Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.

[1]  E. Pansarin,et al.  Floral biology and histochemical analysis of Vanilla edwallii Hoehne (Orchidaceae: Vanilloideae): an orchid pollinated by Epicharis (Apidae: Centridini) , 2014 .

[2]  M. Kozieradzka-Kiszkurno,et al.  Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae) , 2014, Plant Systematics and Evolution.

[3]  M. Stpiczyńska,et al.  Comparative anatomy of floral elaiophores in Vitekorchis Romowicz & Szlach., Cyrtochilum Kunth and a florally dimorphic species of Oncidium Sw. (Orchidaceae: Oncidiinae). , 2014, Annals of botany.

[4]  J. Otero,et al.  nocturnal pollInatIon by Fungus gnats oF the colombIan endemIc specIes, Pleurothallis marthae (orchIdaceae: pleurothallIdInae) , 2014 .

[5]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[6]  M. Stpiczyńska,et al.  Anatomy and ultrastructure of spur nectary of Gymnadenia conopsea (L.) Orchidaceae , 2014 .

[7]  M. Stpiczyńska.  Anatomy and ultrastructure of osmophores of Cymbidium tracyanum Rolfe (Orchidaceae) , 2014 .

[8]  W. M. Whitten,et al.  Convergent evolution of floral signals underlies the success of Neotropical orchids , 2013, Proceedings of the Royal Society B: Biological Sciences.

[9]  B. Gravendeel,et al.  Taxonomy, molecular phylogenetics, reproductive isolation, and niche differentiation of the Specklinia endotrachys species complex (Orchidaceae: Pleurothallidinae) , 2013 .

[10]  B. Gravendeel,et al.  A new Specklinia (Orchidaceae: Pleurothallidinae) from Costa Rica and Panama , 2013 .

[11]  R. DeSalle,et al.  Monophyly, Divergence Times, and Evolution of Host Plant Use Inferred from a Revised Phylogeny of the Drosophila Repleta Species Group , 2022 .

[12]  B. Gravendeel,et al.  A reconsideration of the empusellous species of Specklinia (Orchidaceae: Pleurothallidinae) in Costa Rica , 2012 .

[13]  M. Stpiczyńska,et al.  Comparative structure of the osmophores in the flowers of Stanhopea graveolens Lindley and Cycnoches chlorochilon Klotzsch (Orchidaceae) , 2012 .

[14]  Günter Gerlach,et al.  Asynchronous Diversification in a Specialized Plant-Pollinator Mutualism , 2011, Science.

[15]  E. Pansarin,et al.  Reproductive biology of Trichocentrum pumilum: an orchid pollinated by oil-collecting bees. , 2011, Plant biology.

[16]  I. Jamieson,et al.  Multimodel inference in ecology and evolution: challenges and solutions , 2011, Journal of evolutionary biology.

[17]  C. Darwin.  On the Various Contrivances by which British and Foreign Orchids are Fertilised by Insects: INTRODUCTION , 2011 .

[18]  P. Cribb.  The orchid collections and illustrations of Consul Friedrich C. Lehmann , 2010 .

[19]  R. Peakall,et al.  Pollinator specificity, floral odour chemistry and the phylogeny of Australian sexually deceptive Chiloglottis orchids: implications for pollinator-driven speciation. , 2010, The New phytologist.

[20]  Stacey D. Smith.  Using phylogenetics to detect pollinator‐mediated floral evolution , 2010 .

[21]  E. L. Borba,et al.  Morphological and histological characterization of the osmophores and nectaries of four species of Acianthera (Orchidaceae: Pleurothallidinae) , 2010, Plant Systematics and Evolution.

[22]  D. Grimaldi,et al.  Lord of the flies: pollination of Dracula orchids , 2010 .

[23]  S. Johnson,et al.  Pollination Efficiency and the Evolution of Specialized Deceptive Pollination Systems , 2009, The American Naturalist.

