana A. CUEVAS1,2 1 Department of Biology, Faculty of Natural Sciences, University of Puerto Rico, PO Box 70377, San Juan PR 009368377, USA; email: [email protected] 2 Center for Applied Tropical Ecology and Conservation, PO Box 23341, University of Puerto Rico, San Juan, PR 00931-3341, USA Invasive plant species are often generalists in their mutualistic interactions, specialists on widespread taxa, or are adept at self-pollination, or vegetative reproduction. Their invasiveness may be due to release from enemies, such as competitors, herbivores or seed predators. Or they may be superior competitors as is often suggested for plants that invade islands. From a single naturalized population 30 years ago, the autogamous Spathoglottis plicata has spread across the island of Puerto Rico. Inflorescences, though, often become deformed and abort flowers and fruits. An orchid-specialist, endemic florivorous weevil, Stethobaris polita, attacks the flowers and reduces reproductive success. Ants that forage at extrafloral nectaries sometimes attack the weevils, but have no effect on plant reproductive success. Plants protected from weevils had 4-6 higher fruit set than those treatments that allowed weevil access. Weevil abundance is weakly density dependent. The frequency of weevils is positively associated with at the number of open flowers on an inflorescence, and the more flowers in the neighborhood, the more weevils that are observed. White and pink morphs exist, and the weevils prefer the pink morphs both in the field and in laboratory choice experiments. Although S. plicata encounters new enemies in Puerto Rico and suffers reduced reproductive success, it remains invasive. With weevil preference for pink flowers, we expect white-flowered morphs to become relatively more frequent until balanced by density dependent effects. 13 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 What determines worldwide orchid species richness? Pavel KINDLMANN1,3, James D. ACKERMAN2, Raymond L. TREMBLAY2, Iva TRAXMANDLOVÁ1, Luboš MATĚJÍČEK3 1) Department of Biodiversity Research, Global Change Research Centre AS CR, Brno, Czech Republic; email: [email protected] 2) Department of Biology, University of Puerto Rico, 100 Carr. 908, Humacao, Puerto Rico, 00791-4300. 3) Institute for Environmental Studies, Faculty of Science, Charles University, Prague, Czech Republic The dependence of the number of orchid species in a location on its area, and habitat heterogeneity has been studied previously by Ackerman on a data set from the Caribbean archipelago. The relative importance of latitude and that of the total size of a location vs. the size of protected areas in the location as factors determining the number of orchid species in that location was studied by Schödelbauerová et al. The importance of connectivity with other habitats for determination of the number of orchid species in a location was, however, never tested. Here we reanalyze the dataset on the numbers of orchid species in the Caribbean archipelago that was previously used by Ackerman and add the connectivity to the analysis. We first fit the relationship between the number of orchid species in each island and the area of this island by a power function. Then we correlate the residuals with habitat heterogeneity and negative exponential connectivity with other potential orchid habitats using a stepwise linear regression. Habitat heterogeneity is measured (i) as the altitude of the highest point in the island, (ii) as the mean slope in the island, measured on 1x1km pixel scale. We then discuss the relative importance of individual factors. 14 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 Flora Malesiana. Orchids of New Guinea Ed de VOGEL¹, André SCHUITEMAN² and Jaap VERMEULEN¹ ¹ Netherlands Centre for Biodiversity Naturalis (section NHN), Leiden University, the Netherlands ² Royal Botanic Gardens, Kew, Great Britain This project started in 1996 with the production of a Demo CD using Linnaeus 2 from ETI, Amsterdam. First author André Schuiteman was full time implementer; Ed de Vogel coordinated and contributed part time; Jaap Vermeulen was co-author of the last CD on Bulbophyllum. In 2001-2010 six CD-ROM’s were produced, treating all recognized 2716 species and 65 taxa of lower rank. The last CD appeared exactly 100 year after the first installment of Schlechter’s Die Orchideeen von Deutsch Neu Guinea. The actual project lasted 13 years. All texts including the descriptions are in English; Latin descriptions of c. 2000 species known only from the type(s) were translated. Illustrations comprise almost 4500 drawings, most of types described by R. Schlechter and J.J. Smith. More than 3450 digital photographs are present, including 1000 sets of Peter Jongejan stereo images in Vol. 6, to be viewed in 3D with a stereo viewer. Volume 6 covers 635 Bulbophyllum species in 33 sections. The division of Bulbophyllum in sections, including some new ones, was revised by Vermeulen. The CD’s contain more than 2400 distribution maps; Vol 2 features a limited number of hand-made maps; subsequent CD’s have digitally constructed maps for all species. and more than 2050 photographs of types are included. Many one month inventory trips were made for this project: in Indonesian New Guinea (3), in PNG (12), acquiring 4500 live orchids for Port Moresby Botanical Garden and Leiden Hortus Botanicus, adding many distribution data and images of species so far never photographed, and some 250 undescribed species. The project ran almost entirely on funding from the Cheng Kim Loke Foundation, the Pacific Science Foundation, and Conservation International. Serious impediments were caused by the 9-11 incident and changing exchange rates. A book with a selection of c. 700 species of New Guinea orchids is published in 2011 just before WOC 25, giving an example for every orchid genus and section in New Guinea, supplemented by species of horticultural and biological importance. A presentation will be given of the major features of the Bulbophyllum CD-ROM. 15 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 Orchids of the cloud forests of southwestern Colombia and opportunities for their conservation Jorge E. OREJUELA-GARTNER Full Professor Autonomous University of the West Environmental Study Group for Sustainable Development-GEADES Director Cali Botanical Garden, Cali, Colombia; email: [email protected] Orchids are fascinating examples of the natural world and key elements in the conservation of biodiversity. Colombia may well be the richest country in the world in orchid species. Nonetheless the orchid rich areas of the country are seriously threatened by human activities such as deforestation, illegal trade of valued species and more recently global warming. Based on inventories made in several cloud forests of southwestern Colombia, but mainly in the La Planada nature reserve in Nariño, the following results appear: A total of nearly 400 species of 85 genera are reported, 30% of the species found are endemics, seventeen new records for Colombia, and twenty species new to science are reported from these Andean forests in recent years. More than fifteen genera are recognized for their economic value and twenty-five additional have potential for sustainable economic use. Unfortunately there are twentyfive species reported in the Red Data Book for Colombia, which was based only on the analysis of eighteen genera. There are many opportunities for the conservation of ecosystems rich in orchids through habitat protection, increased knowledge of species and their distribution, coordination of in situ and ex situ efforts and the involvement of communities in sustainable rural tourism projects. The value of conservation programs along altitudinal gradients in the Andes is emphasized given the key links of orchid conservation efforts with watershed protection, the maintenance of environmental goods and services and meeting the goals of adaptation to climate change. 16 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 The Orchidaceae family species in the Northern Urals (Pechoro-Ilychsky Biosphere Reserve) Irina KIRILLOVA Russia, 167982, Komi, Syktyvkar, Kommunisticheskaya, 28; Institute of Biology, Komi Scientific Centre, Ural Branch, Russian Academy of Science; e-mail: [email protected] The Pechoro-Ilychsky Biosphere Reserve is located in southeast of the Komi Republic between 62° and 63° northern latitude in middle and northern taiga subzones. Its territory covers a part of western macroslope of the Northern Ural Mountains and adjoins the Pechora lowland. The total area of the protected territory numbers 721.300 hectares. In direction from west to east the territory of the reserve is divided into three landscape areas: plains, foothills and mountains. Orchid flora in the Pechoro-Ilychsky reserve is counted 20 species from 12 genera. According to the classification by Dressler (1993), the orchids of the reserve belong to 6 tribes and 4 subfamilies of the family Orchidaceae. High diversity of orchids can be explained by extensive area of this territory, low human influences and significant diversity of ecotopes. The most orchid species occur in spring bogs and limestones within the foothills region of the Northern Urals. The half of orchid species is included in the Red Data Book of the Komi republic (2009) and three species are included in the Red Data Book of Russia (2008). The age structure, number and density of orchid populations have been studied in the Pechoro-Ilychsky reserve during 10 years. The most populations were found to be normal and full-member. Within the age spectrum of species mainly immature or generative less adult vegetative individuals were dominated. 17 SESSION: WORLD-WIDE DISTRIBUTION, DIVERSITY Monday, May 30 Revealing the decline and expansion of orchids of NW European Russia Peter G. EFIMOV1 1 Herbarium, Komarov Botanical Institute of the Russian Academy of Sciences, Prof. Popov str. 2, Saint-Petersburg, 192288, Russia An approach to reveal the dynamics of the taxa for incompletely investigated territories was developed to study the decline/expansion rates of the orchids of NW European Russia (Leningrad, Novgorod and Pskov Regions, an area approx. 195000 sq. km.). The method was based on the comparison between the numbers of the grid cells where a certain taxon was recorded in various time intervals using specially designed software. More than 9000 records representing data as completely as possible were used, but the territory remained insufficiently and unevenly studied both spatially and over time. The study revealed the statistically significant (p<0,01) decrease for Coeloglossum viride, Corallorhiza trifida, Cypripedium calceolus, Gymnadenia conopsea, Herminium monorchis, Malaxis monophyllos, Neotinea ustulata and Orchis militaris and the similarly significant increase for Dactylorhiza baltica, D. fuchsii and Platanthera chlorantha. The trend was inconstant over time for several taxa. Of them, Gymnadenia conopsea displayed significant decline only since the middle of XX century, and Orchis militaris and Epipactis atrorubens, displayed significant extinction only in the end of XIX and beginning of XX century. The reasons for the revealed dynamics are discussed. Parallels between the dynamics of orchids and land use in different periods of time in Russia are provided. 18 SESSION: CONSERVATION GENETICS Monday, May 30 Adaptation in Dactylorhiza allopolyploids – a story from beyond genetics Ovidiu PAUN1,2, Richard M. BATEMAN2, Michael F. FAY2, Mark W. CHASE2 1 Department of Systematics and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; email: [email protected] 2 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, U.K. Hybridization and polyploidy are potent forces that have regularly stimulated plant evolution and adaptation. Dactylorhiza majalis s.s., D. traunsteineri s.l. and D. ebudensis are three allopolyploid species of a polyploid complex formed through unidirectional hybridization between the widespread diploids D. fuchsii and D. incarnata. Differing considerably in geographical extent and ecological tolerance, the three allopolyploids together provide a useful system to explore genomic responses to allopolyploidization and reveal their role in adaptation to contrasting environments. Analyses of gene expression show a significant increase in number of gene variants expressed in the allopolyploid lineages (including novel alleles), providing clear evidence of increased biological complexity following allopolyploidization. The epigenetic variation released by genome doubling has been restructured in species-specific patterns that reflect their recent evolutionary history, and have an impact on their ecology and evolution, hundreds of generations after their formation. The stable epigenetic divergence between the allopolyploids is largely responsible for persistent ecological differences, which then set the stage for species-specific genetic patterns to accumulate in response to further selection and/or drift. Using NGS technologies, we are currently comparing in detail mRNA expression levels between the allopolyploids and their parents. During this investigation we have been constructing reference transcriptomes for the two diploid parents, which contribute important genomic resources for Orchidaceae in general. In addition to stabilizing the allopolyploid genome, genetic and epigenetic alterations are key determinants of adaptive success of the new polyploid species after recurrent allopolyploidization events, potentially triggering reproductive isolation between the resulting lineages. 19 SESSION: CONSERVATION GENETICS Monday, May 30 Pollination in Ophrys: insights from population genetics and genomics Helena C. COTRIM1,2, Filipa A. MONTEIRO2 Maria S. PAIS2 1 present address: University of Lisbon. National Museum of Natural History, Botanical Garden. R. Escola Politécnica 58, 1250-102 Lisboa, Portugal / and Faculty of Sciences, Centre for Environmental Biology (CBA); email: [email protected] 2 University of Lisbon, Faculty of Sciences, Center for Biodiversity, Functional & Integrative Genomics (BioFIG), Plant Systems Biology Laboratory. Edificio ICAT. Campo Grande, 1749- 016 Lisboa, Portugal One of the most fascinating pollination mechanisms is sexual deception a process nearly exclusive to orchids and particularly studied in Ophrys. Pollination is a key step that that triggers tactile, visual or pheromone cues to the next orchid generation. A dual outlook of Ophrys pollination is presented: 1) a population genetics view of gene flow within a small set of species and 2) a one species genomic pollination insight. A small set of co-occurring Ophrys species (Pseudophrys sect) was studied by plastidial and nuclear neutral molecular markers allowing to address the degree of reproductive isolation and premating barriers. Population genetics illustrates the value of premating barriers where interspecific gene flow occurs increasing genetic diversity. Labella transcriptome of in-field O. fusca unpollinated and pollinated flowers was analysed at 2 and 4 days after pollination (DAP). Statistical analysis revealed 140 unigenes differentially expressed. Transcripts were grouped into functional categories after putative function assignment. Preliminary results reveals that main gene expression changes are related to secondary metabolism, energy processes, stress and defense-responses. Results will allow getting an overview on the regulation mechanisms after pollination and give a valuable contribute to the knowledge on the early post pollination mechanisms in Ophrys and ultimately in orchid biology. 20 SESSION: CONSERVATION GENETICS Monday, May 30 Genetic differentiation among Gymnadenia conopsea and G. densiflora, and their conservation genetics in anthropogenic vs. seminatural habitats Walter DURKA1, Christiane STARK1 1 Helmholtz-Centre for Environmental Research-UFZ, Department of Community Ecology, Theodor-Lieser-Str. 4, 06120 Halle, Germany Gymnadenia conopsea and G. densiflora are two taxa with considerable variability, which complicates their identification and subsequent management. We assessed ITS sequences, microsatellite variation and chromosome numbers on the European and morphology in Germany. ITS analysis revealed strong divergence between the taxa indicating that the two taxa are no sister species. This was confirmed by microsatellite data. Chromosome numbers showed that G. conopsea was either diploid or tetraploid, while G. densiflora was diploid throughout. Despite significant differences in the mean values of diagnostic morphological characters, discriminant analysis showed that morphological variability is substantial aggravating assignment on the individual level. Non-reclaimed successional sites in former brown coal mines or quarries may represent habitat relevant for conservation. However, because such sites may have been colonized by only a few founders, it is unkown whether such populations are comparable to natural populations with respect to genetic variation, reproductive fitness and thus conservation value. We assessed genetic variation and reproduction in 32 populations of G. conopsea and G. densiflora in seminatural grassland and anthropogenic habitats. Our results revealed reduced genetic diversity, increased inbreeding and lowered fruit set for the populations in the post-mining area, whereas no such effects were detected for the quarry populations. The reduced genetic diversity had no effect on the general plant performance. The results suggest that anthropogenic habitats may comprise valuable habitat for endangered species but also emphasize the importance of population connectivity in order to provide gene flow and prevent founder effects. 21 SESSION: CONSERVATION GENETICS Monday, May 30 A comparative study of two leafless Vanilla species V. roscheri Rchb. and V. humblotii Rchb. f. from the South West Indian Ocean, using reproduction biology and genetic analyses Rodolphe GIGANT1,2, Laurence HUMEAU2, Thierry PAILLER2, Alexandre DE BRYUN1,2, Guillaume VISCARDI3, Brigitte CHURCH4, Valérie GUIOT3, Michel GRISONI1, Pascale BESSE2 1 Cirad, UMR C53 PVBMT Université de la Réunion-Cirad, 7 chemin de l’Irat, Ligne Paradis 97410 Saint Pierre, île de la Réunion, France 2 Université de la Réunion, UMR C53 PVBMT Université de la Réunion-Cirad, 15 avenue René Cassin, BP7151, 97715 Saint Denis messag cedex 9, île de la Réunion, France 3 Conservatoire Botanique National de Mascarin, Antenne de Mayotte, Route Nationale 2, 97670 Coconi, Mayotte, France 4 Ezemvelo KwaZulu Natal Wildlife, P.O. Box 13053 Cascades, 3202 KwaZulu-Natal South Africa Within the Vanilla genus, a primitive lineage in the Orchidaceae family, the leafless species V. roscheri, endemic to the African East Coast and nearby islands, and V. humblotii, endemic to the Comoros Archipelago, are threatened by habitat destruction and human pressures. We combined crossing pollination experiments, field observations and microsatellite markers analyses to study the reproduction biology as well as population genetics, on populations of V. roscheri near Lake Sibaya (iSimangaliso Wetland Park, South Africa) and V. humblotii from Mayotte Island (France). The two species have a pollination-dependant sexual reproduction associated with vegetative propagation. Our results appeared paradoxical at first sight, with a high fruit set but a very limited genetic diversity revealed for V. roscheri, and, a much lower fruitset but a relatively high genetic diversity for V. humblotii. The high fruit set observed in V. roscheri could be due to the situation of the population within a large nature area with a well-preserved fauna of pollinators. Because of the presence of anarchic cultures and degraded forest systematically close to the populations of V. humblotii, the situation is completely different in Mayotte with certainly less pollinators than originally. The poor genetic diversity revealed within the populations of V. roscheri, suggests a founder effect (bottleneck), in accordance with the southernmost range distribution of this population with regards to the species general geographical distribution. In Mayotte, forest fragmentation led to the division of a former large and diverse V. humblotii population into several well-differentiated small subpopulations, endangered by their low population size and the habitat fragmentation. 