Дисертації з теми "Orchid, pollination"

Dracula orchids are hypothesized to rely on mushroom mimicry for pollination. These orchids look and smell like mushrooms and are pollinated by mushroom-associated flies in the family Drosophilidae. Dracula includes over 130 species, representing a significant radiation, yet there has never been a systematic study of their pollination biology. Elucidating the processes and mechanisms of pollination in these flowers will broaden our understanding of mimicry within the Orchidaceae, a family well known for its diverse pollination strategies, as well as add to the growing literature on the evolution and maintenance of communication signals. In this study we demonstrate the co-occurrence of the mimics and the putative mushroom models, which is important for evolution by natural selection. We also showed that the resemblance to mushrooms is in fact adaptive, a requisite for floral mimicry. We did this by determining that insect visitors are required for pollination and subsequent fruit set with a hand pollination experiment. We also measured increased visitation rates to the orchids when adjacent to mushrooms. The mechanisms whereby plants attract pollinators can be diverse and often multi-modal, particularly in deceptive systems. Dracula orchids are no exception, with both visual and olfactory signals contributing to the overall success in attracting visitors. We used a series of experiments, first selectively masking the visual and olfactory cues successively, and then using 3D-printed artificial flowers to further disentangle these cues and determine their effect in combination. Upon confirmation that both play a role, we dissected each aspect further. We utilized the artificial flowers to determine the roles of color, contrast, and pattern and employed gas chromatography-mass spectroscopy to identify the volatile signals. The results show that fine-scale contrast is critical to the visual component and that these flowers produce the volatile `mushroom-alcohol' (1-octen-3-ol) in their labella. Finally, we specifically address the hypothesis of brood-site mimicry by using a combination of field observations, insect collections, and rearing studies. The flies gain shelter, a rendezvous location, and food from the flowers. However, no mushroom visiting flies hatched from the flowers, suggesting this may be a brood-site mimicry. This dissertation includes previously unpublished co-authored material.
2015-09-29

The Neotropical cloud forest inhabiting orchid Dracula felix has long been postulated to be a fungal mimic due to the form of its lower labellum and attraction to it by drosophilid flies that are often found feeding on fungal fruiting bodies in the surrounding area. The low number of co-occurring flowers in the area combined with the high number of fruiting fungi appears to have driven the evolution of the orchid genus Dracula to mimic these co-occurring fungi so that pollinators may be recruited. Over several years of working with these orchids we have noticed a particular lapping behavior by the pollinating flies on the labella and sepals of the Dracula flowers. In this study we have first surveyed floral yeasts and molds associated with Dracula flowers and then investigated the role of these fungi in attracting pollinators and offering a food reward to retain them for pollination purposes. In addition to the floral yeasts, leaf endophytes and root associated fungi were cultured and identified, and their frequencies were determined.