[24]  E. L. Borba,et al.  Self-incompatibility and myophily in Octomeria (Orchidaceae, Pleurothallidinae) species , 2009, Plant Systematics and Evolution.

[25]  R. Peakall,et al.  Implications of pollination by food and sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae) , 2009 .

[26]  L. Harder,et al.  Darwin's beautiful contrivances: evolutionary and functional evidence for floral adaptation. , 2009, The New phytologist.

[27]  W. M. Whitten,et al.  Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia. , 2009, Annals of botany.

[28]  M. Ritchie,et al.  When are vomiting males attractive? Sexual selection on condition-dependent nuptial feeding in Drosophila subobscura , 2009 .

[29]  D. Maddison,et al.  Mesquite: a modular system for evolutionary analysis. Version 2.6 , 2009 .

[30]  T. Markow,et al.  Reproductive ecology of Drosophila , 2008 .

[31]  M. Sazima,et al.  Reproductive biology of Cyrtopodium polyphyllum (Orchidaceae): a Cyrtopodiinae pollinated by deceit. , 2008, Plant biology.

[32]  K. Vi.  Elaiophore Structure and Oil Secretion in Flowers of Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb.f.) Garay & Pabst (Oncidiinae: Orchidaceae) , 2008 .

[33]  Jeffrey C. Hall,et al.  Neurogenetics of courtship and mating in Drosophila. , 2008, Advances in genetics.

[34]  A. Rambaut,et al.  BEAST: Bayesian evolutionary analysis by sampling trees , 2007, BMC Evolutionary Biology.

[35]  W. M. Whitten,et al.  Molecular phylogenetics of Maxillaria and related genera (Orchidaceae: Cymbidieae) based on combined molecular data sets. , 2007, American journal of botany.

[36]  M. Dicke,et al.  AGGREGATION PHEROMONES OF Drosophila immigrans , , 2007 .

[37]  Maria Clara Estanislau do Amaral,et al.  Biologia reprodutiva e polinização de duas espécies de Polystachya Hook. no Sudeste do Brasil: evidência de pseudocleistogamia em Polystachyeae (Orchidaceae) , 2006 .

[38]  A. Cocucci,et al.  First confirmed case of pseudocopulation in terrestrial orchids of South America: Pollination of Geoblasta pennicillata (Orchidaceae) by Campsomeris bistrimacula (Hymenoptera, Scoliidae) , 2006 .

[39]  M. Sazima,et al.  Biologia floral, melitofilia e influência de besouros Curculionidae no sucesso reprodutivo de Grobya amherstiae Lindl. (Orchidaceae: Cyrtopodiinae) , 2006 .

[40]  P. Kindlmann,et al.  Mechanisms and evolution of deceptive pollination in orchids , 2006, Biological reviews of the Cambridge Philosophical Society.

[41]  Octavio Albores-Ortiz,et al.  POLINIZACIN DE DOS ESPECIES SIMPTRICAS DE STELIS (PLEUROTHALLIDINAE, ORCHIDACEAE) , 2006 .

[42]  A. Smithson.  Pollinator limitation and inbreeding depression in orchid species with and without nectar rewards. , 2006, The New phytologist.

[43]  T. Markow,et al.  Evolutionary genetics of reproductive behavior in Drosophila: connecting the dots. , 2005, Annual review of genetics.

[44]  M. Symonds,et al.  The mode of evolution of aggregation pheromones in Drosophila species , 2005, Journal of evolutionary biology.

[45]  A. Widmer,et al.  Orchid diversity: an evolutionary consequence of deception? , 2005, Trends in ecology & evolution.

[46]  L. Ascensão,et al.  Comparative structure of the labellum in Ophrys fusca and O. lutea (Orchidaceae). , 2005, American journal of botany.

[47]  M. Blanco,et al.  Pseudocopulatory pollination in lepanthes (orchidaceae: pleurothallidinae) by fungus gnats. , 2005, Annals of botany.