22 SESSION: MYCORRHIZA Monday, May 30 A biological comparison between non-photosynthetic (albino) and photosynthetic individuals in Cephalanthera damasonium suggests why green orchids rarely shift to mycoheterotrophy M. ROY1,2, C. GONNEAU1, A. ROCHETEAU1, D. BERVEILLER3, B. SCHATZ1, J.-C. THOMAS4, C. DAMESIN3, M.-A. SELOSSE1 1 Centre d’Ecologie Fonctionnelle et Evolutive, UMR 5175, CNRS 1919 route de Mende, 34093 Montpellier cedex 5, France 2 Present address: Université de Toulouse; UPS; UMR5174 EDB (Laboratoire Evolution et Diversité Biologique); 118 route de Narbonne, F-31062 Toulouse, France 3 Laboratoire 4 d'Ecologie, Systématique et Evolution, Université Paris-Sud, Bât. 362, 91 405 Orsay cedex, France Département de Biologie, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris, France Heterotrophy relying on fungi as an organic carbon source (mycoheterotrophy) arose several times by plants, and even more frequently among orchids than among any other angiosperm family. Nevertheless, only 400 mycoheterotrophic plant species are described, and only 1% of orchids species present such a habit. Interestingly, photosynthetic orchids closely related to mycoheterotrophic species can partly rely on a fungal carbon source (so-called mixotrophic), or even completely for some albino individuals. Such albino punctually appear in photosynthetic populations but they do survive for several years and are able to reproduce. Nevertheless, such albino variants never invade populations, and are generally rare within populations, occurring in rare populations. We investigated a mixed population of albino and green variants of Cephalanthera damasonium for several years to compare their relative fitness and determine what conditions could influence albino individuals’ frequency and then select complete mycoheterotrophy from a mixotrophic species. When comparing several fitness estimators, albino always tend to have a lower fitness than green individuals. Our result underline many physiological limitations leading to albino shoot loss during fruiting season. Nevertheless, the albino “phenotype” could still be maintained by mutation or reproduction with green variants. Even if albino individuals are limited by their physiology or other ecological parameters, they permit to underline what traits or in what conditions a shift to complete mycoheterotrophy can be selected. Such traits or ecological conditions can be compared with what was selected in mycoheterotrophic species. We discuss our results in the light of mycoheterotrophy ecology and evolution by plants. 23 SESSION: MYCORRHIZA Monday, May 30 Specialised symbioses and their role in orchid rarity Ryan D. PHILLIPS1, Rod PEAKALL1, Matthew D. BARRETT23, Kingsley W. DIXON23 and Stephen D. HOPPER43 1 Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia 2 Kings Park and Botanic Garden, The Botanic Garden and Parks Authority, West Perth, 6005, Western Australia 3 School of Plant Biology, The University of Western Australia, Nedlands, 6009, Western Australia 4 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK The role of specialized plant-pollinator and plant-mycorrhiza relationships in plant species rarity is poorly understood and has rarely been tested. We tested the role of these symbioses in rarity in Drakaea, a genus endemic to south-western Australia, where five of the nine extant species are rare and endangered. Drakaea are pollinated by sexual deception of male thynnine wasps and use a specific morphotype of Tulasnella mycorrhizal fungus. Using sequencing of the ITS region and germination experiments, all Drakaea were shown to utilize a single, widespread species of Tulasnella. Further, in situ baiting demonstrated that germination frequency in both common and rare Drakaea was similar to that exhibited by co-occurring orchid genera. Baiting for pollinators and DNA barcoding demonstrated that most Drakaea species rely on a single pollinator species. Comparative pollination rate and pollinator abundance data was used to test for general trends between common and rare Drakaea. Fruit set was high in most species but low in a subset of rare species. Baiting for pollinators across south-western Australia demonstrated that rare species tend to have rare pollinators that can limit their expansion into suitable habitat. At regional scales, some pollinators of rare species were more widespread than the orchid demonstrating that they are not limiting their distribution. While there are trends within Drakaea, considering the ecology of each species demonstrates that the causes of rarity are idiosyncratic. Species rarity appears to result from different combinations of the abundance of pollinators, habitat specialisation and the evolutionary history of each Drakaea species. 24 SESSION: MYCORRHIZA Monday, May 30 Specificity of mycorrhizal associations in photosynthetic terrestrial orchids: an open question Lorenzo PECORARO, Tiiu KULL Estonian University of Life Sciences, Riia St. 181, Tartu 51014, Estonia Mycorrhizae have a pivotal impact on orchid growth and metabolism in several plant life stages. Molecular methods, applied over the last few years, have allowed to improve the knowledge of these intimate symbioses between fungi and orchids, but there is still more to be learned about their physiology and the degree of mycorrhizal specificity, especially as far as the photosynthetic terrestrial orchid species are concerned. Some photosynthetic orchids, such as Cypripedium sp., Cephalanthera sp. and Dipodium hamiltonianum, even when sampled over a wide range, have a single dominant mycorrhizal fungus. On the contrary, some other species, such as Orchis militaris, O. purpurea and O. simia are characterized by a low level of fungal specificity. We have investigated the diversity of mycorrhizal fungi in eight green orchid species (Cephalanthera damasonium, C. longifolia, Himantoglossum hircinum subsp. adriaticum, Ophrys bertolonii, Orchis morio, O. pauciflora, O. provincialis and O. tridentata) growing in three protected areas of Central Italy, by means of morphological and molecular methods. Phylogenetic analysis revealed that orchids were predominantly associated with fungi belonging to Tulasnella, Ceratobasidium and Sebacina and indicated an high degree of mycorrhizal specificity. However, fungal species not commonly collected from orchid roots (Bjerkandera adusta, Phlebia acerina, Schizophyllum commune, Terfezia sp.) were also identified, but their trophic role is unclear. The identification of mycorrhizal symbionts and the deep analysis of their trophic relationship with orchids will be of critical importance to understanding the ecology of these delicate plants and to conservation efforts. 25 SESSION: MYCORRHIZA Monday, May 30 Stable isotopes as a tool to study nutrient exchange between orchids and fungi Gerhard GEBAUER1 1 BayCEER – Laboratory of Isotope Biogeochemistry, University of Bayreuth, 95440 Bayreuth, Germany In the mycorrhizal symbiosis, plants usually provide their fungal partners with carbohydrates from photosynthesis and receive mineral nutrients, which are more efficiently taken up from the soil through the fungal mycelium. Within the Orchidaceae this mutualistic arrangement has been modified in different directions. Based on carbon isotope signatures and the identity of their mycorrhizal associates, it is now clear that terrestrial orchids can utilize at least four nutritional pathways: (1) Autotrophy, where green orchids have carbon isotope signatures indistinguishable from surrounding autotrophs and mainly associate with fungi of the polyphyletic Rhizoctonia group. (2) Obligate mycoheterotrophy, where orchids have lost the ability to photosynthesize, are specialized on either ectomycorrhizal or wood-rotting saprotrophic fungi, and are enriched in 13C similar to their host fungi. (3) Partial mycoheterotrophy, where green orchids have carbon isotope signatures intermediate between those of autotrophs and mycoheterotrophs and associate with fungi forming ectomycorrhizae. (4) 13C depletion in another group of green orchids from the tribes Orchideae and Cranichideae mainly associated with ceratobasidioid and tullasnelloid fungi indicates a carbon transfer from the plants back to their mycorrhizal fungi. This presentation provides evidence that the nutrition of green orchids belonging to the groups (2) and (4) is not a static mode but a flexible mechanism related to the local light climate and to leaf chlorophyll concentrations. Detailed mechanistic knowledge about the nutrient exchange between orchids and fungi, fungal partners involved in this matter exchange and underlying physiological mechanisms is of essential importance for improved orchid conservation concepts. 26 SESSION: MYCORRHIZA Monday, May 30 North American Orchid Conservation Center – a concept for a national focus on orchid conservation and restoration Dennis WHIGHAM1, Barbara FAUST2, Holly SHIMIZU3, Gary KRUPNICK4, Frank CLEMENTS5, Melissa MCCORMICK1, John KRESS4 1 Smithsonian Environmental Research Center, Edgewater, MD 21037, USA 2 Smithsonian Gardens, Washington, DC 20013, USA 3 U.S. Botanic Garden, Washington, DC 20024, USA 4 National Museum of Natural History, Smithsonian Institution, Washington, DC 20013, USA 5 National Zoological Park, Smithsonian Institution, Washington, DC 20013, USA Many orchid species are known to be rare and threatened with extinction and international efforts (e.g. CITES) have focused on the illegal trade of orchids and the conservation of threatened species. No single organization in North America focuses on the conservation and restoration of native orchids and no single entity is devoted to educating the public about the importance of orchids in an evolutionary and ecological context. In addition, organizations mandated to identify and protect threatened and endangered orchids on public lands rely almost completely on habitat conservation as a management tool. While habitat management is important, ecological attributes of orchids (e.g., obligatory relationships between orchids and fungi) dictate that it alone will not result in successful conservation or restoration. Units within the Smithsonian have joined the U.S. Botanic Garden to obtain a grant to plan for the establishment of the North American Orchid Conservation Center (NAOCC). Research, education and outreach will be key components of NAOCC and the education and outreach components will have a national focus that will include botanic gardens that will conserve orchid biodiversity through collections of seeds, mycorrhizal fungi and living collections representative of regional genetic diversity. This presentation will focus on the status of the effort to design and implement NAOCC and encourage discussions among Congress participants to enhance the effort to fulfill the NAOCC concept. 27 SESSION: POLLINATION Tuesday, May 31 Biotic resource needs of specialist orchid pollinators Robert W. PEMBERTON Research Associate, Florida Museum of Natural History and Fairchild Tropical Botanical Garden, C/O 2121 SW 28th Terrace, Fort Lauderdale, Florida 33312, USA; email: [email protected] Orchid pollinators have highly varied life histories with complex biotic resource requirements, about which we have limited knowledge. Among the specialist orchid pollinators are insect predators and parasitoids with specific prey types such as aphids and subterranean scarab larvae; oligolectic bees that collect pollen from limited sources such as bellflowers; euglossine bees and butterflies that collect particular plant chemicals for reproduction and self defense; oil-collecting bees that provision their brood cells with floral oils from a few plants; bees that collect rare floral resins to construct their nest and brood cells; moths and butterflies that require specific larval host plants; mosquitoes and horse flies that need blood; and fungus gnats and carrion flies tied to fungi and dead animals. Loss of critical biotic resources and relationships can reduce the abundance of orchid pollinators and/or their effectiveness. Protection of large, plant rich, pesticide-free orchid habitats is key to conserving essential pollinator resources. 28 SESSION: POLLINATION Tuesday, May 31 Differences in pollen viability in Mediterranean orchids Giuseppe PELLEGRINO, Francesca BELLUSCI Alessia LUCA, Aldo MUSACCHIO Dept. of Ecology, University of Calabria, Rende Italy Current research involving pollen viability has been evaluated in a relatively low number of orchid species. In the present study, we focused on 6 related Mediterranean orchid genera (Anacamptis, Barlia, Orchis, Dactylorhiza, Ophrys and Serapias) that are characterised by different types of deceptive pollination. The aim of our study was to examine the pollen viability duration of several members of the deceptive EuroMediterranean orchid guild, which is particularly associated with infrequent pollinator visits. We evaluated the in vitro germination ability of increasingly aged pollinaria of 10 food, 7 sexually and 3 shelter deceptive species. We have also performed pollination experiments on 2 food, one sexually and one shelter deceptive species and checked the percentage of embryonate seeds derived from the increasingly aged pollinaria. All of the examined species showed long-term viability that ranged from 8 to 35 days. Species with the same deceptive pollination strategies exhibited the same pollen viability trends. Interestingly, pollen viabilities of species groups with different deception types have shown significant differences, with sexually- and shelter- deceptive species exhibiting a lower lifespan than food deceptive ones. In addition early flowering species exhibited a prolonged capacity for in vitro germination, fertilization and embryonate seed development than late flowering species. This study confirms the prolonged germination and fertilization capacities of orchid pollinaria, demonstrating a clear relationship between pollen viability and pollination system. We propose that this relationship is attributed to the different types of reproductive barriers, pre- or post-zygotic, that characterize Ophrys and Serapias and the food deceptive species, respectively. 29 SESSION: POLLINATION Tuesday, May 31 Challenges in pollinator restoration with considerations for orchid conservation Myles H.M. MENZ1, 2, 3, Ryan D. PHILLIPS1, 2, 3, Kingsley W. DIXON1, 2 and Rod PEAKALL3 1 Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, Fraser Avenue, West Perth 6005, Australia 2 School of Plant Biology, The University of Western Australia, Crawley 6009, Australia 3 Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia Over 90% of the worlds flowering plants rely on biotic pollination for reproduction. In some habitats, where pollinators may have been lost, or have greatly reduced population sizes, efforts may need to be implemented to restore pollinators. In these cases, there are a number of considerations that need to be taken into account prior to initiating a restoration project. These considerations include: (i) structural characteristics of plant–pollinator networks; (ii) selecting plants to restore pollinators; (iii) ecological requirements of pollinators; (iv) landscape structure and facilitation of pollinator movement; and (v) variation among biogeographic regions. The depth to which each of these needs to be understood is reliant on the expected outcomes of the restoration project. Orchids, as one of the worlds’ most diverse plant families, display many different pollination strategies spanning from generalised food reward and food deception, to the highly specialised sexual deception. When considering orchid conservation, intimate knowledge of the ecological requirements of the pollinators will be essential. Restoration challenges may be particularly acute when dealing with species that are typically reliant on one or only a few pollinator species. This talk will first explore the general principles of pollinator restoration before evaluating the issues for pollinator restoration of a rare sexually deceptive orchid. 30 SESSION: POLLINATION Tuesday, May 31 Orchids of the genus Broughtonia: a small Caribbean group with contrasting reproductive strategies Ángel VALE1, Danny ROJAS1, Julio C. ÁLVAREZ2, Luis NAVARRO, Yosvanis Acanda ARTIGA 1 Departamento de Biología Vegetal, Universidad de Vigo, Campus Lagoas-Marcosende, 36200-Vigo, Spain. 2 Facultad de Biología, Universidad de La Habana, Calle 25 e/ J e I, Vedado, CP 10400, La Habana, Cuba. The genus Broughtonia s. l. comprises six species that occur in the Greater Antilles and the Bahamas. The phylogeny of the group confirms three clades each circumscribed to a group of islands. Nevertheless floral traits have converged in independent lineages at different islands. This suggests independent adaptations to bee-wasp or hummingbird pollination, probably linked to the absence/presence of intrafloral nectar. Deceptive pollination is ancestral in Broughtonia while the nectariferous habit arose twice and independently within the Cuban and Jamaican clades. Furthermore, the nectar of the Cuban B. ortgiesiana is similar to the nectar of bird-pollinated plants, while the nectar from Jamaican orchids is more diluted. Considering hummingbird energy requirements and foraging behavior, we propose the Jamaican orchids represent a case of quasi-deceptive pollination. Additional data about the reproductive consequences of deception, quasi-deception and rewarding are presented and discussed. Other evolutionary aspects of Broughtonia pollination are compared at the island level. The three Cuban Broughtonia present clear premating reproductive isolation barriers (including temporal, spatial and behavioral) and no hybrids has been reported. Jamaican species overlap their blooming season and distribution, forming a natural hybrid zone which apparently occupies an extensive area. Other aspects of this phenomenon in progress are addressed, with an emphasis on the importance of hummingbirds in the inter-taxa flow of pollen. Natural hybrids between B. domingensis (the only Broughtonia that occurs in Hispaniola) and a co-occurring species of Psychilis reinforce this new and complex hybrid scenario within the Broughtonia Alliance which requires to be explored. 31 SESSION: POLLINATION Tuesday, May 31 Retrospective spatial analysis and the concept of chance as tools for understanding orchid pollination Anne DAMON1, Javier VALLE-MORA1 1 El Colegio de la Frontera Sur (ECOSUR). Carretera Aeropuerto Antiguo km. 2.5, Tapachula, Chiapas, México. The pollination strategy of many orchid species are unknown and are difficult to study, due to the inaccessibility of epiphytes, rare species with small scattered populations, rare and ephemeral pollinator visits, and deceit strategies that limit pollinator interest. Many orchid species are consistently seen to have low levels of fruit set but the possibility of increasing fruit set manually does not necessarily imply that the natural process of pollination is deficient. Furthermore, although the pollinators of many orchid species are relatively frequent, percentage fruit set remains low. The suggestion that orchids are constantly struggling against pollinator limitation is an unlikely scenario for this highly diverse and successful plant family. We suggest that orchids have specialized in maximizing seed production resulting from the naturally few, ‘lucky’, rare or chance encounters between flower and pollinator. Chance, as a biological concept, could be conceived as a situation in which there is no factor, or combination of factors, that increases the likelihood that an event will take place, and no way of predicting that event, which is so rare in space and time that it is statistically improbable. We found that the spatial presentation of orchid flowers in terms of aggregation, spatial distribution, spatial relationship to the host plant, visibility and height above the ground all have a very limited effect on the visitation rate of the euglossine bee pollinators of the miniature twig epiphytes Notylia barkeri Lindl.and the Centris bee pollinators of Erycina crista-galli (Rchb.f.) N.H.Williams and M.W.Chase. We discuss these and other interesting and novel results. 