A presente dissertação pretende contribuir para o aumento do conhecimento numa área específica da polinização em orquídeas, designada como polinização deceptiva (sexual deception) usando a espécie Ophrys fusca como modelo. Tendo por base 100 labelos de 100 plantas diferentes, colhidas no seu habitat natural, recorreu-se a técnicas de transcritómica e metabolómica com o objectivo de (1) analisar a expressão génica por microarrays de cDNA após a polinização; (2) proceder à caracterização metabólica por cromatografia gasosa e espectrometria de massa (GC-MS). Os resultados obtidos permitiram contribuir para a compreensão dos mecanismos de polinização por sexual deception, nomeadamente no que respeita às características do labelo (ex. pigmentação, emissão de compostos), dos processos de senescência ou da biologia floral das orquídeas. A construção de um chip de cDNA para O. fusca permitirá realizar hibridações com outras espécies de Ophrys, possibilitando a determinação do grau de conservação dos mecanismos genéticos na polinização por sexual deception; ABSTRACT:This work aims at contributing to the knowledge on orchid pollination biology, through the study of the peculiar pollination mechanism of Ophrys fusca by sexual deception. In this mechanism, Ophrys labellum mimics the female sex pheromones thereby deceiving male pollinators that attempt to copulate with the orchid labellum. Labellum transcriptome analysis by a custom-made cDNA microarrays allowed to verify gene expression modulation of post pollination changes. Processes involved in labellum morphology, petal senescence and pollination biology were adressed. A metabolic profiling by gas-chromatography mass- spectrometry was focused on compounds involved in Ophrys-pollinator crosstalk, in order to determine their dynamics after pollination. By means of both transcriptional and metabolic analysis, the work here presented gives an important contribution towards the understanding of orchid pollination biology by deceit. The custom-made cDNA chip may be useful for performing cross-species hybridization to track differences on transcripts modulation thereby disclosing the genetic basis underlying sexual deception; RESUMO:A família das orquídeas (Orchidaceae) inclui mais de 22 000 espécies, representando cerca de 10% das plantas com flor. A sua extraordinária diversidade floral reflecte a importância das relações planta-polinizador na evolução das orquídeas, sendo as diferentes estratégias de polinização consideradas como uma das razões para a diversificação e especiação na família. Os mecanismos de polinização em orquídeas sempre intrigaram os cientistas, incluindo Darwin. Uma das estratégias mais fascinantes na biologia destas plantas é a capacidade de polinização deceptiva, ocorrendo em cerca de 1/3 das espécies. A presença de uma pétala modificada, o labelo, tendo como função principal atrair insectos polinizadores, é igualmente uma das características mais distintivas das orquídeas. Os mecanismos de polinização deceptiva mais comuns incluem a imitação de flores que apresentam néctar (food deception), ocorrendo em 38 géneros; e a imitação de insectos-fêmea (sexual deception), abrangendo 18 géneros. O género Ophrys sempre foi considerado um modelo para estudo de polinização deceptiva, nomeadamente do caso de flores sexualmente deceptivas (sexual deception). As flores deste género emitem substâncias químicas idênticas às feromonas libertadas pelas fêmeas sexualmente receptivas dos insectos polinizadores, bem como apresentam características morfológicas (ex: forma, cor, pilosidade) que mimetizam o corpo dos insectos-fêmea. Os estudos desenvolvidos em polinização de orquídeas têm sido desenvolvidos nas áreas da biologia celular, micromorfologia, genética populacional, análise química e na determinação funcional de determinados genes, bem como em estudos bioquímicos e fisiológicos. Contudo, para a compreensão global de um processo são necessárias técnicas que permitam obter dados a larga escala. Este trabalho pretende constituir um contributo para o conhecimento dos mecanismos regulados pela polinização em orquídeas. Para tal, uma espécie de orquídea selvagem abundante em Portugal, Ophrys fusca Link, foi usada como modelo de estudo do mecanismo de polinização deceptiva (sexual deception). Para atingir o presente objectivo, foram aplicadas duas técnicas diferentes: a análise da expressão génica por microarrays de cDNA e a caracterização metabólica por cromatografia gasosa e espectrometria de massa (GC-MS). O labelo foi seleccionado como foco do estudo, dada a sua importância na emissão de compostos importantes na comunicação com o insecto polinizador e no processo geral de polinização em orquídeas. A amostragem foi efectuada em 100 labelos de 100 plantas diferentes no seu habitat natural, e seleccionaram-se dois tempos de estudo: 2 dias após a polinização (DAP) e 4 DAP.A análise do labelo por microrrays de cDNA permitiu verificar a modulação da expressão génica após a polinização. Com este estudo de larga escala conseguiu-se verificar que aos 2 DAP, o evento de polinização é reconhecido como uma resposta a um stress e aos 4 DAP, detectaram-se genes que indicam a mobilização de nutrientes bem como uma nova síntese proteica, necessária para a progressão específica da senescência do labelo. A polinização despoleta processos de proteólise, mobilização de nutrientes como o fosfato, carbono e azoto, e desactiva mecanismos energeticamente dispendiosos, como a fotossíntese e fotorespiração bem como as principais vias metabólicas que permitem manter a vitalidade do labelo. Os transcritos identificados revelam processos importantes do metabolismo secundário envolvidos em características do labelo (ex. pigmentação, emissão de compostos), em proteólise a larga escala (ex. proteases cisteínicas) e dirigida (ex. fosfatases e quinases), stress e defesa, além de vias associadas à mobilização de nutrientes. Inicialmente, a expressão génica de diversos transcritos descritos em situações de stress e de patogenicidade (ex. GST, proteínas Lea5, metalotioneínas tipos 2 e 3, quitinases, proteínas PR, proteases cisteínicas, RNases) indicam que a modulação da transcrição é regulada por vias não específicas de reconhecimento do evento de polinização, semelhantes a uma situação de stresse abiótico e/ou biótico. Contudo, aos 4 DAP, verificou-se a transcrição de genes associados à síntese proteica, indicando a activação de um novo processo de tradução de proteínas específicas que irão dirigir o labelo para a morte celular irreversível. A análise do perfil metabólico dos extractos dos labelos foi focada em compostos da cutícula, especificamente alcanos e alcenos, descritos como responsáveis por despoletar o comportamento de pseudocópula dos machos polinizadores. Os resultados demonstram que, após a polinização, a quantidade total dos compostos não diminui, estando de acordo com resultados anteriores observados em Ophrys sphegodes. Esta observação poderá dever-se à função que estes compostos desempenham, nomeadamente como parte integrante das camadas das ceras prevenindo a desidratação. A análise do labelo após a polinização por microarrays permitiu a identificação de transcritos, nomeadamente duas sequências de stearoil ACP desaturase (SAD), envolvidos nas vias biossintéticas dos compostos da cutícula, importantes na interacção Ophrys-polinizador. A subexpressão destes transcritos em conjugação com a manutenção da produção do odor após a polinização indica que a correlação entre os níveis de expressão dos genes com os seus produtos de síntese não pode ser directamente efectuada. O estudo do labelo após a polinização por técnicas de proteómica irá permitirá a detecção das enzimas bem como alterações pós tradução importantes na regulação das proteínas. Esta abordagem irá possibilitar a compreensão da regulação das proteínas após o evento de polinização. O presente estudo permitiu obter uma visão geral no labelo dos mecanismos regulados pela polinização, contribuindo para a compreensão da polinização por sexual deception recorrendo a técnicas de Ó’micas. A análise do labelo através de técnicas de transcritómica e metabolómica após a polinização permitiu dar um importante contributo para a compreensão dos processos de senescência, características do labelo (ex. pigmentação, emissão de compostos), bem como da biologia floral das orquídeas. Além disso, a construção de um chip de cDNA construído especificamente para a orquídea em estudo irá permitir a realização de hibridações com outras espécies do mesmo género, possibilitando o estudo da conservação dos mecanismos genéticos na regulação dos eventos pós-polinização de orquídeas com flores sexualmente deceptivas; ABSTRACT:Orchidaceae family includes more than 22,000 species of plants, representing around 10 % of all flowering plants. The extraordinary floral diversity in orchids reflects the importance of plant-pollinator associations in their evolution, and pollination biology is regarded as a driving force in orchid diversification and speciation. Pollination biology in Orchidaceae has long intrigued evolutionary biologists, and interest in orchid pollination dates back to Darwin. The most fascinating in orchid biology is pollination by deception, occurring in approximately 1/3 of the species, being food (38 genera) and sexual (18 genera) deception the most common types. Sexual deception mechanism was first described in the European Ophrys genus by Pouyanne in 1917, and in this mechanism, Ophrys orchids mimic their pollinators’ mating signals, and are pollinated by male insects during mating attempts. Studies on orchid pollination have mainly focused on cell biology, population genetics, micromorphology, chemical analysis and gene-function studies, as well as biochemical and physiological studies on flowers. A general approach towards the understanding on orchid pollination biology, as well as in the events following pollination, by means of high throughtput techniques is lacking. The study here presented intends at contributing to the knowledge on post pollinationregulated mechanisms in the sexual deceptive orchid Ophrys fusca Link, a common bee orchid in the Mediterranean, natural occurring in Portugal. To accomplish such goal, two different approaches were assigned: a transcriptional analysis and a metabolic profiling. Transcriptomics and metabolomics were both used to gather insights on the post pollination changes occurring in Ophrys fusca labellum. To access pollination-enhanced events, two time points were considered for analysis: 2 and 4 days after pollination (DAP). Labellum transcriptional analysis allowed probing gene expression modulation of post pollination changes. The first response to pollination appears to be a stress response (2DAP) and later at 4DAP, nutrient mobilization occurs and de novo protein synthesis is induced for senescence progression. Pollination sets off proteolysis, remobilization of nutrients such as phosphate, carbon and nitrogen from labellum and deactivates energy-consuming processes (e.g. photosynthesis, photorespiration) and major metabolic pathways related to labellum upholding. Transcripts identified by microarray analysis reveal pivotal processes associated with secondary metabolism responsible for labellum traits (e.g. pigmentation, compounds emission involved in pollination), proteolysis, stress and defence, and remobilization of nutrients associated with pollination induced-senescence. Labellum transcriptional regulation seems to be mediated by non-specific stress-related pathways, disclosed by the expression of several stress- and pathogen-related transcripts (GST, antimicrobial snakin proteins,Lea5 protein, metallothioneins types 2 and 3, chitinases, PR protein, Cys proteases, RNases), until the newly protein synthesis is achieved for senescence progression. Metabolic profiling in labella extracts was focused on cuticular compounds (alkanes and alkenes), known to trigger the pseudocopulatory behaviour of male pollinators. Results show that post pollination machinery does not rely on an abrupt decrease of odour production, which is in agreement with previous reports on other Ophrys species, probably due to compounds function as part of the desiccationpreventing wax layers. Through labellum gene expression analysis, transcripts related to biosynthetic pathways of cuticular compounds, involved in Ophrys pollinator attraction, were identified: stearoyl ACP desaturases (SAD). Down regulation of these transcripts along with maintenance of odour production may indicate that correlation between RNA level and its by-products cannot be directly made. Thus, a labellum post pollination proteomics approach will allow tracking enzymes responsible for alkenes’ production, thereby giving a more comprehensive walkthrough of their regulation on pollination event. Such observations could adjoin some awareness on the genetic basis of pollinator attraction. By combining both transcriptional and metabolic profiling analysis to study post pollination events in a sexually deceptive orchid, the work here presented gives an important contribution for the understanding of this peculiar pollination system.