[48]  L. L. Jackson,et al.  Aggregation pheromones inDrosophila borealis andDrosophila littoralis , 1988, Journal of Chemical Ecology.

[49]  L. L. Jackson,et al.  Ester and ketone components of aggregation pheromone ofDrosophila hydei (Diptera: Drosophilidae) , 1987, Journal of Chemical Ecology.

[50]  L. L. Jackson,et al.  cis-Vaccenyl acetate as an aggregation pheromone inDrosophila melanogaster , 1985, Journal of Chemical Ecology.

[51]  J. Zimmerman,et al.  Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification , 2004 .

[52]  B. Gravendeel,et al.  Epiphytism and pollinator specialization: drivers for orchid diversity? , 2004, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[53]  D. Janzen,et al.  Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[54]  K. L. Davies.,et al.  Pseudopollen in Dendrobium unicum Seidenf. (Orchidaceae): reward or deception? , 2004, Annals of botany.

[55]  Rodrigo B Singer,et al.  Sexual mimicry in Mormolyca ringens (Lindl.) Schltr. (Orchidaceae: Maxillariinae). , 2004, Annals of botany.

[56]  S. Teixeira,et al.  Lip anatomy and its implications for the pollination mechanisms of Bulbophyllum species (Orchidaceae). , 2004, Annals of botany.

[57]  M. Ayasse,et al.  Pollinator attraction in a sexually deceptive orchid by means of unconventional chemicals , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[58]  Jeremy R. deWaard,et al.  Biological identifications through DNA barcodes , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[59]  Rodger Staden,et al.  Analyzing Sequences Using the Staden Package and EMBOSS , 2003 .

[60]  R. Singer,et al.  The pollination mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): sexual mimicry and trap-flowers. , 2002, Annals of botany.

[61]  Á . Szentesi,et al.  INSECT-PLANT RELATIONSHIP - CHANCE AND NECESSITY , 2002 .

[62]  M. Chase,et al.  Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences. , 2001, American journal of botany.

[63]  J. Semir,et al.  Pollinator Specificity and Convergence in Fly-pollinated Pleurothallis(Orchidaceae) Species: A Multiple Population Approach , 2001 .

[64]  R. Greenspan,et al.  Courtship in Drosophila. , 2003, Annual review of genetics.

[65]  R. Vrijenhoek,et al.  DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. , 1994, Molecular marine biology and biotechnology.

[66]  P. Adams,et al.  Pollination in Australian Orchids: a Critical-Assessment of the Literature 1882-1992 , 1993 .

[67]  R. Dressler.  Phylogeny and classification of the orchid family , 1993 .

[68]  S. Renner,et al.  The role of scent glands in pollination : on the structure and function of osmophores , 1992 .

[69]  K. Curry,et al.  Osmophores, floral features, and systematics of Stanhopea (Orchidaceae) , 1991 .

[70]  T. Markow.  Reproductive behavior of Drosophila melanogaster and D. nigrospiracula in the field and in the laboratory. , 1988, Journal of comparative psychology.

[71]  E. Smets,et al.  Types of floral nectaries and the concepts ‘character’ and ‘character‐state‘—a reconsideration , 1988 .

[72]  L. L. Jackson,et al.  Aggregation pheromones in five taxa of the Drosophila virilis species group , 1986 .

[73]  R. Steele.  Courtship feeding in Drosophila subobscura. II. Courtship feeding by males influences female mate choice , 1986, Animal Behaviour.

[74]  M. Behan,et al.  Single cell responses in female Pieris brassicae (Lepidoptera: Pieridae) to plant volatiles and conspecific egg odours , 1980 .

[75]  H. Carson.  Three flies and three islands: parallel evolution in Drosophila. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[76]  C. H. Dodson,et al.  Orchid Flowers: Their Pollination and Evolution , 1966 .

[77]  E. Reynolds.  THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY , 1963, The Journal of cell biology.