32 SESSION: POLLINATION Tuesday, May 31 Compatible and incompatible pollination of Dendrobium flowers Saichol KETSA1 and Kanjana LUANGSUWALAI2 1 Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand 2 Agriculture Program, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Bangkok 10600, Thailand Open flowers of Dendrobium ‘Miss Teen’ (or ‘Kenny’) were cross pollinated by placing pollinia from five cultivars of Dendrobium namely ‘Miss Teen’, ‘Karen’, ‘Sakura’, ‘Willie’, and ‘Pompadour’. The pollinated flowers were held in distilled water at ambient temperature. The characteristics of ethylene production and post-pollination development were monitored. The pollinated flowers were devided into two groups. The first group was compatible pollination, which consisted of flowers pollinated by pollinia from ‘Sakura’, ‘Willie’, and ‘Pompadour’. In this group, the premature senescence of perianth took place within a day. The second group was incompatible pollination, which consisted of flowers pollinated by ‘Karen’ and ‘Miss Teen’ pollinia. Incompatible pollinated flowers did not exhibit postpollination symptoms. Compatible pollination of ‘Miss Teen’ flowers resulted in an immediate burst of ethylene production and a rapid stimulation of ovary growth followed by an increase in pollen tube length. In contrast, when ‘Miss Teen’ flowers were self-pollinated or pollinated with ‘Karen’ pollinia, these changes did not occur. Pollinia of the cultivars ‘Pompadour’, ‘Sakura’ and ‘Willie’ had greater amounts of auxin and 1-aminocyclopropane -1- carboxylic acicd (ACC) than that of the ‘Miss Teen’ and ‘Karen’ pollinia. Compatible pollination resulted an increment in higher ACC synthase and ACC oxidase activities than that of incompatible pollination. ACC oxidase activities of compatibly pollinated flowers rapidly increased within 12 h after pollination. Nonpollinated flowers showed a low ACC oxidase activity, while, column plus pedicel contained higher ACC oxidase activity than petal plus sepal and lip. 33 SESSION: POLLINATION Tuesday, May 31 Role of stigmatic fluid on ovary growth of pollinated Dendrobium ‘Pompadour’ flowers Saichol KETSA1, Apinya WISUTTIAMONKUL2 1Department of Horticulture, Faculty of Agriculture Kasetsart University, Bangkok 10900, Thailand 2Agricultural Technology Department, Institue of Scientific and Technologiacl Research, Pathum Thani 12120, Thailand Pollination induced an increase in ethylene production concomitant with ovary growth epinasty, drooping, venation and colour fading in Dendrobium ‘Pompadour’ flowers. Removal of stigmatic fluid prior to pollination reduced siginificantly ovary growth, while ethylene production, epinasty, drooping, venation and fading of pollinated flowers were not significantly affected. 1-Methylecyclopropene (1-MCP) treatment had no effect on the ovary growth, whereas NAA treatment increased significantly the ovary growth of pollinated flowers with and without stigmatic fluid removal. However, removal of stigmatic fluid reduced significantly ovary growth induced by NAA treatment. The role of stigmatic fluid on the ovary growth of pollinated flowers will be discussed. 34 SESSION: PHYLOGENETIC RELATIONSHIPS Tuesday, May 31 Effects of light quality on vegetative growth, flowering and expression of FT-like gene in Phalaenopsis Kumala DEWI1, Yohana, M. T. ASTUTI2, Yekti A. PURWESTRI1, Lila NATASAPUTRA3 and Parmi1 1 Faculty of Biology, Gadjah Mada University. Jl. Teknika Selatan, Yogyakarta 55281, Indonesia 2 Faculty of Agriculture, Agriculture Institute “INSTIPER”. Jl. Nangka II, Maguwoharjo, Sleman, Yogyakarta 55282, Indonesia 3 Green Leave Orchid.Jl. Sinoman Tempel III/235, Salatiga, Central Java, Indonesia The effects of LEDs (Light- emitting Diodes) having different colours namely red, blue, red and blue, and white lights on vegetative growth and flower forcing of Phalaenopsis have been evaluated. Phalaenopsis seedlings in the bottle were subjected to different light qualities for either 2 or 4 weeks, and then each seedling was planted in a plastic pot containing sphagnum and grown in the growth chamber under similar light quality for 3 months. For flower forcing, mature Phalaenopsis plants having 4 – 6 leaves were grown for 3 months in the growth chamber under different light qualities. The leaf span, chlorophyll, gibberellin and cytokinin content were determined. In addition, the expressions of FT-like gene in the leaf, axillary bud, flower bud and stalk were examined. The results showed that growth of Phalaenopsis seedlings were better when placed in the growth chamber, especially under blue, red-blue or white LEDs compared to control. Gibberellin and cytokinin content increased in the seedlings subjected to white LEDs. Based on the average of leaf span increment it was suggested that the growth of Phalaenopsis seedlings can be promoted by giving either blue, redblue or white LEDs. From the second experiment, it was found that flower forcing in Phalaenopsis can be obtained in plants that had just finished flowering without LEDs application. The expression of FT-like gene in the leaf as well as flower bud and stalk suggests that this gene involves in flower regulation of Phalaenopsis. 35 SESSION: PHYLOGENETIC RELATIONSHIPS Tuesday, May 31 An intriguing morphological variability of Platanthera s.l. Peter G. EFIMOV1 1 Herbarium, Komarov Botanical Institute of the Russian Academy of Sciences, Prof. Popov str. 2, Saint-Petersburg, 192288, Russia The morphology of 34 species of the genus Platanthera s.l. was investigated by the means of light and electron microscopy, described and illustrated. Particular attention was paid to the structure of gynostemium and pollinaria. The species investigated represent 10 major lineages of the genus, all being recognized as sections or segregate genera in various taxonomical treatments. A considerable morphological variability was confirmed for Platanthera s.l. Of them, the most notably variable are tuberoids (root-like, stoloniferous, spherical, etc.), lip (entire, lobed, fimbriate etc.), rostellar lateral lobes (flat, concave, bursicle-like), lateral stigma lobes (flat, protruding, encircling spur entrance etc.), viscidia (orbicular, lanceolate, sub-linear, deeply concave, reduced) etc. The morphology of several taxa confirm that they do not belong to Platanthera s.l., of them Neolindleya displays relation to Galearis s.l. and several east asiatic taxa (P. latilabris, P. clavigera, P. biermanniana etc.) – to subtribe Habenariinae. Usually, the morphological variability confirms the monophyly of the lineages within Platanthera s.l., as Tulotis, Stigmatosae-clade, Gymnadeniopsis etc. Some of them were previously confirmed by molecular phylogenetic studies (Hapeman, Inoue, 1997; Bateman et al., 2009) and may be used for delimitation of the groups within Platanthera s.l. The morphological polymorphism in Platanthera s.l. (especially gynostemium structure) seems to be higher than in many other large genera of the subtribe Orchidinae, as Orchis, and led to the recognition of several segregate genera within Platanthera s.l. However, less polymorphism revealed for molecular markers gives evidence for morphological (in particular, gynostemium) plasticity and subsequently quick morphological evolution in this group. 36 SESSION: PHYLOGENETIC RELATIONSHIPS Tuesday, May 31 Where have the hexaploids vanished? The role of breeding barriers in coexistence of Gymnadenia conopsea cytotypes Jana JERSÁKOVÁ1,2, Stefan DÖTTERL3, Sílvia CASTRO4, Pavel TRÁVNÍČEK5,6, Jana RAUCHOVÁ5,6, Barbora KUBÁTOVÁ7, Vladislav ČURN7, Jan SUDA5,6 1 Faculty of Science, University of South Bohemia, České Budějovice, Czech Rep. 2 Department of Biodiversity Research, Global Change Research Centre AS CR, , Brno, Czech Rep. 3 Department of Plant Systematics, University of Bayreuth, Bayreuth, Germany 4 Faculty of Science, Charles University, Prague, Czech Rep. 5 Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice, Czech Rep. 6 Centre for Funct. Ecol. and Dpt. of Botany, University of Coimbra, Coimbra, Portugal 7 Faculty of Agriculture, University of South Bohemia, České Budějovice, Czech Rep. Polyploidy has played a key role in plant diversification. However, it is still largely unknown how prezygotic and postzygotic barriers contribute to reproductive isolation among cytotypes in mixed-ploidy populations. We investigated the origin and maintenance of contact zones of five sympatric cytotypes in Gymnadenia conopsea agg. (Orchidaceae). To investigate pre-mating barriers, we determined cytotype spatial distribution, flowering phenology, floral morphology, and visual and olfactory cues leading to assortative mating. Gas chromatography coupled with electroantennography was used to identify scent compounds with physiological activity in two main pollinators. Importance of visual and olfactory cues was also assessed in the field by analysing pollinator’s behaviour on artificial arrays. Artificial crosses were used to asses post-mating barriers. Our study demonstrated that the coexistence of Gymnadenia cytotypes is partly maintained by differences in floral phenology and spatial segregation. However, cytotypes with overlapping flowering hybridize due to similarities in floral colour and spur length, the lack of assortative behaviour of pollinators and the absence of gametic incompatibility. Though both pollinator species physiologically detected scent differences, they did not use this cue to discriminate the cytotypes in the field. Crossing experiments revealed that minority ploidies largely originate from heteroploid crosses, with most progeny being 6x, whereas backcrossing of 6x with parents results in very low seed quality. The co-occurrence of dominant cytotypes in mixed populations is thus maintained by temporal and spatial segregation and limited reproduction of hybrids. The absence of a barrier to pollen discounting as well as the scarcity of 6x plants is discussed. 37 SESSION: PHYLOGENETIC RELATIONSHIPS Tuesday, May 31 Conservation significance of the renewed assessment of the molecular phylogenetic and mycorrhizal relationships in Pterostylidinae (Cranichideae) Mark Alwin CLEMENTS1, Christopher Garry HOWARD1, Joel Tupac OTERO2, Joseph MILLER1 1Centre for Australian National Biodiversity Research, GPO Box 1600, Canberra, ACT, 2601, Australia 2Departamento de Ciencias Biológicas and Instituto de Estudios Ambientales IDEA – Palmira,Universidad Nacional de Colombia Sede Palmira, Palmira, Valle, Colombia. The conservation of threatened orchids can be impeded by both taxonomic uncertainty and a lack of knowledge regarding mycorrhizal fungi associations. A recent reassessment of species representatives of all taxonomic groups within subtribe Pterostylidinae (Cranichideae) based on analyses of nuclear and plastid DNA sequence datasets has provided a solid foundation for research into species delimitations where doubts still exists about the status of many described or undescribed taxa including some nationally threatened species (4 critically endangered; 9 endangered; 1 extinct; 11 vulnerable). It was revealed that none of the presently proposed classification systems truly accounts for the underlying phylogenetic signal. Strong support for three major groups, clades A–C, containing ten easily identifiable, monophyletic groups well supported by morphological synapomorphies: Clade A - Speculantha (including Petrorchis) and Diplodium (where there is no support for continued recognition of Taurantha, Crangonorchis, Linguella and Eremorchis); Clade B - true Pterostylis; and Clade C - contains the morphologically disparate Bunochilus, Hymenochilus, Oligochaetochilus, Pharochilum, Plumatichilos, Stamnorchis and Urochilus (including Ranorchis). Parallel molecular studies of associated mycorrhizal fungi, based on 200 isolates collected from 41 host species covering the three major Pterostylidinae clades, provided a means to unravel the complex relationships between these organisms. Three levels of association were identified; high, intermediate and broad facilitating a high predictive probability of fungal associations for all Pterostylidinae species. This new knowledge of fungal associations can now be applied to the development of Management Plans for the conservation and propagation of these threatened species. 38 SESSION: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Tuesday, May 31 Methods of studying field germination and seedling physiology: present potential and drawbacks Hanne N. RASMUSSEN Forest & Landscape, LIFE faculty, University of Copenhagen, Rolighedsvej 32, DK-1958 Frederiksberg C, Denmark; email: [email protected] Requirements for orchid seed germination are not identical to those of seedling growth and development. Both processes are of course crucial for population survival. Dormancy release and fungal requirement during germination depend much on the orchid species in question, and varies with conditions under which the seeds developed on the mother plant. Observation of germinating seeds in a natural substrate is inhibited by their small size. The numerous experiments in vitro with immature seeds can be misleading regarding the behavior of natural seeds in the field. Mature seeds germinated in vitro, however, may yield some indications to wild behavior, if growth conditions are manipulated suitably. The seed package technique for germination in the field has, however, yielded considerable information concerning spatial distribution and timing, and identity of participating fungi. In seedling physiology we are also fairly dependent on observations in vitro; seedlings in near-natural settings are difficult to manipulate, and natural seedling often too sporadic for correllation with growth conditions. The assumption that all orchid seedlings depend on mycophagy covers great specific differences in strategy, concerning when, if and how much photosynthesis contributes to seedling survival in the orchid species under study. Experiments in vitro, especially symbiotic and on series of minimal media, have much to offer in supporting and extending field observations of substrate and site preferences. 39 SESSION: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Tuesday, May 31 Seed propagation and re-introduction of the U.S. federally endangered Hawaiian endemic, Platanthera holochila (Hbd.) Kral. (Orchidaceae) Lawrence W. ZETTLER1, Erin M. WOOD1, Lynnaun J.A.N. JOHNSON1,2, Anna K. KIRK1,3, Steve PERLMAN4 1 Biology Department, Illinois College, Jacksonville, Illinois 62650 USA 2 Department of Biological Sciences, Western Illinois University, Macomb, Illinois 61455 USA 3 Department 4 National of Entomology, Michigan State University, East Lansing, Michigan 48824 USA Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, Hawaii 96741 USA Known for its natural beauty, Hawaii harbors a distinctive flora found nowhere else on Earth. Of its 1,159 native plant species, 9% are now extinct and ca. 53% are at risk of extinction. Three orchid species are endemic to the archipelago, one of which, Platanthera holochila (Hbd.) Kral., is U.S. Federally-listed as endangered. Fewer than 50 individuals of this species remain on three islands (Kauai, Maui, Molokai), and only one plant occurs on Kauai. During a 30-year period, repeated attempts to cultivate P. holochila from seed were unsuccessful despite coordinated efforts by specialists and commercial orchid growers alike. In 2004, the technique of in vitro symbiotic seed germination was applied to P. holochila for the first time, with promising results. However, the fungus used for this purpose originated from Florida, not Hawaii, raising concerns that the release of mycotrophic seedlings would also result in the release of an exotic fungus. Subsequent attempts to germinate seeds with mycorrhizal fungi acquired from P. holochila protocorms in Hawaii were unsuccessful, prompting germination trials using asymbiotic media. Of the 24,940 seeds initially sown on agar, a sizeable number (200+) of leaf-bearing seedlings were obtained in vitro. In March 2011, 85 of the largest seedlings were deflasked and promptly flown to Hawaii. Eight seedlings were re-introduced into Alakai Swamp on Kauai, and 9 were taken to Kamakou Preserve on Molokai. The remaining seedlings are currently in cultivation (e.g., Lyon Arboretum) and will likely be reintroduced in situ within the coming year. 40 SESSION: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Tuesday, May 31 Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers Laura L. COREY1, Lillian L. MOLLER-JACOBS1, April Y. ROSS1, Lawrence W. ZETTLER1, Larry W. RICHARDSON2 1 Biology Department, Illinois College, 1101 W College Avenue, Jacksonville, Illinois 62650 USA 2 Florida Panther National Wildlife Refuge, U.S. Fish & Wildlife Service, 3860 Tollgate Blvd., Suite 300, Naples, Florida 34114 USA Epiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms), which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet - harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate - harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the anamorphic genus Ceratorhiza Moore, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may have triggered the germination process in situ. 41 SESSION: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Tuesday, May 31 Limitations to orchid distribution inferred from in situ germination experiments Tamara MALINOVÁ1,2, Jana JERSÁKOVÁ1,2, Jakub TĚŠITEL1, Marc-André SELOSSE3 1 Faculty of Science, University of South Bohemia, České Budějovice, Czech Rep. 2 Department of Biodiversity Research, Global Change Research Centre AS CR, , Brno, Czech Rep. 3 Centre d’Ecologie Fonctionnelle et Evolutive, Université Montpellier II, Montpellier, France Reasons for orchid natural rarity and habitat specialization have attracted researchers attention for years, and are sometimes explained by limited distribution of the seeds, narrow specialization to mycorrhizal symbionts, obligate nutrient donors during seedling development, or unfavourable abiotic conditions. We focused on a rare and declining mountain meadow species, Pseudorchis albida and four forest habitat specialized Epipactis species to reveal the key factors influencing their distribution at germination stage. We performed sowing experiments introducing seeds to (i) vacant sites similar to occupied habitats, and to (ii) putatively unsuitable sites where the focus species does not occur. We analyzed soil parameters and mycorrhizal fungi in seedlings and adults using barcoding with nuclear ribosomal DNA (ITS). We revealed generally high germination rate in both meadow and forest orchid species, although the amount of advanced mycorrhizal seedlings was low (around 0.6% for P. albida and around 7% for Epipactis species). Distribution of either saprophytic fungi from Tulasnellaceae (in case of P. albida) or ascomycetes that usually form ectomycorrhizas on trees (in Epipactis species) does not represent a key limiting factor to orchid establishment at regional scale, but is more pronounced at micro-site scale. Similarly, the soil abiotic parameters do not influence the early germination stage. We suggest that several factors, at least, contribute to orchid rarity: lower probability for seeds to reach distant sites, high mortality at early germination stage and low probability to find the mycorrhizal fungus in heterogeneous environment. 