There is considerable evidence that pollinator shifts can explain many differences in flower morphology between closely related plant species, but the extent to which pollinator shifts can explain the maintenance of among-population variation in floral traits within species is poorly known. In this thesis, I combined comparative and experimental approaches to examine the evolution of floral traits in the moth-pollinated orchid Platanthera bifolia. More specifically, I investigated (1) the relationship between flower and pollinator morphology, (2) phenotypic selection on morphology and phenology in populations in contrasting environments, (3) components of prezygotic reproductive isolation among divergent populations, and (4) the adaptive and functional significance of two correlated floral traits. A study of Scandinavian of P. bifolia populations revealed that spur length was positively correlated with proboscis length of local pollinators, which suggests that variation in spur length reflects adaptive evolution in response to geographically variable pollinator-mediated selection. A phenotypic selection study on Öland, SE Sweden, suggested that disruptive selection on spur length contributes to the maintenance of a bimodal distribution of spur length in mixed habitats, but provided very limited evidence of divergent selection on plant morphology and flowering phenology in grassland and woodland habitats. Field experiments revealed strong reproductive isolation between divergent populations on Öland, due to differences in spatial distribution, flowering phenology, and pollinators, and among-population incompatibility. The results suggest that prezygotic reproductive isolation contributes to the maintenance of population differentiation in floral traits in P. bifolia. A field manipulation experiment demonstrated that spur length but not perianth size affects pollination success and seed production. This suggests that among-population differentiation in perianth size may be the result of a genetic correlation with spur length. Taken together, the results of this thesis suggest that pollinator-mediated selection can shape the evolution of intraspecific floral variation.

Terrestrial orchids make up one of the most threatened groups of plants in Australia and the genus Arachnorchis is listed as the fourth most threatened. The process of propagation and re-introduction of terrestrial orchid plants to the wild has proven difficult, and so far, nearly impossible for some species. This may be partly because terrestrial orchids form complex relationships with mycorrhizal fungi and in genera like Arachnorchis the dependency on the fungus appears acute. Arachnorchis has long been considered by amateur growers of terrestrial orchids as one of the most difficult groups to propagate and maintain in cultivation. This lack of knowledge on how to grow Arachnorchis species hinders attempts made by conservation authorities to supplement threatened wild populations in order to achieve a more sustainable future for those species. Natural pollination was absent, but artificial pollination achieved 100% capsule production. Individuals were self-fertile, although seed viability was greater for cross-pollinated samples. This study attempted to track the fate of as many Arachnorchis species as possible from germination through to deflasking and re-emergence, and so destructive and potentially destructive measurements at earlier stages were avoided. This thesis examines germination and subsequent growth of up to eight species of Arachnorchis, but concentrated on A. phaeoclavia, A. tentaculata, A. fulva, A. robinsonii and A. venusta. Two of these are common species: A. pha eoclavia and A. tentaculata, and three carried a threatened classification of "rare" or " endangered": A. fulva, A. robinsonii and A. venusta. This study monitored the fate of individuals of the endangered A. fulva in the field and showed that large reproductive plants re-emerged and flowered each year, whereas smaller individuals might be absent in one or more years and were less likely to flower. Germination of all species concentrated on using symbiotic culture (using mycorrhizal fungi), since germination is known to be more rapid, resulting in healthier, more robust seedlings than when plants are grown asymbiotically. Tests using A. fulva and A. venusta, two threatened species, showed similar viability to A. tentaculata and A. phaeoclavia, more common species. Germination was maximised by examining the viability of seeds before and after treatment with surface-sterilising solutions required for aseptic culture. The highest levels of germination, with limited contamination, were achieved using 0.5% available chlorine for 3 minutes. The most effective fungal isolates (>65% germination) were obtained from common species like A. phaeoclavia and A. tentaculata, but there was no correlation between germination and time of year or life stage of the orchid. Collar collection was shown to be non-fatal to robust orchid plants, with large reproductive individuals (at the time of collar collection) re-emerging in the next year and producing a flower bud. Collar collection from small, weedy individuals could be fatal to the plant and isolation of an effective fungus was unlikely. Cross-inoculating seeds with fungi isolated from a different orchid species was not recommended, since the symbiosis failed in all experiments, as late as Stage 4 protocorm development. A range of substrates was used to produce strong seedlings capable of surviving the transfer to nursery conditions with minimal loss. More than 81% of seedlings survived deflasking from non-agar substrates, while the best result from agar was 55%. Some substrates reduced the time involved from seed to plants in the field to as little as 4 months, but aftercare became critical. Sucrose promoted tuberisation, but led to tuber deaths during dormancy. Potting mixes were tested in the nursery and a free-draining loam mix based on a mix used by the Australasian Native Orchid Society was the best medium for deflasking of seedlings. Watering during dormancy should be avoided. The choice of propagule for re-introduction was examined and the best survival to re-emergence was obtained by planting out actively growing seedlings in autumn. Identification of cultures using classical morphology grouped cultures as belonging to the form-genera Epulorhiza and Moniliopsis and suggested that most cultures contained more than one fungus. Identification of the most useful fungal cultures was attempted using molecular techniques such as sequencing the ITS region and mitochondrial DNA. One effective culture, CALAPHAER18 SHTX (cultured from a single monilioid cell) was identified as Serendipita vermifera (Oberwinkler) Roberts. All other cultures tested were mixtures of fungi. The use of specific primers designed to amplify a sequence present in the identified isolate (CALAPHAER18 SHTX) showed that nine mixed cultures also contained a fungus most closely related to Serendipita vermifera. Specific primers also showed that Rhizoctonia solani was not present in any of the 10 isolates from Arachnorchis plants. The molecular work showed that, although the sequenced endophytes from Arachnorchis were all most closely related to Serendipita vermifera, three dist inct groups of fungi were present and these associated with separate species of Arachnorchis. Future work with Arachnorchis species will require the isolation of single fungus cultures and further examination of the development of the orchid plant. In particular, the process of tuberisation and growth in vitro on various non-agar substrates should be investigated further.