42 SESSION: IN SITU GERMINATION AND SEEDLING PHYSIOLOGY Tuesday, May 31 The embryology of Gastrodia nantoensis: embryo development Yung-I LEE1 1 Botany Department, National Museum of Natural Science, No 1, Kuan-Chien Rd., Taichung, Taiwan, ROC Gastrodia is an achlorophyllous orchid with an underground tuber. It could be noticed only when the flowers or capsules elongate above the ground. After successful pollination, the capsules become mature only about one month. It is interesting to study why this orchid could complete the procedure of seed production within such a short period (compared with other orchids). Except for the early works on Gastrodia elata, little information is available about the embryo development. In this study, developing capsules of Gastrodia nantoensis are collected for histological and histochemical investigation. The suspensor of Gastrodia nantoensis consists of a single cell, derived from the larger basal cell after the first mitotic cell division of zygote. Throughout the embryo development, the suspensor is not enlarged further. During the early stages of embryo development, starch grains are aboundant in the embryo proper and the suspensor cell, suggesting the suspensor plays a role in nutrient uptake for the developing embryo. As the seeds mature, starch grains have disappeared while protein and lipid bodies are present in the embryo proper cells. In the mature seed, an apical zone with relatively small cells can be observed in the embryo proper. Nile red staining indicates that a cuticular layer is mainly present on the surface walls of the embryo proper and the lateral walls of the suspensor cell, but is absent from the suspensor cell wall adjacent to the micropylar end. Besides, cuticular material is also found to accumulate on the innermost layer of testa. 43 SESSION: POPULATION DYNAMICS Thursday, June 2 25 years in life of Cypripedium calceolus genets Tiiu KULL 1 Estonian University of Life Sciences, Riia 181 Tartu 51014 Estonia; email: [email protected] Individual genets of rhizomatous long-lived perennial orchids as clonal plants may survive for hundreds of years. However, very few data is available on genet dynamics for a longer period. A dataset of 40 clones during more than 25 years in two Cypripedium calceolus populations was analyzed. The death rate has been very low – less than 1% per year. The speed of growth of clones diameter has been steady (in average 5–10 mm per year) although frequent temporal reductions in diameter have been recorded that are probably connected to rhizome branch dormancy. Although larger clones have produced generally more fruits during the study period, the production of fruits per flower is smaller in clones with more ramets and flowers. This can be a consequence of deception in pollination system. We have also established a positive correlation between the average height of flowering ramets in the clone and the production of fruits per flower. A detailed knowledge of the processes of genet life serves as a basis for the understanding of population dynamic mechanisms. 44 SESSION: POPULATION DYNAMICS Thursday, June 2 Risk aversion and specificity in euglossine bee pollinated plants: do pollinator population stabilities, bee abundances, and flowering phenologies matter? James D. ACKERMAN1,2, David W. ROUBIK3 1 Department of Biology, University of Puerto Rico, PO Box 70377, San Juan PR 00936-8377, USA, [email protected] 2 Center for Applied Tropical Ecology and Conservation, PO Box 23341, University of Puerto Rico, San Juan, PR 009313341, USA 3 Smithsonian Tropical Research Institute, Balboa, Panama Asymmetrical and nested pollination networks are characterized by the rarity of reciprocal specificity. The importance of multiple interaction partners may be based on ameliorating risks of extinction caused by high variance in pollinator service or in the availability of pollinator rewards. From the perspective of plants, specificity should then be related to long-term pollinator population stability. Alternatively, it may be viewed as a sampling problem based on either pollinator abundance or the duration of flowering. We tested these ideas for a community of euglossine bees and flowering plants of lowland central Panama. We compared bee population stability indices from seven continuous years of census data with pollinator and flowering phenology data at 37 plant species and found that pollinator specificity was not related to pollinator population stability or pollinator abundance, and no relationship existed between length of flowering season and the stability index of pollinators. A strong relationship did exist between length of flowering and specificity: the longer the flowering season, the lower the specificity. While risk aversion may not be based on pollinator population stabilities or abundances, it may still be important to the generalist-specialist continuum through variability in flowering duration. 45 SESSION: POPULATION DYNAMICS Thursday, June 2 Conservation of Isotria medeoloides, a tale of dormancy and fungi Melissa K. MCCORMICK1, Dennis F. WHIGHAM1, John P. O’NEILL1 1Smithsonian Environmental Research Center, P.O. Box 28, Edgewater, MD 21037, USA. Isotria medeoloides is considered one of the most endangered orchids in the United States and is distributed along the East Coast of the US, with most populations in the northeast. Challenges to conservation of this orchid are many. Almost all populations in the mid-Atlantic region are very small and dynamic (i.e., many individuals fail to produce a leaf in a given year, becoming “dormant”), making it very difficult to accurately assess population size. The incidence of dormancy seems to reflect population health, with high rates of dormancy indicating declining populations. Leaf production in I. medeoloides and its sister species I. verticillata is initiated in the fall with development of a large overwintering bud. We have found that bud damage contributes to the incidence of dormancy but does not fully explain it. Seed germination is another challenge for I. medeoloides conservation. We have identified mycorrhizal fungi as members of Russula and Lactarius in the Russulaceae, a family of obligately ectomycorrhizal fungi. This implies that management to conserve I. medeoloides may need to promote trees that host the mycorrhizal fungi needed by the orchids. We are using seed packets to assess germination in the field. These fungi have proven difficult to culture, complicating efforts to germinate seeds in the laboratory for reintroduction and so far no attempts have successfully produced symbiotic seedlings. 46 SESSION: POPULATION DYNAMICS Thursday, June 2 Vegetative dormancy, shrinkage, and fitness in two orchids Richard P. SHEFFERSON1, Tiiu KULL2, Kadri TALI2, Kimberly M. KELLETT1 1 Odum School of Ecology, University of Georgia, 140 E. Green St., Athens, Georgia 30602 USA 2 Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Riia 181, Tartu 51014 Estonia Vegetative dormancy has been described in many plant families, but its evolutionary context remains unclear. Plant shrinkage, or regression, is a similarly mystifying phenomenon that is only now being studied from an evolutionary perspective. We present evidence that dormancy functions as an adaptive response to environmental stress in long-lived plants. We defoliated, shaded, or observed plants in two populations of Cypripedium calceolus and three populations of Cephalanthera longifolia in Estonia in 2002, and then monitored through 2007. We analyzed the resulting demographic data with life table response experiment (LTRE) analysis, and by assessing correlations between dormant episodes and lifespan during the study. Treatment had negative consequences on in Cypripedium, but had mixed consequences in Cephalanthera. was most sensitive to the impacts of treatment on fertility in small individuals and growth from dormancy. Stasis had impacts on occasionally, while regression was unimportant. Defoliation caused increased levels of dormancy in both species, while shading did not. Further, the observed lifespan of Cypripedium plants that became dormant in years two or three of the study was significantly higher than plants that had not become dormant, although this pattern was not repeated in Cephalanthera. Dormancy was not associated with any changes to flowering or shoot numbers, but was associated with increased leaf numbers in Cypripedium in the long-term. Dormancy had strong impacts on survival, and even impacted leaf growth. Thus, dormancy likely functions as an adaptive buffer to survival against environmental stochasticity. Regression, in contrast, seems less important in this system. 47 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 Seed quality of endangered terrestrial orchid species Coeloglossum viride (L.) Hartm in Estonia Mirjam METSARE,1 Kadri TALI1 1 Institute of Agricultural and Environmental Sciences, University of Earth Sciences, Riia 181, Tartu, Estonia Coeloglossum viride (L.) Hartm., a tiny short-lived terrestrial that has decreased over 90% in Estonia, is strongly dependent on its seed quality to be able to persist in natural populations. In a three-year study we analysed seed quality and in vitro germination of this species. Seed quality, (incl. proportion of empty seeds, germination and enzyme activity in embryo) varies greatly over years. Germination potential decreases to zero in 18 months when stored at +5 C. Very old seeds from herbarium specimen still show some enzymatic activity after treatment. 48 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 Mycorrhiza assisted conservation of two commercially important and endangered orchids of India: Dactylorhiza hatagirea D.Don (Soo) and Vanda coerulea Griff Ex Lindl. Simmi AGGARWAL1, C. NIRMALA2, Shanuja BERI3, Sachin RASTOGI4 and Alok ADHOLEYA5 1 Department of Botany, Panjab University, Chandigarh- 160 014, India 2 Department of Botany, Panjab University, Chandigarh- 160 014, India 3 Biotechnology and Bioresources Division, The Energy and Resources Institute, IHC, Lodhi Road, New Delhi -110003, India 4 Biotechnology and Bioresources Division, The Energy and Resources Institute, IHC, Lodhi Road, New Delhi -110003, India 5 Biotechnology and Bioresources Division, The Energy and Resources Institute, IHC, Lodhi Road, New Delhi -110003, India The technique of symbiotic seed germination-using fungi to cultivate orchid seedlings in vitro leading to their reintroduction in situ has considerable potential for conservation evidenced by studies mostly in Australia and North America. However, its use has yet to be fully realized throughout the world. On the Indian subcontinent, which harbours a considerable number of orchid species, symbiotic germination has been virtually unexplored. In the present studies, we provide a protocol for the symbiotic seed germination and eco-restoration of two rare and endangered orchids, Dactylorhiza hatagirea (D.Don) Sooa terrestrial species highly sought after for its use in traditional medicine and Vanda coerulea Griff Ex Lindl which is a floriculturally significant epiphyte used to progenate a vast variety of hybrids. Seeds were obtained from the mature un-dehisced capsules and sown on oat meal agar medium with the fungus isolated from the roots of mature D. hatagirea and V. coerulea plants in situ. Using molecular characterization techniques, cultures were assignable to the teleomorph genus Ceratobasidium and Rhizoctonia. For D. hatagirea, seeds inoculated with the fungus resulted in 90% germination within 10 days of inoculations and healthy protocorms were obtained in 40 days. Seedlings with well-developed root, tubers and leaves were obtained after 3 months. These were transferred to their natural habitat at Lahul Spiti in North West India. For V. coerulea, all the seeds germinated within 5 wks of culture and very healthy, dark green protocorms were obtained in 5-month-old cultures. Seedlings with 1-2 roots and 2-3 leaves were obtained in 8 months. These were acclimatized in the green house for the next one year and introduced to their natural habitat at Manipur in North East India. 49 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 Symbiotic germination of 3 species of orchids susceptible to genetic erosion, in Soconusco (Chiapas, Mexico) Vincenzo BERTOLINI1, Anne DAMON1 1 El Colegio de la Frontera Sur (ECOSUR). Carretera Aeropuerto Antiguo km. 2.5, Tapachula, Chiapas, México. Soconusco region (southeast Mexico) has almost a quarter of the orchid species registered in Mexico and 37 threatened species (NOM-059-SEMARNAT-2001), many with severely reduced and non-viable populations. We chose 2 of the most threatened species, Rossioglossum grande (Lindl.) Garay & G.C. Kenn. and Cuitlauzina convallarioides (Schltr.) Dressler & N. H. Williams and a rare species recently discovered in the region, Rhynchostele bictoniensis (Bateman) Soto Arenas & Salazar, to study the mycorrhizal fungi and bacteria associated with the roots, isolate them and use them to induce seed germination in the laboratory. Long term we hope to develop protocols using symbiotic germination for the sustainable, mass production of rare and over exploited species under rustic conditions in rural communities, and produce plants vigorous and resistant enough to survive reintroduction into selected sites in the wild; some plants will be cultivated for their flowers for locally produced craft products. We isolated 10 strains of rhizoctonious fungi from R. grande and 3 from C. convallarioides, and tested for the promotion of germination of seeds from the same species. For R. bictoniensis, we used 9 strains of Rhizoctonia isolated from other orchid species. For R. grande, at 8 months in cultivation, 4 strains of Rhizoctonia promoted germination with 70% survival of the protocorms and production of rhizoids. For C. convallarioides, at 2 months, 1 strain was pathogenic and for the other 2 strains the seeds have only swelled with no development of chlorophyl. For R. bictoniensis 2 strains have promoted germination and, at 3 months, the protocorms are green. 50 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 The seed productivity of several species of orchids in the Komi Republic Dmitry KIRILLOV Russia, 167982, Komi, Syktyvkar, Kommunisticheskaya, 28; Institute of Biology, Komi Scientific Centre, Ural Branch, Russian Academy of Science; e-mail: [email protected] The peculiarities of the seed and vegetative propagation of the two species of the orchids (Cypripedium calceolus L. and Dactylorhiza cruenta (O. F. Muell) Soó) have been studied in the Komi Republic. The seed productivity has been defined and the morphometric characteristics of the fruits and seeds are presents. C. calceolus reproduces by seeds and vegetatively. Capsule C. calceolus up to 4.5 cm long and about 0.70.9 cm in diameter, containing in average 7758.1 seeds (min – 489, max – 20449). The seed length of this species ranges from 0.7 to 1.5 mm, width ranges from 0.2 to 0.3 mm. From 0.2 to 5.2% of seeds are lacking embryos. Population seed yield varies from 3-7 thousands seeds per sq. m in the limestones to 2144 thousands seeds per sq. m in the swamps. D. cruenta is characterized by an active seed reproduction. Seed productivity in one fruit fluctuates from 3563 to 12765 (mean 8416), individual plant produces 88-143 thousands seeds, the crop may have 106268128869 seeds per sq. m. The seed length of D. cruenta ranges from 0.4 to 0.6 mm, width ranges from 0.1 to 0.3 mm. From 6.4 to 21.3% of seeds of this species are lacking embryos. Vegetative reproduction was not observed. 51 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 Effect of banana extract on the growth and development of plantlets from protocorms of Cymbidium spp and Dendrobium lituiflorum Lindl. Vera Yurngamla KAPAI1, Shivani VYAS1, Satyakam GUHA2, Priyanka KAPOOR3 & I. Usha RAO1* 1 Department of Botany, University of Delhi, Delhi 110 007, India 2Hansraj 3Gargi College, University of Delhi, New Delhi 110 007, India College, University of Delhi, New Delhi 110 049, India *Corresponding author’s e-mail: [email protected]; Orchids exhibit a wide range of diversity in size, shape and color of their flowers. They are among the most threatened of all flowering plants due to over exploitation and habitat loss. Clonal mass scale propagation of orchids using tissue culture techniques is thus, very important for ex situ conservation. The effect of a natural additive, banana extract (BE; 1%-20% v/v) was studied on regeneration from protocorms of Cymbidium aloifolium, C. bicolor and Dendrobium lituiflorum by supplementing it to plain agar-agar medium (without Knudson C salts) or KC medium with or without sucrose (2%w/v). Incorporation of salts of KC medium along with higher percentages of BE [10% and 20% (v/v)] promoted protocorm regeneration and root formation. The presence of sucrose in the medium further enhanced root length and percentage rooting irrespective of the presence of salts of KC medium. The incorporation of salts of KC medium and sucrose were found to be obligatory apart from BE for prolific growth. The addition of BE produces stout shoots with broad leaf laminae and thick roots. Pre-hardening was carried out by culturing the plantlets in ¼ KC major salts without sugar and was transferred to plastic trays filled with coir. In vitro raised plantlets of these orchids exhibited successful survival under greenhouse conditions. Scanning electron microscopy analysis showed elliptical stomata and granular wax deposition on the abaxial leaf surface during acclimatization. 52 SESSION: SEED STORAGE AND GERMINATION, EX SITU PROPAGATION Thursday, June 2 Conservation through in vitro propagation and acclimatization of the endangered orchid Laelia speciosa Irene ÁVILA-DÍAZ1, Martha Mireya ORTEGA-LOEZA2 and Rafael SALGADO-GARCIGLIA3 1 Facultad de Biología de la Universidad Michoacana de San Nicolás de Hidalgo. Edif. R planta baja. Ciudad Universitaria, C.P.58040 Morelia, Michoacán, México; email: [email protected] 2 Instituto Tecnológico Superior de Coalcoman. Coalcoman de Vezquez Pallares, Centro, Álvaro Obregón 580, C.P.60840.Coalcoman Michoacán, México. 