Orientador: Marlies Sazima
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-04T22:48:57Z (GMT). No. of bitstreams: 1 Pansarin_EmersonRicardo_D.pdf: 5740977 bytes, checksum: b8a38716f8e2e6697ba77e6ff49e8303 (MD5) Previous issue date: 2005
Resumo: Neste trabalho foi realizada a revisão taxonômica, o estudo químico e micromorfológico das ceras epicuticulares, bem como estudados os processos de polinização e os polinizadores de representantes sul-americanos do gênero Cleistes. Foi, também, elaborada uma hipótese filogenética da subtribo Pogoniinae com base em caracteres morfológicos e moleculares, através do seqüenciamento das regiões ITS (nrDNA), e rps16 e trnL-F (cpDNA). O gênero Cleistes apresenta 17 espécies distribuídas entre as Américas Central e do Sul, ocorrendo principalmente em regiões de cerrado do Brasil central. Três dessas espécies foram descritas durante a elaboração do presente trabalho e estão aqui apresentadas. Através do estudo da hipótese filogenética de Pogoniinae foi verificado que essa subtribo apresenta dois clados bem sustentados. Um clado é norte-americano-asiático, e inclui os gêneros Pogonia, Isotria e as espécies norte-americanas de Cleistes. O outro é centro-sul-americano e inclui as espécies de Cleistes distribuídas entre as Américas Central e do Sul. Com o desenvolvimento do presente estudo, incluindo a maioria das espécies de Cleistes nas análises, pode ser demonstrado que esse gênero é parafilético, concordando com os dados publicados por Cameron & Chase (1999). As espécies norte-americanas de Cleistes estão mais relacionadas com os gêneros norte-americano-asiáticos Isotria e Pogonia do que com as espécies centro-sul-americanas de Cleistes. A subtribo Pogoniinae também é parafilética. O saprofítico gênero Pogoniopsis está mais relacionado com representantes dos gêneros Galeola e Cyrtosia (Galeolinae) do que com os demais gêneros pertencentes à subtribo Pogoniinae. Duckeella, um gênero endêmico da Amazônia e irmão das Pogoniinae, não apresenta os caracteres sinapomórficos que define essa subtribo, devendo ser transferido para a subtribo Duckeellinae. Espécies de Cleistes apresentam micromorfologia das ceras epicuticulares existentes nas folhas variando desde lisas, com grânulos, ou até formando placas. As ceras epicuticulares de Cleistes são constituídas principalmente por álcoois, ácidos e ésteres. A composição química das ceras epicuticulares das folhas de espécies de Cleistes é muito variável entre espécies, podendo ser usada na delimitação específica dentro do gênero. Espécies de Cleistes, assim como as demais Pogoniinae, são polinizadas principalmente por abelhas, embora C. libonii apresente beija-flores do gênero Phaethornis como co-polinizadores. Espécies sul-americanas de Cleistes apresentam picos de floração, em que todos os botões maduros de cada planta abrem simultaneamente no mesmo dia. As flores das espécies estudadas são pouco duráveis (geralmente um dia), e oferecem néctar aos polinizadores. O néctar é produzido em nectários glandulares da base do labelo das flores. Para os gêneros norte-americano-asiáticos esses nectários estão ausentes e as abelhas são atraídas às flores por engano. Esse estudo sustenta a hipótese de evolução de flores de engano para flores de néctar
Abstract: The taxonomic revision, the micromorphological and chemical study of the epicuticular waxes, as well as the pollination processes and pollinators of the genus Cleistes were reported. A phylogenetic inference within Cleistes and among genera of subtribe Pogoniinae was also established, based on morphology and nrDNA (ITS) and cpDNA (trnL-F and rps16) sequence data, and using maximum parsimony. The genus Cleistes includes 17 species distributed among Central and South America, occurring mainly in ¿cerrado¿ areas of Central Brazil. Three of these species are new and were described in the present study. The phylogenetic study of Pogoniinae showed that this subtribe presents two well-supported clades. One clade is North-American-Asiatic and includes the genera Isotria, Pogonia and the North-American species of Cleistes. The other clade is Central-South-American and includes the species of Cleistes occurring in Central and South America. The genus Cleistes, in agreement to Cameron & Chase (1999), is paraphyletic. The North American species of Cleistes are more related to the North American-Asiatic genera Isotria and Pogonia than to the remaining species of Cleistes. The subtribe Pogoniinae is also paraphyletic. The saprophytic genus Pogoniopsis is more related with Galeola and Cyrtosia (Galeolinae) than to the remaining genera currently recognized within subtribe Pogoniinae. The Amazonian genus Duckeella, sister of all remainder of Pogoniinae, lacks the synapomorphic characters that define this subtribe, and should be transferred to the subtribe Duckeellinae. Species of the genus Cleistes present micromorphology of the epicuticular waxes of the leaves varying from a singular film to forming granules or platelets. The epicuticular waxes in Cleistes are constituted mainly by alcohols, acids and esters. The chemical compounds of epicuticular waxes of Cleistes were very variable among species, and may be used for specific delimitation within the genus. Species of Cleistes, as for remaining Pogoniinae, are pollinated mainly by bees, although C. libonii is co-pollinated by hermit hummingbirds. The South American species of Cleistes presents flowering peaks, in which all mature buds of each plant flowering simultaneously on the same day. The studied species produced short-lived flowers (generally one day), which offer nectar as reward produced by two nectariferous glands on the basis of the lip. For the North American-Asiatic genera these nectariferous glands are absent and the floral visitors are attracted by deceit. This study supports the evolution of deceptive flowers to nectar flowers
Doutorado
Biologia Vegetal
Doutor em Biologia Vegetal

Comparative studies on endangered species and their more common congeners can shed light on the mechanism of species rarity, and enable conservationists to formulate effective management strategies. I compared the breeding and pollination systems of the endangered Geodorum eulophioides and its two more common sympatric congeners in subtropical China. Geodorum eulophioides and G. recurvum were self-compatible, both depending on Ceratina cognata for fruit production, while G. densiflorum can autonomously self. Although the floral visitation frequency of G. eulophioides was the highest among the three, its natural fruit set was similar to that of G. recurvum, but both lower than that of G. densiflorum. These results partially explain the difference in species abundance. Coupled with severe habitat loss and degradation, the extremely low pollinator visitation and natural fruit set of G. eulophioides calls for rapid establishment of ex-situ collection, in conjunction with improving in-situ habitat. Natural hybridization tendencies were also studied among species.

Les angrecoïdes constituent le groupe d'orchidées épiphytes le plus diversifié dans les Afrotropiques, comprenant environ 800 espèces. Bien que beaucoup d'attention leur aient été porté, certaines énigmes taxonomiques subsistent au sein des angraecoïdes, et les facteurs à l'origine de leur diversification rapide sont encore inconnus. Les angraecoïdes présentent une remarquable diversité en termes du nombre chromosomique, en faisant un système très approprié pour explorer l'impact des changements caryotypiques sur la cladogenèse, les taux de spéciation/extinction et la diversification morphologique dans le contexte des fluctuations climatiques en Afrique tropicale depuis le Miocène. En outre, grâce au large éventail des longueurs d'éperon nectarifère que ces orchidées présentent, elles ont fait l'objet, depuis Darwin, de recherches approfondies dans le cadre des interactions plantes-animaux. Ici, sur base de nouveaux arbres phylogénétiques produits en utilisant ITS-1 ainsi que cinq marqueurs plastidiques et englobant environ 40 % des espèces, nous fournissons un nouveau cadre taxonomique pour les principales lignées d'Angraecinae. De plus, le cadre taxonomique des angraecoïdes est mis à jour avec, notamment, la description de trois nouveaux genres et six nouvelles espèces. Cette nouvelle hypothèse phylogénétique nous a permis d'étudier si les changements des caryotypes et des pollinisateurs ont pu être les moteurs de la radiation évolutive des angraecoïdes. La reconstruction des états ancestraux du nombre chromosomique révèle une histoire caryotypique dominée par la dysploïdie descendante en Afrique tropicale continentale, où environ 90 % des espèces dérivent d'au moins un changement inféré de n = 17–18 à n = 25 au Miocène moyen. L’examen des intervalles de position du nectar par rapport au pollen dans les Afrotropiques a révélé qu'environ 3 % de la flore des angiospermes de Madagascar est probablement pollinisée par des sphinx, alors que cette proportion est d'environ 1,6 % en Afrique continentale. Les nombreux changements de guilde de pollinisateur vers la sphingophilie ayant eu lieu chez les angraecoïdes seraient à l’origine d’environ 31 % des espèces, y compris certaines lignées ayant les taux de spéciation les plus élevés. En dehors du domaine de la sphingophilie, de nouveaux exemples possibles d’ornithophilie, de phalénophilie et de pollinisation par des tipules à long proboscis/microlepidoptères sont discutées. Des perspectives de recherche concernant l'évolution génomique chez les angraecoïdes et l'impact et les mécanismes des changements des sites de fixation des pollinies sont suggérées. Enfin, certaines priorités pour l’observation de nouveaux pollinisateurs sur le terrain et les frontières de l’alpha et bêta-taxonomie chez les angraecoïdes sont présentées.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

The objective of seed orchard management is to maximize the genetic gain while maintaining sufficient level of genetic diversity in the orchard’s crops; however, balancing these two parameters is a challenging task. Eight polymorphic nuclear microsatellite DNA markers were used to construct the full pedigree of 801 Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) bulk seeds from 49 orchard parents and 4 external supplemental mass pollination (SMP) donors. Parental balance curves indicated that 80% of the gametes were produced by 23, 45 and 37% of females, males and clones, respectively. Contamination was found to be 10.36% with overhead cooling applied. Selfing rate was found as 15.23% due to the over representation of few orchard clones. The aggregate SMP (internal & external) success rate was estimated to be 15.02%. The female, male and clonal effective population size (Ne) was estimated to be 6.49, 26.00 and 13.70, respectively. The reduction of Ne is mainly the result of unequal parental contribution and to a minor extent by the co-ancestory of the orchard’s parents. The seed crop’s genetic worth was estimated to be 10.23 and -1.07 for volume and wood density, respectively. High correlations between visual reproductive assessment methods F2 (r = 0.90 P < 0.01) and M3 (r 0.77, P < 0.05) with DNA analysis give credence to the visualize assessment methods. The result of this study indicated that the seed orchard deviated from the ideal conditions affecting the expected genetic diversity and gain estimates.

Background and Aims Sexual deception is a species-specific pollination strategy commonly found in Orchidaceae. Sexually deceptive orchids lure male insect pollinators by mimicking the sex pheromones and/or appearance of female insects, which elicit copulatory behaviour with the flower by the male insects. This specialised pollination strategy has recently been found in a Pterostylis species in Australia. Pterostylis orchids also occur in New Zealand, although very few studies have been done on this genus, and no such specialised insect pollination strategy has been documented in New Zealand. Methods I investigated the breeding system and pollinators of three Pterostylis spp. to determine whether sexual deception may be operating in P. oliveri, P. irsoniana and P. venosa growing in native beech forests in Arthur's Pass. We also investigated the floral headspace volatiles of P. oliveri to determine which compounds are present, and which may be responsible for pollinator attraction. Key Results Breeding system experiments suggest that P. oliveri and P. irsoniana are self compatible, but exclusively dependent on insects for pollination. Only male fungus gnats (Diptera: Mycetophilidae) were found carrying pollinia attached to their thoraxes in traps set up over the flowers. Insect identification and ITS DNA analysis of the pollinia showed that each orchid species was pollinated by a specific fungus gnat species; Mycetophila latifascia males found with pollen of P. oliveri; Morganiella fusca males found with pollen of P. irsoniana; and Tetragoneura sp. males found with pollen of P. venosa. Field tests of an unidentified compound found in headspace volatiles of P. oliveri did not attract any Mycetophila latifascia males. Conclusions These results indicate that pollination via sexual deception may be operating in these three Pterostylis spp. However, further floral volatile analyses are required to confirm whether the flowers emit volatile compounds that resemble the sex pheromones of the specific pollinators.

Pollinator visitation patterns should affect pollination success and mating patterns in flowering species. In the orchid family, about one third of the species do not provide any reward for their pollinators. Pollination by deceit is typically associated with low fruit set but may increase the chance of cross-pollination since the pollinator should soon leave the individual plant when there is no reward in the flowers. This may be beneficial if self-fertilisation results in inbreeding depression. I studied the mating patterns of one rewarding and one deceptive orchid in two closely related genera by tracking the fate of stained pollinia. I also conducted controlled crosses to estimate inbreeding depression. The results show that the deceptive orchid Dactylorhiza lapponica has lower pollination success, but higher cross-pollination rate (ca. 90%) than the nectariferous orchid Gymnadenia conopsea (ca. 18% cross-pollination). The results further suggest that in G. conopsea, nocturnal visitors mediate higher geitonogamous pollination rate (ca. 100%) than diurnal visitors (ca. 60%). In both study species, fruits produced from cross-pollination were heavier than fruits produced from selfing. Inbreeding depression for fruit mass did not differ significantly between the two species (δ = 0.21 in D. lapponica and δ = 0.29 in G. conopsea). These data support the hypothesis that pollination by deceit can enhance cross-pollination. A literature study including several rewarding and non-rewarding orchid species indicated lower geitonogamy in the deceptive orchids, but the difference was not statistically significant.

Unlike other rosaceous fruit crops such as apple and cherry, commercial raspberry cultivars are largely self-fertile and can set fruit in the absence of pollinators. However, their floral morphology often prevents complete self-pollaintion. Incomplete pollination yields unmarketable small or crumbly fruits. Insect visitation is therefore essential to maximizing raspberry yield. Honey bees are typically used to pollinate commercial raspberry; however, escalating prices for hive rentals coupled with increasing acreage encourage evaluation of other manageable pollinators. Bumble bees (Bombus spp.) and several mason bees (Osmia spp.) are promising raspberry pollinators. Five bee species were evaluated and compared for their single-visit pollination efficacies on raspberry. From this a pollinator effectiveness index was created and an estimation of the minimum number of visits required to maximize fruit set was calculated. This estimation was then experimentally verified. Finally, in an attempt to synchronize their brief activity period with raspberry bloom, winter management options aimed at delaying the emergence of the mason bee, O. lignaria, were investigated. All five bee species proved excellent pollinators of raspberry. None of the alternative manageable species greatly outperformed honey bees. For this reason honey bees remain the most economical and practical option for open-field raspberry pollination. The adoption of alternative manageable bees could still be justified in other production systems, such as high-tunnel or greenhouse grown raspberry, which hamper honey bees’ ability to forage effectively. The pollinator effectiveness score for honey bees suggested that as few as two visits can achieve maximum fruit set. This estimate was confirmed through experimentation on three different red raspberry cultivars. For two of these cultivars, just one visit yielded drupelet counts similar to openly-pollinated flowers. This information can be used to help refine stocking density estimates for honey bees on raspberry. Wintering bees at 0° or -3° C rather than 4° C effectively delayed emergence of O. lignaria by more than a month without any impact on post-winter performance. These results suggest winter storage at near freezing temperatures is a viable management option for the use of O. lignaria with later-blooming crops.