3 Instituto de Investigaciones Químico-Biológicas de la Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3. Ciudad Universitaria, C.P.58040 Morelia, Michoacán, México A multidisciplinary project on the endemic and endangered Mexican epiphytic orchid L. speciosa that includes biological research and work with local people in communities was carried out in order to reach a sustainable management. This time we focused on in vitro propagation as the alternative, and developed systems for efficient sexual and asexual in vitro reproduction. Acclimatization is a critical stage and a main study factor of in vitro propagation. Relationship of the stomata opening during ex vitro acclimatization, and the time of in vitro culture (0, 5, 10, 15, 20, 25, 30 days) in greenhouse conditions (pre-acclimatization), on the survival and development of plantlets during the ex vitro acclimatization were carried out. Furthermore the effect of different levels of nutrients (100%, 75%, 50%, 25% and 0% -strength salts) and sucrose (0, 10, 20, 30, 40 g/L) in the Murashige and Skoog medium (MS) on the same parameters were measured. Plantlets that spent 20 days in greenhouse conditions before ex vitro acclimatization, displayed the best growth with a survival frequency of 97.5%, related with the highest stomatal opening. Plantlets developed on MS containing 100% -strength salts (with 20 days of pre-acclimatization, 40 g/L sucrose) had the highest frequency (100%) of survival and vigor when acclimatized. Micropropagated plants are kept under greenhouse conditions in order to use them as a source in Mexican religious art; this way, diminishing the impact on L. speciosa wild populations and promote its sustainable management; a central issue for an orchid that has been used for centuries in many different ways. 53 SESSION: TAXONOMY Thursday, June 2 Genetic variation and cultivar identification in Cymbidium ensifolium Hui-zhong WANG 1 ·Jiang-jie LU 1 ·Xu HU 1·Jun-jun LIU 2 ·Yu-jun SHI3 1Hangzhou Key Laboratory for Systems Biology of Medicinal and Ornamental Plants, Hangzhou Normal University, Hangzhou 310018, China. E-mail: [email protected] 2 Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, Victoria, V8Z 1M5 Canada 3Zhejiang Gongshang University. Cymbidium ensifolium (L.), a popular flowering species with many cultivars, is commercially important in horticulture. However, little is known about its genetic diversity and conservation genetics. Understanding of the genetic diversity and relationships among the cultivars is a prerequisite for future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C. ensifolium cultivars using DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of the genetic diversity with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% of them were polymorphic with a wide genetic variation as shown by Nei’s gene diversity (H = 0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar, Sijiwenhan, was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters, with limited correlation (r = 0.221, p < 0.004) with their geographical origins. A further analysis of genetic diversity revealed that about 69% of the total genetic variation in this species was due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in situ conversation are important for future plan of the C. ensifolium species conservation. 54 SESSION: TAXONOMY Thursday, June 2 Colour-scent associations in a tropical orchid: three colours but two odours Thierry PAILLER 1, Roxane DELLE-VEDOVE 2, Nicolas JUILLET BARTHES 2, Laurent DORMONT 2, Bertrand SCHATZ 2 1,2 , Jean-Marie BESSIÈRE 3, Claude GRISON 2, Nicolas 1 UMR53, Laboratoire des Peuplements Végétaux et Bioagresseurs en Milieu Tropical, 15 Avenue René Cassin, BP 7151, 97415 St Denis de la Réunion messag cedex 9, France 2 Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR CNRS 5175, 1919 route de Mende, 34293 Montpellier cedex 5, France 3 3 Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier cedex 5, France Colour and scent are the major pollinator attractants to flowers, and their production may be linked by shared biosynthetic pathways. Species with polymorphic floral traits are particularly relevant to study the joint evolution of floral traits. We used in this study the tropical orchid Calanthe sylvatica from Réunion Island. Three distinct colour varieties are observed, presenting lilac, white or purple flowers, and named respectively C. sylvatica var. lilacina (hereafter referred as var. lilacina), C. sylvatica var. alba (var. alba) and C. sylvatica var. purpurea (var. purpurea). We investigated the composition of the floral scent produced by these colour varieties using the non-invasive SPME technique in the wild. Scent emissions are dominated by aromatic compounds. Nevertheless, the presence of the terpenoid (E)-4,8-dim- ethylnona1,3,7-triène (DMNT) is diagnostic of var. purpurea, with the volatile organic compounds (VOC) produced by some individuals containing up to 60% of DMNT. We evidence specific colour-scent associations in C. sylvatica, with two distinct scent profiles in the three colour varieties: the lilacina-like profile containing no or very little DMNT (<2%) and the purpurea-like profile containing DMNT (>2%). Calanthe sylvatica var. alba individuals group with one or the other scent profile independently of their population of origin. We suggest that white-flowered individuals have evolved at least twice, once from var. lilacina and at least once from var. purpurea after the colonisation of la Réunion. White-flowered individuals may have been favoured by the particular pollinator fauna characterising the island. This study prove that colour-scent associations may be complex, depending on pollination ecology of the populations concerned. 55 SESSION: TAXONOMY Thursday, June 2 Sympodial and monopodial types of growth: why it’s important in orchid biology and ecology? Irina TATARENKO Open University, Walton Hall, Milton Keynes, UK One of the classical characters used in orchid morphology and systematic is sympodial and monopodial type of growth (Pfitzer, 1889). It is rarely mentioned in recent species descriptions (Genera Orchidacearum, 2000-2000), however considered as a basic in morphological classifications of orchids (Smirnova, 1986; Tatarenko, 1996, 2007; Kolomeytceva, 2006). This paper looks at the relationships between the type of orchid shoot growth and species reproduction ability, duration of individual life, spatial structure of the populations and habitat requirements. 56 SESSION: TAXONOMY Thursday, June 2 Taxonomic uncertainty in species identifications and its potential consequences David L. ROBERTS1 1 Durrell Institute of Conservation & Ecology, University of Kent, Marlowe Building, Canterbury, Kent, CT2 7NR. The scientific community is facing a taxonomic crisis. Linnean shortfall, a euphemism for the hole in our knowledge of biodiversity, cannot be estimated to within an order of magnitude. Faced with the vast number of species yet to be discovered and accelerating extinction rates, the task of cataloguing Earth’s biodiversity is immense. Central to this is the need for species identification, not only to meet this challenge but is also by society as a whole. Border authorities may be interested in identifying species controlled under CITES, agriculturalist in pest species, building developer in those that are nationally protected species, as well as the amateur naturalist communities’ general interest. Misidentification can however have major consequences. For example, the US FWS have drafted a multi-million dollar recovery plan for a species based on an uncertain sighting. Acceptance of unconfirmed or verifiable sightings can lead to both false-positives and false-negatives. Such errors have serious implications for conservation practice and allocation of resources. To avoid such errors we rely on expert opinion for the verification of a taxon’s identity. For some species, routine assessments can result in accuracies as high as 95%. For others more experience is required, and in some cases inconsistent identification can be over 40%. Here we discuss the issues around taxonomic uncertainty, how it may be measured and give an example of how it can be applied in extinction models. Specifically a Bayesian analysis of the Ghost Orchid (Epipogium aphyllum Sw.) that allows for sightings of uncertain validity. 57 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Potential impact of insect herbivores on orchid conservation Marilyn H. S. LIGHT 1, Michael MacCONAILL 1 2174 2 rue Jolicoeur, Gatineau, Québec, Canada J8Z 1C9 When an orchid is deliberately or inadvertently relocated, it is likely to encounter a range of biological challenges to long-term survival including a complex and dynamic insect community which could alter conservation expectations yet there have been few studies of the phytophagous insects associated with wild orchids. We have investigated the assemblage of such insects associated with terrestrial orchids being monitored in our long term studies in Gatineau Park, Québec, Canada, where aphids, leafminers, moths, thrips, weevils, and whiteflies were found to be injurious to orchids although in different combinations and with varying impact according to the orchid host, habitat and year. Loss of seeds and even complete desiccation of plants was observed. Some infestations such as with the leafminer, Parallelomma vittatum (Diptera: Scathophagidae) seemed to be in equilibrium with parasitoids thus minimizing potential impact. A likely climate-related asynchrony of parasitoid and leafminer led to an outbreak in 2009 which heavily impacted the introduced E. helleborine while a phenological shift in a thrips primary host, Trillium grandiflorum (Melanthiaceae), in 2010, contributed to severe herbivory in habitats where both the orchid and trillium occurred. If climate change can lead to changes in insect abundance and impact on orchids, it would be useful to investigate the potential impact of phytophagous insects before assisted migration is considered as a conservation measure. 58 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Conservation and reintroduction of the native orchids of Singapore – the next phase Tim Wing YAM, Peter ANG, Felicia TAY, Weijing SOH Orchid Breeding and Conservation, Singapore Botanic Gardens, National Parks Board, Singapore Some 226 species of native orchids have been recorded in Singapore. However, of these 178 are considered to be extinct, and only five are common. The orchid conservation programme aims to monitor existing species, explore ways to conserve their germplasm, and increase their number for subsequent reintroduction into appropriate habitats, including roadside trees, parks and nature areas. From 1999 to 2008, we carried out experiment and have successfully propagated and re-introduced 5 species of native orchids, namely Grammatophyllum speciosum Blume, Bulbophyllum vaginatum Rchb.f., Bulbophyllum membranaceum Teijsm. & Binn., Cymbidium finlaysonianum Lindl. and Cymbidium bicolor Lindl. spp. pubescens (Lindl.) Du Puy & Cribb. More than 80% of the plants are growing well and all of them have flowered. From 2009 to 2012, we expand our reintroduction effort by planting a larger quantity of native species. Altogether fifteen species (Bulbophyllum blumei (Lindl.) J.J.Sm., Bulbophyllum medusa (Lindl.) Rchb.f., Bulbophyllum membranaceum Teijsm. & Binn., Bulbophyllum purpurascens (T.&B.) J.J.Sm., Bulbophyllum vaginatum Rchb.f., Coelogyne mayeriana Rchb.f., Coelogyne rochussenii De Vr., Cymbidium finlaysonianum Lindl., Dendrobium aloifolium (Blume) Rchb.f., Dendrobium leonis (Lindl.) Rchb.f., Grammatophyllum speciosum Blume, Phalaenopsis cornu cervi (Breda) Bl. & Rchb.f., Cymbidium bicolor Lindl. spp. pubescens (Lindl.) Du Puy & Cribb, Thrixspermum amplexicaule Rchb.f., and Vanilla griffithii Rchb.f.) will be planted. Ultimately we hope that the reintroduced species would act as catalysts in the restoration of at least part of the original ecosystem. For example, pollinators may be attracted to come back to pollinate the flowers. Orchid seeds that are formed naturally after flowers are pollinated may be blown to the proper environment where appropriate mycorrhizal fungi are present. And we hope that we will be able to see natural populations of native species sprouting up all over the island! 59 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 The orchid conservation strategy and research in China Jing CAI1,3, Yong Fu YU2, Sheng Li ZHAO2, Si Peng LEI1,2, Lai Qiang HUANG1,2,3, Yi Bo LUO4, Wen Chieh Tsai5, Yu Yun Hsiao5, Hong Hwa Chen5, Xun XU6, Jun WANG6, Jian WANG6, Zhong Jian LIU1,2,3 1 National Orchid Conservation Center, Orchid Conservation and Research Center of Shenzhen, Wangtong Rd., Shenzhen 518114, China 2 Orchid Conservation Committee of China Wild Plant Conservation Association, Wangtong Rd., Shenzhen 518114, China 3 The Center for Biotechnology and BioMedicine, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China 4 Institute of Botany, Chinese Academy of Sciences, Beijing, China 5 Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan 6 Beijing Genomics Institute Shenzhen（BGI）, Beishan Rd., Shenzhen 518055, China As we all know China is one of the countries that has the largest diversity of orchids species in the world. There are 1300 orchid species in 148 genera found in China. However, due to the illegal exploration for gardening and medicine use, many orchid species is closing to extinction in the wild. To protect the endangered orchids, we set up more than 160 reservoirs and one conservation center. Most of the endangered species survived and resurrected in the reservoir where there is free of human intervention. And in the garden of the national conservation center, many species proliferate quite well. In the national orchid conservation center, we also have a lot of botanists and geneticists carrying out scientific research on the pollination biology, molecular ecology and molecular phylogeny on Chinese orchids. One of the most interesting discoveries we made is the self-pollination mechanism of Holcoglossum. We found for the first time that this orchid can put the pollinia to the column by bending the stock of its pollinia. Our findings were published in Nature in 2006. We also carried out successful wild restoration of the endangered species Paphiopedilum armeniacum. In 2009, the conservation center launched a scientific project to sequencing the genome of Phalaenopsis equestris in collaboration with Tsinghua University, Institute of Botany of Chinese Academy of Sciences, Beijing Genomic Institute Shenzhen and National Cheng Kung University. 60 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Conservation recommendations from a large survey of French orchids Philippe FELDMANN1, Daniel PRAT1,2 1 Société Française d’Orchidophilie, Commission scientifique, 17 Quai de la Seine, 75019 PARIS, France 2 Université Claude Bernard – Lyon 1, UMR LEHNA, 6 rue Raphaël Dubois, 69622 VILLEURBANNE CEDEX, France The French Atlas of Orchids published in autumn 2010 is a large-scale and intensive survey of orchid populations throughout the France territory. The project was directed by the French Orchid Society and involved 3000 specialists in orchids. Overall, more than 110.000 stations were referenced and 160 taxa were reported with their range on 154 maps. This project is original by synthesizing, at large scale, much accurate naturalistic information from local surveys, thanks to a participative approach and on the basis of scientific objectives. Orchid stands have been recorded for more than 20 years, which revealed demographic trends at the scale of the country. These data were suitable to apply IUCN regional guidelines in order to establish the French Red List of orchid species. This is one of the few applications of IUCN criteria at a regional scale in a plant family. Every taxon has been investigated and classified within an IUCN categorization. This revealed than about 1 on 5 species is threatened (one as RE, 4 as EN and 23 as VU). The main extinction risk consists of population size decrease, mostly due to habitat change. Further investigations are needed for the 33 taxa classified as Data Deficient. Most threatened species benefit already of protection regulations. Some recommendations will be drawn in order to conserve orchid species, paying attention to patrimonial and threatened species. 61 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Rural, sustainable cultivation of native orchids in Soconusco (Chiapas, Mexico) Anne DAMON1 1 El Colegio de la Frontera Sur (ECOSUR). Carretera Aeropuerto Antiguo km. 2.5, Tapachula, Chiapas, México The Program for the Sustainable Cultivation of Soconuscan Orchids” was set up in 2000, as a strategy to motivate the sustainable exploitation of attractive species and to prevent further plundering of scarce orchid populations in the region of Soconusco, in the biodiversity-rich state of Chiapas, in the southeast of Mexico, which provides habitat to almost a quarter of Mexico’s orchid species and 37 endangered species. The main objectives of the program are to rescue plants (fallen after high winds, on pruned branches and felled trees, from areas designated for development etc.), to restore populations and to conserve both the orchids and their habitats. To that end, strategies include: 1. Research into key aspects of orchid ecology, particularly the two bottlenecks of pollination and symbiotic seed germination, 2. Training farmers to sustainably cultivate orchids and produce craft products from the flowers, 3. Training farmers to propagate and cultivate orchids for the restoration of populations in selected natural sites and 4. An extended program of Environmental Education in conjunction with the Regional Botanical Garden “El Soconusco” (Director Anne Damon, ECOSUR). The program is linked to the important regional collection of orchids maintained at two sites at different altitudes. In conjunction with the National Council for Protected Areas (CONANP) we are negotiating the necessary permits (UMAs: SEMARNAT) to legalize orchid production in 5 communities in key areas in Soconusco and ensure financial return for the orchid producers. The program requires support and funding to be able to achieve its objectives. 62 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Use and conservation of Vanilla planifolia J. in México Victor Manuel SALAZAR-ROJAS1, Braulio Edgar HERRERA-CABRERA1, Adriana DELGADO-ALVARADO1, Marcos SOTOHERNÁNDEZ2, Fernando CASTILLO GONZÁLEZ3, Jorge CAMPOS-CONTRERAS4, Juana CERVANTES-VARGAS1 1Colegio de Postgraduados en Ciencias Agrícolas–Campus Puebla, Programa de Estrategias para el Desarrollo Agrícola Regional. Km. 125.5 Carr. Fed. Méx.-Pue. Col. La Libertad. Puebla, Pue 2 Colegio de Postgraduados en Ciencias Agrícolas – Campus Montecillos, Programa de Botánica. Km. 36.5 Carr. Fed. Méx.-Tex. Montecillo, Edo. de México, México 3 Colegio de Postgraduados en Ciencias Agrícolas – Campus Montecillos, Programa de Genética. Km. 36.5 Carr. Fed. Méx.-Tex. Montecillo, Edo. de México, México 2 Universidad Nacional Autónoma de México– Unidad de Biología, Tecnología y Prototipos (UBIPRO), Facultad de Estudios Superiores, Iztacala. Av. De los Barrios No.1, Los Reyes Iztacala, Tlalnepantla Edo. de México The use and conservation of Vanilla planifolia Jack. in Mexico requires comprehensive and detailed analysis of its genetic variation and the issues that determine and affect such variation in vanilla. For this reason it is proposed, to study the variation in the contents of phytochemicals that define the vanilla aroma quality by HPLC (High Performance Liquid Chromatography), to identify genetic variation at infraspecific level among 14 microsatellite loci, and to study assessment criteria which determine the attitude of stakeholders around the use and conservation of vanilla germplasm in the Totonacapan region, Mexico, through multiattribute analysis method, AHP (Analytic Hierarchy Process). The results showed the existence of six phytochemical groups (chemotypes) within V. planifolia germplasm, each with specific aromatic characteristics. The 14 loci analyzed were polymorphic for vanilla chemotypes with variable sizes, and genetic clustering of accesions corresponded with the phytochemical grouping (chemotypes). It was noted that the process of economic and cultural valuation of users, has built the aromatic variation in vanilla germplasm, and conservation has allowed at the regional level. Thus the data indicate that in the possible center of origin of vanilla, there is genetic and phytochemical variation, fundamental to the design of a breeding program for optimizing the benefits of vanilla production for their users and contribute to the conservation of the primary gene pool of Vanilla planifolia. 