Cette thèse aborde l’écologie et l’évolution des relations qu’entretiennent les orchidées du genre Ophrys avec leurs pollinisateurs. L’approche comparative et la combinaison (i)

d’analyses chimiques de phéromones sexuelles et de parfums floraux, (ii) d’analyses génétiques avec des outils moléculaires, et (ii) de tests de comportement réalisés sur les insectes in situ nous ont permis d’éclairer certains aspects méconnus de ces interactions inter-spécifiques. La pollinisation des orchidées du genre Ophrys est assurée par des mâles d'abeilles ou de guêpes solitaires qui opèrent une tentative d’accouplement (pseudocopulation) sur le labelle des fleurs. L'attraction des pollinisateurs est généralement hautement spécifique, régie par un mimétisme des signaux (chimiques, visuels, tactiles) des femelles des espèces d'insectes concernés. Malgré cette spécificité, des hybrides se forment occasionnellement en conditions naturelles, témoignant de la perméabilité partielle des barrières d'isolement reproductif entre espèces. Au cours de

ce programme de recherche, nous avons entrepris l’étude des interactions Ophryspollinisateurs en mettant l’accent sur trois aspects spécifiques, à savoir (i) la sélection des caractères floraux par les pollinisateurs, (ii) l'isolement reproductif entre espèces d'Ophrys sympatriques, et enfin (iii) l'évolution des caractères floraux au sein d'un complexe d'espèces-soeurs d'Ophrys associées à différents pollinisateurs. Les principaux

résultats de ce travail sont repris ci-dessous, ponctués de références aux articles qui rassemblent l’intégralité des études réalisées.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

It has been debated whether pollination success in nonrewarding plants that flower in association with nectar-producing plants will be diminished by competition for pollinator visits or, alternatively, enhanced through increased local abundance of pollinators (the magnet species effect). We experimentally evaluated these effects using the nonrewarding bumblebee-pollinated orchid Anacamptis morio and associated nectar-producing plants at a site in Sweden. Pollination success (estimated as pollen receipt and pollen removal) in A. morio was significantly greater for individuals translocated to patches of nectar-producing plants (Geum rivale and Allium schoenoprasum) than for individuals placed outside (similar to20 m away) such patches. These results provide support for the existence of a facilitative magnet species effect in the interaction between certain nectar plants and A. morio. To determine the spatial scale of these interactions, we correlated the visitation rate to flowers of A. morio with the density of sympatric nectar plants in 1-m(2) and 100-m(2) plots centered around groups of translocated plants, and at the level of whole meadows (similar to0.5-2 ha). Visitation rate to flowers of A. morio was not correlated with the 1-m(2) patch density of G. rivale and A. schoenoprasum, but showed a significant positive relationship with density of these nectar plants in 100-m(2) plots. In addition, visitation to flowers of A. morio was strongly and positively related to the density of A. schoenoprasum at the level of the meadow. Choice experiments showed that bees foraging on the purple flowers of A. schoenoprasum (a particularly effective magnet species) visit the purple flowers of A. morio more readily (47.6% of choices) than bees foraging on the yellow flowers of Lotus corniculatus (17% of choices). Overall similarity in flower color and shape may increase the probability that a pollinator will temporarily shift from a nectar-producing "magnet" plant to a nonrewarding plant. We discuss the possibility of a mimicry continuum between those orchids that exploit instinctive food-seeking behavior of pollinators and those that show an adaptive resemblance to nectar-producing plants.

The reproductive outcome of plants is often determined by a multitude of interacting factors operating at both the plant level and the population level. For many plants, fruit production and the subsequent release of seeds are paramount for the persistence of the species. Understanding the processes that influence variation within and among populations is therefore crucial for the successful long-term management of threatened plants. While abiotic factors such as resource availability and environmental conditions can influence seed production directly through their effects on plant growth, biological interactions such as those between plants and pollinators or herbivores can be equally important. The relative intensity and direction of such interactions are often determined by the nature of the plants themselves, or by characteristics of the plant population or the habitat in which it occurs. This thesis examines the processes that influence spatio-temporal variation in the reproductive success of two terrestrial orchids, Caladenia rigida and Caladenia tentaculata. The study was carried out over three years (2005 – 2007), in several populations located in the Mount Lofty region of South Australia. A detailed investigation of the pollination strategy employed by C. rigida revealed that this species is a generalist, being pollinated by a suite of food-seeking insects, possibly attracted by the presence of small amounts of nectar. Successful pollination and seed release for C. rigida was highly variable across space and time. Furthermore, both measures were consistently higher than for the sexually deceptive species, C. tentaculata, leading to the suggestion that the highly specialised pollination syndrome of the latter species may place it at a reproductive disadvantage. Pollination success of C. rigida was influenced by the height of flowers, but not by the local density of conspecifics. Small populations of C. rigida did not produce capsules when environmental conditions were stressful, suggesting that resource availability may indirectly restrict reproductive success by limiting the availability of pollinators. Poor seed quality in some populations may also be attributed to reduced population size. Both orchid species were subject to intense levels of vertebrate florivory and capsule predation, leading to significant reductions in seed output. A herbivore exclusion experiment was carried out to help elucidate the size and type of herbivores, and video surveillance identified birds as a predominant florivore in some populations. The intensity of florivory varied within and among populations, as well as among years, in response to several factors including flower height, the local density of conspecifics, concealment amongst neighbourhood vegetation and proximity to the habitat edge. Spatio-temporal variation in seed release was thus the net outcome of processes acting on both mutualistic and antagonistic interactions. This work provides valuable baseline data of factors that influence the reproductive ecology and, hence, population dynamics of Caladenia species. Implications for the conservation and management of threatened populations are discussed, with respect to both short-term and long-term goals. The thesis is presented as a series of five manuscripts. Two of these have been published, and the remaining three have been prepared for submission as publications.
Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2010

Thesis (M.S.)--University of Georgia, 2004.
Directed by C. Ronald Carroll. For abstract see http://getd.galib.uga.edu/public/reynolds_alexander_200408_ms/reynolds_alexander_200408_ms.pdf. Includes bibliographical references (leaves 95-104).