63 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Conservation and improvement of indian Vanilla species Minoo DIVAKARAN1, Nirmal BABU K2 and Peter K V 3 1 Providence Women’s College, Calicut – 673 009, Kerala, India; email: [email protected] 2 Indian Institute of Spices Research, Calicut – 673 012, Kerala, India; email: [email protected] 3 Director, World Noni Research Foundation, Chennai; email: [email protected] Vanilla, Vanilla planifolia Andrews (syn. Vanilla fragrans Salisb.) Ames, the source of natural vanilla is native to Mexico and Central America, but now cultivated in other parts of the globe. The history of cultivated vanilla suggests that cultivated vanilla outside Mexico originated from a single clone leading to monoculture, susceptibility to diseases and hampering breeding programmes. The Linear Dependence of Stability on Diversity indicates that reduction in the number of species will result in lower stability of the genus. Presence of important agronomic characters in few species of Vanilla, V.andamanica, V.aphylla and V.pilifera, is reported. V.aphylla is tolerant to Fusarium wilt and has longer flower life than the cultivated vanilla. V.pilifera flowers were fragrant, and V.andamanica showed signs of insect pollination. The species were amenable to interspecific hybridization. Suggesting the possibility of converging the useful genes from wild species into cultivated vanilla. This orchid can be grown as a mixed crop increasing the socio-economic status of farmers. In vitro conservation of Vanilla and its related species was standardized paving a way for conservation of endangered species. Utilizing techniques viz., synthetic seed, slow growth and cryopreservation, helped in the conservation of vanilla germplasm. The protoplast isolation and fusion technology developed indicates its amenability to genetic manipulation studies and possibility of transfer of useful traits through the production of somatic hybrids. Pollen from two asynchronously flowering species of Vanilla viz., cultivated V. planifolia and its wild relative V. aphylla, were cryopreserved, retrieved and viability tested both in vitro and in vivo and the system can be used for conserving the haploid gene pool of Vanilla in cryobanks for their subsequent utility in crop improvement. Polymorphic bands expressed in the RAPD profiles were used to estimate the level of genetic diversity and interrelationships among different collections of Vanilla planifolia and few related species. Studies revealed that there are very limited variation within collections of V. planifolia, but the species are diverse The techniques standardized in this study open up new vistas in the improvement an important orchid of vanilla. 64 SESSION: ENDANGERED SPECIES, THEIR CONSERVATION AND RESTORATION Friday, June 3 Neottia nidus-avis (L.) Rich. in Kyiv: distribution, ecology, conservation Ivan PARNIKOZA Kyiv historical architectural memorial “Kyiv Fortress”, Hospitalna str., 24a, Kyiv, 01133, Ukraine; email: [email protected] Among the species currently known from the Ukraine’s capital, Kyiv, Neottia nidus-avis remains all but the least studied. During 2001-10, we studied the N. a current distribution, growing conditions, flowerage dynamics, as well as the effects of natural and anthropogenic factors of the environment. N.a has up to date been only reported from the right bank of the Dnieper in deciduous stands of Holosievo, Lysa Hora, Teremki, as well as in mixed forests of Lisnyky reserve and Svyatoshyn forestry. The species grows under 40-80% crown density and in 1-80% grass canopy. Within a population, the species forms local aggregations which flowerage terms probably depend on the total aggregation size (the number of dormant individuals). Small loci develop a potential sufficient for one to several years. A Lysa Hora locus that was active there in 2005 didn’t flower during the next 5 years. Similar behavior was observed for most loci in Holosievo as well, though some aggregations flowered every year up to 2009-10. An outburst of the number of flowering individuals was recorded there in 2001, but it has never repeated for the time of study. After population number fluctuations, no locus of the Svyatoshyn population flowered in 2010. The species is indicative of primeval forests and does not occur in artificially afforested areas. It suffers from increased recreational load, lumbering and timber transportation, which, alongside with successional changes, inflict damage to soil and mulch. Conservation of the species requires inclusion of its growth habitats into the strictly protected areas. Author thanks N. Shevchenko, M. Bogomaz, and M. Shernyshenko for their help in carrying out the study. 65 SESSION: MODELLING AND DATA ANALYSIS Friday, June 3 A primer of Bayesian modelling for the analysis of orchid data María-Eglée PEREZ1,2, James ACKERMAN2,3, Raymond L. TREMBLAY2,4 1 Department of Mathematics, University of Puerto Rico, Río Piedras campus, San Juan, PR 00931-3355 USA. 2 Center for Applied Tropical Ecology and Conservation, PO Box 23341, University of Puerto Rico, Río Piedras campus, San Juan, PR 00931-3341, USA 3 Department of Biology, PO Box 23360, University of Puerto Rico – Río Piedras campus, San Juan, PR 00931-3360, USA. 4 Department of Biology, 100 Highway 908, University of Puerto Rico, Humacao campus, Humacao, PR 00792, USA The use of Bayesian methods in ecology has recently increased. One of the reasons for this is the flexibility of Bayesian hierarchical models, which allows one to include information both on prior knowledge about the parameters of the model and on the relationships between them. Here we present some previously published examples of the application of Bayesian Hierarchical Models in the analysis of orchid data. Two of these examples are related to the analysis of dormancy length and life cycle of multiple species of Caladenia. In these particular examples, the use of Bayesian methods allowed efficient use of a relatively small dataset for attaining useful insights on the stage transitions in the life cycle of Caladenia. The other examples are related to the estimation of fitness for two orchid species, Caladenia valida and Tolumnia variegata, where we used Bayesian statistics to investigate the effect of variation in morphological characteristics across reproductive bouts on the distribution of individual fitness. 66 SESSION: MODELLING AND DATA ANALYSIS Friday, June 3 Consequence of temporal morphological variation; the emasculation of individual fitness Raymond L.TREMBLAY,1,2,, Maria Eglée PEREZ,2,3,, James D. ACKERMAN2,4. 1 Department of Biology, 100 Highway 908, University of Puerto Rico, Humacao campus, Humacao, PR 00792, USA. 2 Center for Applied Tropical Ecology and Conservation, PO Box 23341, University of Puerto Rico, Río Piedras campus, San Juan, PR 00931-3341, USA 3 Department of Mathematics, University of Puerto Rico, Río Piedras campus, San Juan, PR 00931-3355 USA. 4 Department of Biology, PO Box 23360, University of Puerto Rico – Río Piedras campus, San Juan, PR 00931-3360, USA In fitness studies, it is usual to measure individual morphological characteristics at the beginning of the study, and assume they are fixed through time. However, morphological characters, especially in orchids, can be highly variable in time; ignoring this variability could lead to underestimating or overestimating individual fitness. Using simulated data we fit models that do or do not take into account the variability of morphological characters. We used Bayesian hierarchical models, as they can naturally incorporate information on relationships between variables in the model. In an attempt to construct models with some veracity, we based our simulation on six years of data on the relationship between overall height of plants and fruit set probability from a population of an Australian orchid, Caladenia valida. Moreover we simulated the effect of increased variation in morphological characteristics on predicting fitness. Our results show that character variability has an effect on the estimation of individual fitness. 67 SESSION: MODELLING AND DATA ANALYSIS Friday, June 3 Darwin’s “uniform green carpet”: modelling the theoretical limits to the dispersal of orchid seeds David L. ROBERTS1, Gil BOHRER2 1 Durrell Institute of Conservation & Ecology, University of Kent, Marlowe Building, Canterbury, Kent, CT2 7NR. 2 Department of Civil & Environmental Engineering & Geodetic Science, The Ohio State University, 2070 Neil Avenue, Columbus, OH 43210, USA. Three years after the publication of On the origin of species…, Darwin in 1862 published On the various contrivances by which British and foreign orchids are fertilised by insects…, in support of his theory of evolution by natural selection. In it Darwin speculated that “… the great grandchildren of a single plant would nearly … clothe with one uniform green carpet the entire surface of the land throughout the globe”, noting that, their “minute seeds within their light coats are well fitted for wide dissemination…”. The notion of exceptional dispersability of their dust-like seeds in air currents is based on the fact that they have successfully colonised islands 1,000s of kms from the nearest seed source. This perception of their immense dispersability still continues, with no mechanistic evaluation of their properties. Here we present the dispersal distances for a range of theoretical orchid seeds and how this interacts with ecological filters. Knowledge of the dispersability, as described by seed characteristics that determine their aerodynamic properties and their interactions with turbulent flow inside and above forest canopies, is fundamental to building and understanding of the evolution of this remarkable family. 68 SESSION: MODELLING AND DATA ANALYSIS Friday, June 3 Population viability in Cypripedium calceolus in sites with different light regimes Anne JÄKÄLÄNIEMI1, Juha TUOMI2 1 Finnish Forest and Park Service, Torangintaival 2, 93600 Kuusamo, Finland 2 University of Oulu, Finland In forests, canopy disturbance and ground disturbances are the key components in releasing space for recruitment. In many plants the sexual reproduction peaks rather in disturbed than in stable environments. Light regime is an important factor influencing changes in forest vegetation composition and many herbs experience increased growth and recruitment in response to increased light during few years after canopy opening. Forests are important habitats for threatened species in Finland and in herbrich forests the spruce dominance decreases the light availability for understory species. Cypripedium calceolus L. is an endangered orchid typical for spruce dominated herb-rich forests. In this study, we explored the response of Cypripedium populations for a single tree removal. We used demographical data of ten experiment and ten control sites from the years 2001-2004 and 2008-2010. Management increased the density of flowering ramets, flowers and capsules, while no effect on the density of vegetative ramets or capsule production was seen. The effect on seedling density varied among habitats. In mesic habitats the increase was significant in all study years after management. Since the development of seedlings from seeds requires many years, it seems that the orchid had a seedling bank, where plants persist between the times of favorable periods of recruitment. In general, the tree removal increased the regeneration of populations, but the amount of success was partly dependent on habitat type. 69 SESSION: MODELLING AND DATA ANALYSIS Friday, June 3 Ex situ conservation of tropical African orchids Tariq STEVART 1, 2, 3, Bonaventure SONKE 1, 4, Murielle SIMO 4, Jean Philippe BITEAU 5, Faustino DE OLIVEIRA 6, Steven DESSEIN3, Claire MICHENEAU7, Vincent DROISSART 1, 2, 4, 8. 1 Missouri Botanical Garden, Africa & Madagascar Department, St. Louis, Missouri, USA. 2 Herbarium et Bibliothèque de Botanique africaine, Université Libre de Bruxelles, Belgium. 3 National Botanic Garden of Belgium, Meise, Belgium. 4 Plant Systematic and Ecology Laboratory, Higher Teacher’s Training College, University of Yaoundé I, Yaoundé, Cameroon. 5 Jardi-Gab, 6 Libreville, Gabon Herbier National de Sao Tomé, Jardin Botanique de Bom Sucesso, Sao Tomé et Príncipe 7 Evolutionary 8 Institut Biology and Ecology, Université Libre de Bruxelles, Belgium de Recherche pour le Développement (IRD), UMR AMAP, Montpellier, France. Orchid conservation represents a significant challenge in Central Africa which is facing increasing destruction of tropical forests through mining and logging activities. In this region, 70% of the epiphyte flora is composed of orchids, but most of these species are impossible to identify because of the lack of fertile material in the field. Species identification is however the first step towards any development of conservation strategies. To propose sustainable actions, a shadehouse cultivation system has been developed in São Tomé in 1997. Apart from allowing ex situ orchid species conservation, this structure has proved to be useful in providing fertile identifiable material, and similar actions have been extended across Central Africa. Until now, 13,000 living orchids collected in the field were put into cultivation in the shadehouses’ network situated in Central Africa (see www.orchid-africa.net/ombriere.asp). Each living specimen is digitalized in a relational database that allows a complete traceability from the sampling locality of living specimens to produce outputs like fertile herbarium specimens, silicagel samples, pictures, etc. Herbarium data are further used to assess the IUCN conservation status of threatened orchids. Our shadehouses’ system is particularly powerful for conducting thorough inventories of targeted areas since most living epiphytes found in the field can be brought into cultivation. We are currently working in sites identified as critical for conservation (e.g. the Coastal forest of Gabon, the island of Sao Tomé, Hills around Yaoundé in Cameroon), and we will start soon to work in West Africa and at Ambatovy site in Madagascar. 70 POSTER SESSION The effect of simulated eutrofication on the growth and competition of Serapias lingua and Plantago lanceolata Milan BALÁŽ, Zuzana KYJOVSKÁ Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, the Czech Republic; email: [email protected] It is widely acknowledged that eutrofication damages orchid populations and causes their vanishing. In principle, the enhanced availability of nutrients can affect the growth of orchids adversely either directly, disturbing physiological functioning of the orchid or its mycorrhizal symbiosis, or indirectly, through the enhanced competition for limiting resources with co-occurring plant species. We aimed to discriminate these mechanisms in a glasshouse cultivation experiment, using 5mM concentration of N in a nutrient solution to simulate eutrofication caused in nature by deposition of atmospheric N, whereas control solution was the same but 10× lower in N. The tuberous orchid Serapias lingua and Plantago lanceolata, an herb often co-occurring with the orchid on its Mediterranean localities, were cultivated in an 11-weeks lasting experiment of substitution design. S. lingua was naturally colonized by its mycorrhizal fungus without artificial inoculation, whereas P. lanceolata was inoculated by arbuscular mycorrhizal fungus Glomus intraradices. When grown in monoculture, the dry mass of P. lanceolata aboveground parts was approx. 5× higher when treated with high N compared to low N solution (F=4 555.61, P<0.001), but no similar effect on dry mass of new tubers was observed for S. lingua (F=0.04, n.s.). When both species were co-cultivated, dry mass of S. lingua tubers was lower compared to monoculture (F=8.51, P<0.05) and this effect was not modified by N availability (F=1.65, n.s.), whereas dry mass of P. lanceolata aboveground parts was higher compared to monoculture (F=234.07, P<0.001), namely when treated with higher N solution (F=61.06, P<0.001). Our results confirm importance of the indirect, by enhanced competition mediated adverse effect of eutrofication on orchids which corresponds well with high efficiency of orchid sites management based on mowing or grazing. 71 Orchids and their conservation in the Vysočina Region (Czech Republic) Kristýna BALÁŽOVÁ Section for the Protection of Natural Resources, Environment Department, Regional Authority of the Region Vysočina, Žižkova 57, 587 33 Jihlava, the Czech Republic The Vysočina Region covers the area of 6 796 km2 (9% of the Czech Republic). In the Region there are two protected landscape areas, eight natural parks and 172 small-size specially protected areas (SPA), covering the total area of 652 km2. Of this total count, the Regional authority manages 119 SPAs of two categories, 54 natural reserves and 65 natural monuments, and in 61 of them some of 25 orchid species growing in the Region can be found. The highest orchid species richness is in Svratecká hornatina in the NE part of the Region, where 18 species (e.g. Orchis ustulata or Spiranthes spiralis) occur. There are also 75 Natura 2000 localities in the Region, where e.g. Cypripedium calceolus or Orchis morio are growing. The majority of SPAs as well as Natura 2000 localities in the Region are private properties. The Vysočina Region supports practical management of these SPAs by approx. 163 thousands euro annually. For each SPA there is a management plan elaborated for 10 years period, and the practical management is applied in accordance to it, depending on available financial resources. On mesophilic meadows, steppe grasslands, and peat meadows the management is based on mowing followed by biomass transportation out of the site, or by sheep or cattle grazing, potentially supplemented by elimination of undesired woody plants or by grass cover disturbance. In forests it is usually aimed to support natural restoration of deciduous trees and of fir tress. When possible, the management is carried out by the property holders. 72 Population biology of three-toothed orchid (Neotinea tridentata (Scop.) R. M. Bateman) on its most important Moravian site over the period 1997-2010 Slavomír DOSTALÍK 1, Vlastimil RYBKA1, Romana ZEMÁNKOVÁ-BARTKOVÁ, Milan BALÁŽ2 1 Sagittaria – sdružení pro ochranu přírody střední Moravy, Lipové náměstí 3, 783 36 Křelov 2 Ústav experimentální biologie, Přírodovědecká fakulta Masarykovy univerzity, Kotlářská 2, 611 37 Brno Neotinea tridentata is tuberous orchid with the centre of its distribution in the Mediterranean region. In the Czech Republic there are seven recent localities and this orchid is treated as critically endangered. The most important site where more than 90% of whole Czech population of this species can be found is National nature reserve Strabišov-Oulehla. Since the end of 80ties of 20th century, management based on the elimination of woody plants and mosaic mowing realized after capsules ripening, followed by biomass removal is applied every year. The local population of N. tridentata is monitored since 1997. We observed a marked increase in number of plants, reaching its maxima in 2002 (695 plants). In the period 2003–2009 the lowest number of all plants was 463 and the mean was 552 plants. There was high interseasonal fluctuation of the number of fertile plants (coefficient of variation calculated from annual means CV=51.3%) and the percentage of fertile capsules from the number of flowers (CV=57.4%), mainly due to low values observed in 2000, 2003, and 2007. These were caused by dry weather during spring months, eventually combined with late frosts in 2007, which strongly damaged the orchids. The effect of weather was observable also on mean values of plant heights and diameters of leaf rosettes, whereas the number of flowers was not affected by weather and its interseasonal variation was low (CV=7.5%). We conclude that local N. tridentata population is in very good condition and we recommend to retain current site management practice. 73 Dynamics and energy performance of flowering and survival of Spiranthes spiralis, a critically endangered species, in its last locality in Bohemian Forest (Sumava), Czech Republic Zdeněk IPSER1, Zuzana BALOUNOVÁ1, Jiří BRABEC3, Jana JERSÁKOVÁ2 1University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Faculty of Agriculture 2University of South Bohemia, Branišovská 31,370 05 České Budějovice, Faculty of Science 3Regional Museum Cheb, Nám.Krále Jiřího z Poděbrad 393/4, 35001 Cheb A population of an endangered orchid species Spiranthes spiralis was discovered in 1980 in the National natural monument Pastviště u Fínů in village Albrechtice, near Sušice. Since 1985 the number of flowering individuals of this population has been regularly monitored. Since autumn 1998 all the specimen found have been permanently marked and biometrically measured. Since the beginning of monitoring, big fluctuations in the number of flowering specimens or in survival of the individual plant rosettes have been observed among the years. Our poster summarizes results (i) on the effect of leaf area on the probability of flowering and the number of flowers, (ii) on the cost of reproduction, (iii) on the survival probability of individual plants over 12 years of monitoring, and (iv) on flowering dynamics of a population over 24 years. 74 Molecular history of Malaxis monophyllos, species with boreal-mountain range disjunction in Europe Edyta JERMAKOWICZ1, Emilia BRZOSKO2Ada WRÓBLEWSKA3, 1 Institute of Biology, University of Bialystok, ul. Świerkowa 20B, 15-950 Bialystok, Polska 2 Institute of Biology, University of Bialystok, ul. Świerkowa 20B, 15-950 Bialystok, Polska 3 Institute of Biology, University of Bialystok, ul. Świerkowa 20B, 15-950 Bialystok, Polska The phylogeography of rare Malaxis monophyllos species, with boreal-mountain range disjunction was analyzed using three cpDNA markers. Species occurs in Boreal part of Europe and along the Alpine, Carpathian and Sudetian mountain with a marked tendency to occupy the natural as well as anthropogenic habitats. We investigated forty-nine populations over the whole its European distribution. Two haplotype groups were distinguished which co-occurred in Europe. The first group contained both the widespread haplotypes and local haplotypes exclusive for Norway. The second group included haplotypes unique for Alps and also widespread through boreal, alpine and highland populations. Analyzes revealed also high average haplotype diversity (hd=0.71) and low nucleotide diversity (π=0.0012) at the species level. By this phylogeographical researches we want to make some main conclusions about elucidate the refugial and evolutionary history of Malaxis monophyllos in Europe, with relation to its range disjunction. 75 Revision of the influence of hypochlorite disinfection on the germination of orchid seeds in vitro Kateřina KMECOVÁ1, Stanislav VOSOLSOBĚ2, Michael MIKÁT3, Jan PONERT2,4 1 Gymnázium a Střední pedagogická škola Znojmo, Pontassievská 3, 669 02 Znojmo, Czech Republic 2 Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Praha 2, Czech Republic 3 Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Praha 2, Czech Republic 4 Prague Botanical Garden, Nádvorní 134, 171 00 Praha 7 – Troja, Czech Republic Sodium and calcium hypochlorites are two major disinfections used in sowing the orchid seeds in vitro. Their effects are notably different, even if they include same concentration of hypochlorite anions. We suppose, that different effect of this disinfections can be explained by their different basicity (pH(NaClO) ~ 12.5, pH(Ca(ClO)2) ~ 11.5). To confirm this hypothesis we tried to prepare solution of NaClO at pH ~ 11.5 and Ca(ClO)2 at pH ~ 12.5 while maintaining the same concentration of ClO -, which had been determined by iodometry. Prepared solutions were used for disinfection of seeds of European terrestrial species Dactylorhiza majalis and Anacamptis morio sown in vitro. During the pH-adjusting of calcium hypochlorite solution by NaOH, complete precipitation of Ca2+ occurs at pH ~ 12 and therefore we obtain simply NaClO solution. When adjusting the pH of NaClO solution by HCl, partial decomposition of hypochlorite proceeds and under pH ~ 10 is the solution unstable. Although we managed to prepare disinfections with complementary pH and the same concentrations (w(ClO-) ~ 3,4 %), we did not achieved comparable germination rates. Application of NaClO at both pHvalues led to seed death, which could possible be explained by instability of ClO -, which concentration in solution decrease even some hours after the pH-adjustment and it could cause different oxidation activity of the solution. This will be confirmed by further experiments. 76 Passport data of orchid seeds in cryobank Galina KOLOMEITSEVA1, Violetta ANTIPINA 1,Tatiana NIKISHINA2, Alexander POPOV2, Alexander SHIROKOV3 1 The Main Botanical Garden RAS, Russia, Moscow 2 Timiryazev Institute of Plant Physiology RAS, Russia, Moscow 3 Botanical Garden of the Nizhny Novgorod State University, Russia, Nizhny Novgorod Cryogenic storage of seeds at extremely low temperature is widely used for the creation of base collections and gene banks of rare and endangered plant species. Cryobank of orchid seeds has been in operation since 2001 in Timiryazev Institute of Plant Physiology and nowadays it has 105 specimens of orchid seeds. Mature seeds were harvested from plant species of the collection of the Main Botanical Garden or places of natural habitat and stored prior to cryopreservation for a different period in refrigerator. The seeds were frozen in plastic cryotubes (1 ml) by direct immersion in liquid nitrogen. The passport of orchid seeds included scanning electron microscopy photo was developed. Each passport contents the following parameters: scientific name of plant sample, inventory number of specimen, taxonomic data (subfamily), conservation status, ecological data (epiphyte, lithophyte, terrestrial plant), area of geographic distribution, cultivation place of generative plants from which seeds were obtained, parameters of greenhouse climatic conditions (temperature, air humidity, light, cultivation method), total number of generative plants, methods of pollination (autogamy, geitonogamy, xenogamy), total number of fruits obtained, duration time of fruit ripening, date of fruits collect, seed type, quantitative data of seeds (length and width of the seed and embryo), evaluation data concerning seed quality (water content, initial rate of seed germination, regrowth percentage after 1 month storage in liquid nitrogen, percentage of abnormal (without embryo) seeds), date of seeds immersion in liquid nitrogen, cryotubes location (number of rack, tube adaptor), total number of cryotubes, place of seeds storage, list of project performers. This research will be continued. 77 Population of Epipogium aphyllum from the archipelago of Kem’-Ludy (White Sea): intensive study vs. conservation Yuriy O. KOPYLOV-GUS’KOV1, Dmitry F. LYSKOV1 1 Moscow State University, Faculty of Biology, Department of Higher Plants, 119992, Vorob'yovy Gory, 1-12, Moscow, Russia Despite a wide geographic range in Eurasia, Epipogium aphyllum (F.W. Schmidt) Sw. – an achlorophyllous mycoheterotrophic orchid – is rare and most of its populations are not stable. The generative shoots exist only a few weeks then die off and decompose. These are the reasons why many aspects of biology of Epipogium are still studied insufficiently. A population of Epipogium on the Bol’shoj Asaf’ev Island (archipelago of Kem’-Ludy, White Sea) is an exceptional example of stable population of this species. This population was discovered about sixty years ago and survived till nowadays. Starting from 2001 till 2008, it contained not less than 30 flowering individuals every year. In 2005, we started our observations on reproductive biology of Epipogium. We have not confirmed bumblebees as pollinators of this species, but damages and absence of pollinia from some flowers can be evidences of entomophily. Potential seed production is high: number of ovules per ovary can reach 4600. However the main way of reproduction is a vegetative propagation via protocorm-like buds forming on stolons. In 2008, we noticed that a path-net made by us in previous years did not disappear. This is a result of intensive walking through territory of population, which is necessary for detailed study of plant. This can be negative for stolons with buds situated in forest litter layer because of their fragility and compression of litter layer. Thus, we decided to suspend our observations and let territory to recover. 78 Vegetative reproduction Dactylorhiza incarnatа (L.) Soo. at protocorm stage in vitro Lavr KRYUKOV, Alexander SHIROKOV Botanical garden of the N. I. Lobachevsky State University of Nizhny Novgorod; Russia, 603062 Nizhni Novgorod, pr. Gagarina, 23; email: [email protected] Current study focuses on identifying of natural potential of Dactylorhiza incarnata vegetative propagation in vitro at the developmental stage "protocorms" under the influence of phytohormones. The optimal phytohormonal conditions for vegetative propagation protocorm were investigated and the maximum effect of vegetative propagation was estimated. Active vegetative propagation of D.incarnata at the initial stage of the primary shoot development in the medium containing indolylbutyric acid (IBA) and 6-benzoaminopurin (6-BAP) was recorded. We have revealed that hormones in equal ratio give pronounced multiplier effect. When the content of phytohormones in the medium was 1 mg/l protocorms formed up to 8-12 primary shoots and its size was the same as in control. The surface of shoots was covered with dense rhizoids. As the сcontent of phytohormones was increased 3mg/l the number of primary shoots formed on protocorms reduced to 3-5 pieces, but its size increased to 2.5 mm and the surface was covered with rhizoids. Under maximum concentration of phytohormones (5 mg/l) protocormes formed 3-5 primary shoots which possessed only a few rhizoids, but the size of shoots was larger than 4.0 mm. In case of IBA dominance (3:1 and 5:1) weak multiplication of primary shoots (2-4 pcs.) was recorded but the shoots had elongated root-like shape and was covered by abundant rhizoids. In other cases the multiplication of primary shoots was less pronounced. 79 In vitro germination of Dactylorhiza incarnata, Dactylorhiza maculata and Cypripedium calceolus Jiří KYNCL1, Hana VEJSADOVÁ2 1 Faculty 2Silva of Agriculture, University of South Bohemia, České Budějovice, Czech Republic Tarouca Research Institute for Landscape and Ornamental Gardening, CZ-252 43 Průhonice, Czech Republic Seed germination rate under in vitro asymbiotic conditions was determined in three critically endangered species of terrestrial orchids (Dactylorhiza incarnata subsp. serotina, Dactylorhiza maculata subsp. maculata and Cypripedium calceolus). The surface sterilization of mature seeds using 70% ethanol and 7.2% calcium hypochlorite was provided. Peptone effect on the percentage of germination was studied. In Dactylorhiza incarnata subsp. serotina (seed collection in 2004), peptone had no significant effect on the germination rate. In Dactylorhiza incarnata ssp. serotina (seed collection in 1998), peptone stimulated the germination rate by 5%. In Dactylorhiza maculata subsp. maculata, there was a turn in the germination rate in the presence of peptone – the reduction of germinated seeds was observed (up to 20%). In Cypripedium calceolus, the influence of ferric salt on the development of protocorms is studied – the assessment of results is under way. Owing to the present results, we have been able to start a pilot study of in vitro germination under symbiotic conditions with two mycorrhizal isolates from roots of D. incarnata subsp. serotina and D. maculata subsp. maculata. 80 Germination course, mycorrhizal partners and nutritious mode of two Listera species Milan KOTILÍNEK1, François-Xavier JOLY2, Tamara MALINOVÁ1,3, Jana JERSÁKOVÁ1,3, Marc-André SELOSSE4 1 Faculty of Science, University of South Bohemia, České Budějovice, Czech Rep. 2 Faculty 3 of Environmental and Life Sciences, University of Rennes 1, Rennes, France Department of Biodiversity Research, Global Change Research Centre AS CR, , Brno, Czech Rep. 4 Université de Montpellier II & Centre d’Ecologie Fonctionnelle et Evolutive, CNRS UMR 5175 Montpellier, France All orchids are dependent on mycorrhizal fungi during germination course, but only some of them require fungal nutrition also at maturity. Species Listera ovata and L. cordata belong to tribe Neottieae, whose members typically exploit nearby trees for carbon via shared ectomycorrhizal fungi. In the Central Europe, L. cordata is growing solely in bog spruce forests, while L. ovata inhabits broader spectrum of forest and non-forest habitats, including open meadows. To date, we have no knowledge on mycorrhizal partners of both species, nor on the level of dependency on fungal nutrition. We investigated (i) seed germination course in 6 populations of L. cordata and 12 meadow and forest populations of L. ovata, (ii) mycorrhizal partners in roots of adult plants and protocorms using rDNA ITS sequencing and electron microscopy and (iii) level of mycoheterotrophy using stable carbon and nitrogen isotopes. Seeds of both species suffered high mortality during two-years of in situ germination. While L. ovata produced few protocorms at both L. ovata and L. cordata localities, L. cordata never reached a protocorm stage. Both adults and protocorms of both species contained mainly saprophytic fungi from Sebacinaceae family and the observed fungal spectra differed according to habitat type. The isotopic signals of both species did not differ from autotrophic plants. We conclude that despite their relatedness to mycoheterotrophic orchids, and their growth in forests, both Listera species associate with saprophytic fungi from rhizoctonia group and are autotrophic in adulthood. 81 A comparative study of the seed germination capabilities of Anacamptis palustris (Orchidaceae), a threatened terrestrial orchid, and other more common Anacamptis species, by asymbiotic culture in vitro Sara MAGRINI1, Alessandro DE CARLI1, Silvano ONOFRI2, Anna SCOPPOLA2 1 Tuscia Germplasm Bank of Viterbo Botanical Gardens, University of Tuscia, Largo dell’Università, I-01100 Viterbo, Italy 2 Department of Ecology and Sustainable Economic Development, University of Tuscia, Largo dell’Università, I-01100 Viterbo, Italy The increasing emphasis on terrestrial orchid conservation has lead to conservation actions for a wide range of threatened Mediterranean species. Many terrestrial orchids are currently at great risk for extinction as a result of a multiplicity of threatening processes. In the present study, we focus on seed germination capability in orchid biology. Specifically: (1) comparisons were made between the germination capability of a threatened species, Anacamptis palustris, that grows in marsh habitats and is strongly affected by human activity, and three other more common species, A. laxiflora, A. morio, and A. papilionacea; and (2) we discuss the impact of germination capability on orchid distribution and conservation. Asymbiotic germination tests were performed with mature seeds, using BM1 medium. In vitro seed germination and protocorm developmental stages were evaluated up to twenty weeks after sowing. The data were processed using germination indices that were statistically analyzed to assess significant differences or correlation. This is the first report of successfully asymbiotic germination of mature seed of A. palustris that was compared with germination results of other Anacamptis species. In all the cultures we could observe all the germination stages, until the development of seedling with shoots, but with significant differences in times and percentages related to the species. Our results show a significant correlation between the rarity of the species and their germinability that demonstrate the presence of intrinsic, biological factors that affect and limit the distribution of A. palustris. 82 An integrated strategy for the conservation of Limodorum (Orchidaceae): the example of the Marturanum Park (Latium, Italy) trabutianum Sara MAGRINI1, Sergio BUONO2, Emanuele GRANSINIGH2, Massimiliano REMPICCI2, Anna SCOPPOLA3 1 Tuscia Germplasm Bank of Viterbo Botanical Gardens, University of Tuscia, Largo dell’Università, I-01100 Viterbo, Italy 2 GIROS, “Etruria meridionale” Section, I-01010 Oriolo Romano (Viterbo), Italy 3 Department of Ecology and Sustainable Economic Development, University of Tuscia, Largo dell’Università, I-01100 Viterbo, Italy Limodorum trabutianum Batt. is a steno-Mediterranean orchid with a highly fragmented distribution area that includes Morocco, Algeria, Portugal, Spain, France, and Italy. Our monitoring of the small population within the Marturanum Regional Park (Barbarano Romano, Viterbo, Italy) has shown a strong demographic decline, about 70% in the last 8 years, highlighting the urgent need to take serious preservation measures. Therefore, an integrated strategy was performed for the conservation of this population. The Marturanum Park was chosen as testing-area where experience in situ/ex situ actions before spreading them to the whole distribution area. In fact, the aim of this research is to test the effectiveness or the limits of these measures in order to extend them to other populations, and perform a successful conservation plan. These in situ measures were carried out: 1) fencing the population area, only during the anthesys/fruiting phases, in order to prevent the damages caused by trampling, grazing, and by the massive presence of wild boars; 2) tourism limitation in the area; 3) population census and monitoring; 4) assessment of present and potential threats. Furthermore, these ex situ actions are thought to be necessary: 6) seed banking to preserve its genetic diversity over time; 7) evaluation of the reproductive success (germination, growth, and development capability) to highlight eventual intrinsic risk factors; 8) in vitro reproduction for conservation and reintroduction. We report here the first results that can provide useful indications for the management and conservation of this threatened orchid. 83 European mediterranean orchid cultivation – from seed to mature plant Jan PONERT1,2, Stanislav VOSOLSOBĚ2 1 Prague Botanical Garden, Nádvorní 134, 171 00 Praha 7 – Troja, Czech Republic 2 Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Praha 2, Czech Republic Terrestrial orchid cultivation is often presented to be more difficult than in epiphytical species. At least in some cases it can be true, but it does not mean that it is impossible. Here we present our years of experiences with some European orchids, mainly tuberous species from mediterranean region. Firstly we summarize possibilities of cultivation in individual orchid groups in relation to mycorrhiza and ecology. In second part we present successful technique for propagating these plants via asymbiotic in vitro sowing on example of genus Dactylorhiza and Ophrys. Seeds are sowing in laboratory into petri dishes with suitable medium. After one to two transplantations (approx one and half year) small seedlings are prepared for potting and cultivating in a glasshouse. Several different substrates are using for ex vitro plant cultivation, but nearly all mediterranean Orchidoideae can be grown in a mixture of Seramis, pumice and perlite with small amount of suitable soil. For a whole time is very important proper temperature adjustment. For cultivation in a glasshouse is important correct soil moisture too. Flowering plants can be obtained during 3 to 4 years. This work has been kindly supported by UTSR. 84 Phytohormonal regulation of orchid protocorm development Jan PONERT1,2, Jan PETRÁŠEK1,3, Helena LIPAVSKÁ1, Stanislav VOSOLSOBĚ1 1 Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Praha 2, Czech Republic 2 Prague Botanical Garden, Nádvorní 134, 171 00 Praha 7 – Troja, Czech Republic 3 ASCR, Institution of Experimental Botany, Rozvojova 263, 165 02 Praha 6, Czech Republic Orchid seeds are very small as is common in many other mycoheterotrophic plants. These seeds contain only small globular embryo without any differentiated organs or their primordiums. After germination, protocorm develop from embryo and this differentiates shoot meristem. Hence in orchids the first specialised meristem formation occurs after germination. We focused on regulation of this process by phytohormones, mainly auxins and cytokinins. Our results show, that auxins support basal part development while cytokinins support apical part development and meristematic regions formation for shoot development. We propose the model of polar auxin localization in protocorm as fundamental process for polarity establishment. This model corresponds to auxin localization in globular stage of Arabidopsis thaliana embryo. According to this, we consider embryos in orchid seeds to be homologous to globular stages of other plants embryogenesis. 