The endangered western prairie fringed orchid, Platanthera praeclara (Sheviak and Bowles), is found in remnant tall grass prairie in southeastern Manitoba, and has a lower level of seed capsule development in comparison to more southern populations in the United States. Pollination of P. praeclara is limited to few select species of sphinx moths, Sphingidae, Lepidoptera, a group that is less abundant in comparison to other lepidopteran families. This study tested the hypothesis that the presence of ultraviolet light sources near orchids would attract more sphinx moths into orchid habitat and increase feeding activity of sphinx moth pollinators, thus increasing seed capsule production. The second part of this study measured orchid nectar quality and quantity during the bloom period and the possible link to pollination success. A significantly larger number of individual flowers and plants developed seed capsules in the ultraviolet light treatment plots (5.13+-0.42% of available flowers; 35.12+-1.74% of total plants) than the control plots (2.78 +- 0.42% of available flowers; 21.76 +- 2.58% of total plants). The intensity of the ultraviolet light treatment may have also had an effect on pollination success. Ultraviolet lights influenced seed capsule production by attracting sphinx moths into experimental plots. Results also indicated that ultraviolet light may be useful to manipulate seed capsule production for other research purposes. Nectar quality and quantity varied considerably during the study. The mean sugar concentration over the sampling season was 23.9+-0.2%; however, values ranged from a low 13% to high of 34%. Nectar sugar concentration decreased by approximately 6% as the flowering period progressed over the season. % of total plants) than the control plots (2.7g + 0.42 %o of available flowers; 2l .7 6 + 2.58 %o of total plants). The intensity of the ultraviolet light treatment may have also had an effect on pollination success.

There are few other structures in nature from which evolution has generated such wide diversity as the flower or inflorescence, and this diversity is commonly attributed to the influence of their animal visitors. By outsourcing their mate choice to pollinators, plants have left themselves - and especially their flowers - subject to the selective forces imposed by the behaviour, cognition and perception of the pollinators that serve them. The orchids provide some of the most remarkable and extreme examples of adaptations to specific animal pollinators. Perhaps one of the most peculiar of these strategies is sexual deception, whereby male insects are lured to the flower by mimicry of the female sex pheromone. This seemingly unlikely strategy has evolved multiple times independently on different continents in different parts of the orchid phylogeny which raises the question of what adaptive advantages might underlie such a strategy. This multidisciplinary thesis studies gene flow and pollinator behaviour in two sympatric sexually deceptive orchids in the genus Chiloglottis. The two species attract their specific wasp pollinators through emission of distinct species - specific semiochemicals. Since floral volatiles play a pre-eminent role in pollinator attraction, Chiloglottis provides an excellent case study for examining the interaction between floral volatile chemistry, pollinator behaviour and the evolutionary dynamics of populations. The thesis begins with a review of floral volatiles and their role in pollinator attraction and plant speciation. The literature is used to develop a research framework of six testable hypotheses under which we might productively explore the influence of floral volatiles on plant evolution. These hypotheses are then explored in the study system over the following chapters. A study of pollinator specificity, neutral genetic differentiation and floral chemistry demonstrates that the chemical mimicry crucial to sexual deception is responsible for reproductive isolation and potentially even speciation. Mating system and paternity analysis provide the first genetic evidence for multiple paternity in orchid broods. Extensive outcrossing is found to predominate and paternity assignment shows evidence for long distance pollen flow supporting the hypothesis that sexual deception promotes outcrossing and so minimizes the potentially deleterious effects of selfing. Lastly, an innovative new method is developed for tracking wasps in the field. Application of this technique to a population of orchid-pollinating wasps reveals detailed information about their movement and mating behaviour. The findings support the conclusion that sexual deception is a superb adaptive solution to the problem flowers face of simultaneously attracting pollinators and persuading them to leave quickly.

Sexual deception, entailing the pollination of flowers through mimicry of female insects, is one of the most remarkable pollination strategies to have evolved. This thesis explores two Australian sexually deceptive orchid systems with contrasting patterns of pollinator exploitation. The first three chapters focus on the genus Cryptostylis, a system with a unique case of pollinator sharing - five Australian species, four of which are largely sympatric, all deceive the same male ichneumonid pollinator. In Chapter One, mark-recapture experiments were used to investigate the consequences of ichneumonid pollination on pollen movement in C. ovata. A high pollinator revisitation rate indicated some potential for self-pollination. In Chapter Two, reproductive barriers contributing to the absence of hybrids between Cryptostylis species were investigated. Pre-pollination barriers, assessed in field experiments, did not prevent hybridisation. Hand cross-pollinations conducted among the four common Cryptostylis species in a greenhouse all produced fruits, however seed mass and the percentage of formed embryos were reduced in hybrids. Major differences in ploidy and chromosome number likely explain this post-pollination fitness reduction. Two Cryptostylis species were found to be self-incompatible, marking the first case of self-incompatibility in the Diurideae. The unique reproductive biology of Australian Cryptostylis, encompassing pollinator sharing, self-incompatibility, and post-pollination reproductive isolation driven by large ploidy differences, may indicate that its mode of diversification may differ greatly to those in other sexually deceptive genera. Chapter Three presents the first phylogeny to encompass both Australian and Asiatic Cryptostylis. An Australian origin of Cryptostylis is supported, with a likely single dispersal event to Asia. Ploidy variation and geographic barriers appear to have played a role in diversification across Cryptostylis. In Chapter Four, the potential presence of pollination ecotypes in Drakaea livida was tested for. Patterns of chemical diversity and pollinator availability across the distribution of the species are investigated. Pollinator choice trials revealed the presence of three discrete ecotypes each attracting its own pollinator species. Patterns of pollinator availability did not correlate with ecotype distribution. Each ecotype possessed a significantly different floral volatile composition. Using Partial Least Squares Discriminant Analysis (PLS-DA), the presence-absence of a subset of taxonomically informative compounds could be used to accurately predict the ecotype of a flower. Different classes of electrophysiologically active compounds were present in different ecotypes. These marked differences in chemical composition between the ecotypes suggest either a long time since their divergence and may hint at a scenario of convergent evolution of floral morphology. In Chapter Five, the ecotype geographic ranges and methods of identifying the ecotypes were investigated. Species distribution modelling predicted each ecotype to have a different core range. Two ecotypes were widespread, while one had a limited distribution within extensively cleared agricultural land, raising conservation concerns. PLS-DA correctly identified the ecotype of a flower when labella extracts were made from pollinated flowers, thereby providing a non-destructive identification technique. The pollinator specificity, morphology, floral chemistry, and ranges of the ecotypes supported them as Evolutionary Significant Units. In conclusion, the ecological and evolutionary consequences of pollination by sexual deception may vary extensively between plant taxa in accordance with their different patterns of pollinator exploitation. The taxonomy, species richness of the pollinator group, and the plant species to pollinator species ratio all influence the evolution and diversification of sexually deceptive orchids.