85 Role of saccharides in Orchideoid Mycorrhiza Jan PONERT1,2, Petr KOHOUT3, Stanislav VOSOLSOBĚ1, Šárka ŠKORPÍKOVÁ1, Helena LIPAVSKÁ1, 1 Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44 Praha 2, Czech Republic 2 Prague Botanical Garden, Nádvorní 134, 171 00 Praha 7 – Troja, Czech Republic 3 Department of Mycorrhizal Symbioses, Institute of Botany, AS CR, Lesni 322, 252 43 Pruhonice, Czech Republic Nearly the only source of nutrition for young developmental stages of orchid seedlings is mycorrhiza. Till this time it is not clear which compounds are transmitted in mycorrhizas as energy and material source and which play a signal role. In many cases, saccharides are not only carbon and energy source, but signal too. In light of this we focused on possible role of saccharides in orchideoid mycorrhizas. We have tested possibilities of different saccharides utilization by protocorms of selected orchid species (Dactylorhiza majalis, Ophrys lojaconoi, Oeceoclades decaryana). Results confirmed ability to utilize sucrose, fructose, glucose, maltose, raffinose, sorbitol and trehalose. Lactose and mannitol are utilized only at a very low level and pure galactose is not metabolised at all. In addition to this, hexoses are able to inhibite germination of some orchid species. Analysis of mycorrhizal fungi endogenous saccharides revealed a wide range of different sugars, often with relatively high levels of mannitol. It is interesting, that orchids are not utilizing mannitol at higher levels although it occurs in mycorrhizal fungi. We suspect a signaling role. Trehalose utilization is also interesting because many other plants contain it in only very low levels for signaling purposes. Orchid ability to utilize it should be connected with mycotrophy. 86 Regeneration Ecology of the Lady’s Slipper Orchid Grace PRENDERGAST1, Alan C. GANGE2 1 Royal Botanic Gardens, Kew, UK. 2 Royal Holloway University of London, UK. The lady’s slipper orchid (Cypripedium calceolus) was reduced to one plant in Britain and recruitment from seed is infrequent, throughout its range. Seedling recruitment is desirable, to maximize resilience to environmental change and understanding germination requirements would facilitate choice of appropriate sites for reintroduction and long-term survival of self-sustaining populations. Experiments were carried out using seeds of C. calceolus and closely related species, to assess the effects of seed ripening time, temperature, nutrients, bleaching treatments and potential symbiotic fungi on in vitro germination. Seedlings were acclimatized using a range of temperatures, compost mixes and commercially available mycorrhizae. Seedling reintroduction and field germination trials were initiated and monitored. Percentage germination was higher from undehisced capsules than for dry seeds and cold stratification improved percentage germination of these seeds. Germination of seeds, from dehisced capsules occurred over a range of temperatures, with a peak at 15 C. Diurnal temperature variation and long bleaching treatments both improved percentage germination. Acclimatized plants survived better at lower temperatures and addition of mycorrhizae improved percentage survival and vegetative proliferation occurred. No field germination was observed. The results suggest that seeds are fully mature before dehiscence, later acquiring dormancy. Prolonged bleaching may removal inhibitory chemicals and germination may naturally occur close to the soil surface where diurnal variation is greatest. Further in vitro trials and field monitoring will be carried out in 2011. 87 Cypripedium calceolus in situ: seed longevity and a requirement for long incubation and cold winters for breaking dormancy Hanne N. RASMUSSEN1, Henrik Æ. PEDERSEN2 1Forest & Landscape, LIFE faculty, University of Copenhagen, Rolighedsvej 32, DK-1958 Frederiksberg C, Denmark; email: [email protected] 2Botanical Garden & Museum, Natural History Museum of Denmark, University of Copenhagen, Gothersgade 130, DK1123 Copenhagen K, Denmark, E-mail: [email protected] Cypripedium calceolus, the European Lady-Slipper, only occurs in two populations in Denmark. One population is stagnant or in slow decline, whereas the other is in rapid increase. In search of an explanation, we carried out a study of genetic diversity, seed set, compatibility and germination, comparing the two sites. The study included lengthy germination experiments in vitro and in situ that are reported below. Mature seeds from within- and among-population crosses were harvested and planted out on the sites in seed packets in November 2005. Samples were subsequently checked about once a year. Germination was first observed in June 2010, following two successive cold and snowy winters, and only in one out of 36 sampled seed packets. This appears to be the first observation of germination in this species in the field with an approximate time scale attached. Seedlings were in stages from broken testa to initial root development. They expanded sideways out through the testa, and there were no root hairs, but the micropylar end of seedlings contained fungal pelotons, indicating that initial infection took place through micropyle. The appearance of the remaining seeds was healthy. This suggest a requirement for successive cold treatments and long incubation times, an interpretation that is supported by asymbiotic germinations in vitro of the same seed lots. 88 Ecological demands of Pseudorchis albida on site management, pollinators and mycorrhizal fungi Zuzana ŠTÍPKOVÁ1, Tamara MALINOVÁ1,2, Kateřina JEŘÁBKOVÁ1, Petr KOHOUT3,4, Martin VOHNÍK3,4 a Jana JERSÁKOVÁ1,2 1 Faculty of Science, University of South Bohemia, České Budějovice, Czech Rep. 2 Department of Biodiversity Research, Global Change Research Centre AS CR, , Brno, Czech Rep. 3 Faculty of Science, Charles University, Prague, Czech Rep. 4 Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice, Czech Rep. Pseudorchis albida is a highly endangered orchid species of the Czech flora growing in nutrient poor Nardus meadows from mountain to alpine level. An alarming, more than 80 % decrease of populations, led us to explore its hitherto unknown ecological demands on site management, pollinators and mycorrhizal fungi. Since 2003 we have followed population dynamics of three P. albida populations in mown and unmown permanent plots. We also analyzed mycorrhizal fungi associated with adults and performed a seed sowing experiment using retrievable seed packets. Annual monitoring showed that plants in mown plots flower more frequently and have smaller leaf area than those in unmown plots which suffer from higher competition of surrounding vegetation. Mowing and disturbance over 7 years have not shown an impact on new recruits so far. The species exhibits high cost of reproduction with 63% probability of being sterile following year. Dormancy typically lasts for 1 year. Pseudorchis albida is a highly reproductive nectariferous species, pollinated by frequently occurring crepuscular moths from Pyralidae and Pterophoridae families, and occasionally by Empis flies (Empididae). Both roots of adults and germinated seeds associate with very similar basidiomycete fungi from Tulasnellaceae family, which are common mycorrhizal partners of meadow orchids. After two years in soil, the seed germination in individual packets ranged from 0 to 70%, but the overall probability for a seed to reach a protocorm stage was very low (0.75%). The species rarity may be caused by limited seed dispersal and high seedling mortality during germination phase. 89 Molecular phylogeny of continental Angraecum species from sects. Dolabrifolia and Pectinaria (Vandeae, Orchidaceae) Murielle SIMO1, Claire MICHENEAU2, Bonaventure SONKÉ1, STÉVART3, 5, 6 2, 3, Vincent DROISSART3, 4, 5, Olivier J. HARDY2 , Tariq 1 Plant Systematic and Ecology Laboratory, Higher Teacher’s Training College, University of Yaoundé I, Yaoundé, Cameroon 2 Evolutionary Biology and Ecology, Université Libre de Bruxelles, Bruxelles, Belgium 3 Missouri Botanical Garden, Africa & Madagascar Department, St. Louis, Missouri, USA 4 Institut de Recherche pour le Développement (IRD), UMR AMAP, Montpellier, France 5 Herbarium et Bibliothèque de Botanique africaine, Université Libre de Bruxelles, Bruxelles, Belgium 6 National Botanic Garden of Belgium, Meise, Belgium Recent molecular studies have shown that the genus Angraecum, the most diverse genus of angraecoid orchids is polyphyletic with a clear-cut phylogenetic pattern between species occuring in Africa, and those occuring in Madagascar and nearby islands. This situation makes particularly difficult the interpretation of evolutionary patterns within the genus, especially for sections that encompasses species in both West Indian Islands and African continent. Our study was conducted on species occuring in tropical Africa, and belonging to sects. Dolabrifolia (African continent exclusively) and Pectinaria (African continent and West Indian Islands). To investigate systematics and biogeography of these continental orchids, phylogenetic relationships were inferred from five plastid DNA regions (matK, rps16, trnC-petN, trnL-F and ycf1), and the nuclear rDNA internal transcribed spacer (ITS). Parsimony analyses conducted on each marker separately provided identical patterns of relationships: (i) sects. Dolabrifolia and Pectinaria form together a well-supported clade, of which Malagasy-Mascarene species of sect. Pectinaria are excluded; (ii) within the African clade, both sections are monophyletic, each receiving a high bootstrap support in all analyses. Although we used a large set of molecular data, the resolution of trees at the species-level was low, especially for the complex A. aporoides – A. distichum – A. bancoense of sect. Dolabrifolia. These results were not expected since most species (including new species) seem to form well-defined groups on the basis of morphology. Further studies, investigating phylogeographic patterns and population genetic structure would be worthwhile to better understand the complex evolutionary history of this group of orchids. 90 Taxonomic revision and phylogenetic analysis of continental African Angraecoid Orchidaceae Tariq STÉVART1,2,3 Vincent DROISSART1,2,4, Claire MICHENEAU5, Murielle SIMO2,5,6, Bonaventure SONKÉ1,5,6, Olivier J. HARDY5, Daniel GEERINCK 2,3, Gregory M. PLUNKETT7 1 Africa & Madagascar Department, Missouri Botanical Garden, St. Louis, Missouri, USA 2 Herbarium et Bibliothèque de Botanique africaine, Université Libre de Bruxelles, Bruxelles, Belgium 3 National Botanic Garden of Belgium, Meise, Belgium 4 Institut de Recherche pour le Développement (IRD), UMR AMAP, Montpellier, France 5 Evolutionary Biology and Ecology, Université Libre de Bruxelles, Bruxelles, Belgium 6 Plant Systematic and Ecology Laboratory, Higher Teacher’s Training College, University of Yaoundé I, Yaoundé, Cameroon 7 Cullman Program for Molecular Systematics, The New York Botanical Garden, USA The infrafamilial classification system for orchids that has been developed over the past decade remains incomplete primarily because of the complexity of Epidendroideae, the last subfamily remaining to be treated. Within this subfamily, the angraecoid orchids are one of the most taxonomically problematic groups. To help overcome this gap, a project recently funded by the U.S. National Science Foundation, will study four key angraecoid groups centered in Central Africa, representing a total ca. 40 taxa: two sections of Angraecum and two morphologically well delimited genera (Ancistrorynchus and Bolusiella). The project involves four major activities: (1) intensive field work, (2) phylogenetic analyses, (3) collections-based taxonomic revisions, and (4) broad data dissemination. Field work will employ an orchid sampling strategy developed in Central Africa that has proven to be an effective method for producing high-quality liquid-preserved specimens, photos, and leaf tissue for phylogenetic analyses. The study includes several innovative elements. (1) Results will provide the scientific community with critical information to explore important evolutionary patterns in Orchidaceae. (2) Our inventory work will focus on the largely unexplored central portion of the Congo Basin. (3) The use of a near-comprehensive sampling of taxa and sequence data from a broad set of DNA markers (both the plastid and nuclear) will allow the production of a robust phylogeny. (4) The phylogenetic framework will provide the information needed to re-interpret generic circumscriptions in the Angraecoids. (5) Finally, we will use a new distribution-modeling method to improve estimates of Areas of Occupancy, thereby increasing the accuracy of threat assessments. 91 Breeding system potential of four orchid species with different pollination syndromes Izabela Tałałaj1, Emilia Brzosko2 1University of Białystok, Institute of Biology, ul. Świerkowa 20B, 15-950 Białystok, Poland; [email protected] 2 University of Białystok, Institute of Biology, ul. [email protected] e-mail: Świerkowa 20B, 15-950 Białystok, Poland, e-mail: Factors enabling and limiting selfing and crossing were analyzed in four orchids that represent a different pollination syndromes: food deception of Cephalanthera rubra, unspecialized pollination of Neottia ovata and specialized pollination by the Lepidoptera of Platanthera bifolia and Gymnadenia conopsea s.l.. In these species high level of self- and cross-compatibility was noted. However, quantity and quality of seeds producing through induced autogamy, geitonogamy and xenogamy were differentiated between species. In P. bifolia the lowest seed number was noted as a result of experimental autogamy and geitonogamy in relation to crossing (P<0.001). Otherwise, in N. ovata after induced autogamy the highest seed number was developed in relation to the remaining treatments (P<0.05). From the studied species the poorest quality of seeds from the particular breeding treatments was observed in G. conopsea s.l. (in average from 13.7% to 58.6% seeds without embryos). The highest quality of all seeds, regardless of the treatment type was documented in C. rubra. Despite self-compatibility of each species, only in G. conopsea s.l. spontaneous autogamy was proved (5%31.4% fruit set without pollinators). Possible relations of the breeding systems in four orchids and their evolutionary consequences are discussed in the context of different pollination syndromes. 92 Effects of geitonogamy and xenogamy on seed quality of rewarding and nonrewarding orchid species Kadri TALI1, Mirjam METSARE1, Kirke PILVIK2 1 Institute of Agricultural and Environmental Sciences, University of Earth Sciences, Riia 181, Tartu, Estonia 2 Institute of Ecology and Earth Sciences, University of Tartu, Lai 40, Tartu, Estonia There is experimental evidence that pollinators visit fewer flowers per inflorescence on nectarless plants. Nectarless orchids generally have lower reproductive success than do rewarding ones. We used one nectarless and one rewarding species (Orchis militaris and Gymnadenia conopsea respectively) to test effects of self- and cross-pollination on seed quality. Manipulations in two populations took place in 2010 and seeds were both TTC-tested and germinated on media. Orchis militaris showed more differentiation between treatments i.e. seed quality depended more on pollination mode than in case of Gymnadenia conopsea. 93 Vegetative morphology and population biology of Bletilla striata (Orchidaceae) Irina TATARENKO1, Katsuhiko KONDO2 1 Open University, Walton Hall, Milton Keynes, UK 2 Tokyo Agricultural University, Tokyo, Japan Bletilla striata is a large terrestrial orchid growing in meadows in Honshu Island (Japan) at the northern boarder of the geographic range. There species has a short winter break in vegetation, however in a warmer climate of Okinawa plants keep foliage leaves all year around. Two populations of the species were studied in Hiroshima Prefecture, Japan. The pollination and seeds production was very much successful only in one population. Seeds germinated rapidly, within 2 months after sowing. It took 1-2 months to develop protocorms, however the protocorms and juvenile plants death rate was significant, especially at early stages of development. Intensity of vegetative reproduction reached about 30% in both populations studied; it demonstrates similar rates in all age groups including juveniles. Large perennial corms form a specific type of “storage rhizome”, regularly branching and forming a dense layer in the turf. High density of growth of the adult plants underground organs as well as well as significant shading by large foliage leaves prevent an establishment of young plants in the area occupied by the adult clone. Spatial structure of the population was characterized by disperse of the young individuals in 2-5 meters diameter around the long-lived and dense clone. 94 Identifying important areas for orchid conservation in Crete Spyros TSIFTSIS1, Ioannis TSIRIPIDIS1, Panayiotis TRIGAS2 1 Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece 2 Laboratory of Systematic Botany, Department of Agricultural Biotechnology, Agricultural University of Athens, GR11855 Athens, Greece Crete is especially rich in orchids and their distribution is well known and documented by many floristic studies. This information, however, has not been used yet to set conservation plan and priorities. In the present study, we used both published and unpublished distribution data together with environmental ones in order to predict orchids’ potential distribution applying the MaxEnt model. The resulted probabilistic maps of species occurrence were used in the Zonation decision support tool in order to identify the important areas for orchids’ conservation in the island. Sites’ prioritization was performed by applying a species weighting scheme, which was based on orchids’ niche breadth. Site scores, calculated by the Zonation analysis, were related to variables corresponding to topography, vegetation and geology using regression tree models. The high importance sites were found on Mts Thripti, Ida and Lefka Ori, as well as at the low altitude areas east of Heraklion and at the easternmost part of the island. Most of the variation in the site scores was explained by geological substrate, latitude and altitude. Based on the regression tree analysis, sites with the highest scores have been found at medium and high altitude areas, which correspond to medium latitudes. These areas are mainly located eastwards of Mt Thripti and they dominated by limestones, ophiolithic rocks and deposits. Our results indicate that areas of the main mountainous ranges of Crete, as well as those located near the north and northeastern coast are of special interest for orchids’ conservation and should be appropriately managed. 95