The morphology, anatomy and pollination biology of Mystacidium Lindl., a small, epiphytic genus of orchids, was investigated within a phylogenetic context. Morphological and anatomical studies were carried out in order to obtain characters for a cladistic analysis of the genus using Cyrtorchis arcuata (Lindl.) Schltr. as an outgroup. The phylogenetic analysis indicated that the genus may not be monophyletic. Two species of the closely related genus Diaphananthe Schltr., D. caffra (H.Bol.) Linder and D. millarii (H.Bol.) Linder, appear to be nested within Mystacidium. Mystacidium species grow in habitats varying from mistbelt forest to dry savanna. Analysis of stable isotope composition (Ȣ¹³C values) of leaves and roots showed that all Mystacidium species, as well as D. caffra and the outgroup C. arcuata, employ CAM photosynthesis. The Ȣ¹³C values were significantly negatively correlated with mean annual rainfall at the collection sites. Breeding system experiments revealed that Mystacidium is dependent on pollinators for fruit set, and that self-pollination results in substantially reduced seed set due to either inbreeding depression or partial self-incompatibility. Field observations revealed that M. venosum Harv. ex Rolfe and M. capense (L.f.) Schltr. are hawkmoth-pollinated, and that M. gracile Harv. and M. pusillum Harv. are pollinated by settling moths. The spurs of the flowers contain dilute, sucrose-dominant nectar. Mystacidium venosum and M. capense showed evidence of nectar reabsorption. Nocturnal emission of scent occurred in all species except M. aliceae H. Bolus and M. brayboniae Summerh., which are unscented, and was composed largely of a combination of monoterpenes and benzoids. Despite substantial variation in spur length (1 - 4.7 cm) among species, no evidence for directional selection on spur length was found in M. venosum, M. capense or M. gracile. Hand pollinations significantly increased fruit set in M. capense in two consecutive seasons at different sites, indicating pollen limitation. Although pollen removal was greater than pollen receipt in M. venosum, M. capense and M. gracile, suggesting transport loss or insufficient visitation, a remarkably high percentage of removed pqllen reached stigmas (35 - 50%). Experiments on M. venosum revealed that flower longevity is reduced by pollination, and that pollinia removed from flowers remained viable for up to 20 days under field conditions. The phylogeny indicated that long-spurred, hawkmoth-pollinated species are basal within the genus, and that shorter-spurred species pollinated by noctuid moths are derived.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

碩士
國立屏東科技大學
農園生產系所
100
Incompatibility often occurs during the process of pollinating Phalaenopsis orchids, which led to hybridization barriers, including flower drop, failed capsule setting or no seeds formation. The purpose of this study is to explore pollen tube growth in the gynoecium and capsule development after self- or cross-pollination of Doritaenopsis Sogo Shiva ‘NPU2’ as female parent. Besides, relation of sporad type of male parents and capsules development after cross-pollination was also investigated. Three days after self-pollination pollen tubes started to germinate. Eight weeks after pollination cristae-like structure were produced in placenta surface. After 11 weeks, seed tunica was observed but mostly without embryo. After 22 weeks, some seeds were observed, which corresponds to previous observations of fertilization 60 days after pollination. Three cases of aborted cross-pollination were observed, including flower drop 7-15 days, 16-22 days, or failed capsule setting about 2 months later, suggesting some different situations of hybrid barriers. Two sporad types of pollen mother cells (PMCs) were revealed for all tested varieties. The first is with more than 60% tetrads, the second consists of tetrads with high frequency micronuclei, depending on different varieties. Phalaenopsis species have a higher tetrad ratio of more than 90%. Despite Some varieties with more than 60% tetrad, but either without pollination success, low pollen viability, cross-incompatibility, or weak pollen tube growth. In two of the four varieties of yellow flower sporad morphology was unrecognized, and other two with high micronuclei ratio. It is speculated that meiosis may be incomplete, or due to their interspecific or intergeneric hybrids nature. If sporad type is known before pollination, choosing the males with higher tetrad ratio may have the chance to improve the success of cross-pollination.

Orchids provide a model system for addressing evolutionary and ecological questions both because of their species diversity, and because the packaging of their pollen into pollinia facilitates the estimation of male and female pollination success. This thesis focuses on the ecology and evolution of pollination systems in the African orchid genus Eulophia, with an emphasis on deceptive pollination, mechanisms promoting cross-pollination, and pollinatordriven speciation. Pollination in the deceptive species E. zeyheriana is shown to depend on flower colour and proximity to the rewarding model species, Wahlenbergia cuspidata (Campanulacae). This study demonstrates the functional importance of colour matching between model and mimic in a floral Batesian mimicry system, as well as the importance of facilitation by the rewarding model [chapter 2]. The pollinaria of the vast majority of Eulophia and Acrolophia species undergo reconfiguration following removal by pollinators, similar to the phenomena first described by Darwin in some European orchids and which he hypothesised to be adaptations to limit pollinator mediated self-pollination. In chapter 3, a less common mechanism – anther cap retention – is described for E. foliosa. Observations of reconfiguration times were compared to the respective visit times by pollinators in a number of orchids (including Eulophia and Acrolophia) and asclepiads. In 18 of 19 species, pollinarium reconfiguration times exceed the average visit times, providing empirical support for Darwin’s cross-pollination hypothesis [chapter 4]. All of the 25 species of Eulophia examined are deceptive, but two of the three species in the small, closely related Cape genus Acrolophia examined in chapter 5 are rewarding. This translates into very high levels of pollen transfer efficiency in the rewarding A. cochlearis relative to the deceptive A. capensis and species of Eulophia. In addition, A. cochlearis exhibits high rates of pollinator-mediated self-pollination, as quantified using a novel method based on levels of inbreeding depression during embryo development. In chapter 6 the evolutionary divergence of long- and short-spurred forms of E. parviflora in response to different pollinators is investigated. This shows that divergence has occurred in floral morphology, scent chemistry and flowering phenology and that this can be attributed to adaptations to the respective bee and beetle pollinators of each form. This thesis also includes case histories of bee pollination in an additional five Eulophia species, and beetle-pollination in two other species of Eulophia with dense inflorescences and slow pollinarium reconfiguration [chapter 7]. In addition, four taxa were found to undergo auto-pollination [chapter 8]. The main conclusions of this thesis are that pollination of food-deceptive species can be enhanced by spatial proximity to, and floral colour matching with, sympatric rewarding species; that selection strongly favours traits that promote cross-pollination; that pollinatorshifts can drive speciation; and that floral adaptations for bee-, beetle-, and auto-pollination are found in South African representatives of Eulophia.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

I investigated how orchid biology, floral morphology, and diversity of surrounding floral and pollinator communities affected reproductive success and hybridization of Cypripedium candidum and C. parviflorum. Floral dimensions, including pollinator exit routes were smallest in C. candidum, largest in C. parviflorum, with hybrids intermediate and overlapping with both. This pattern was mirrored in the number of insect visitors, fruit set, and seed set. Exit route size seemed to restrict potential pollinators to a subset of visiting insects, which is consistent with reports from other rewardless orchids. Overlap among orchid taxa in morphology, pollinators, flowering phenology, and spatial distribution, may affect the frequency and direction of pollen transfer and hybridization. The composition and abundance of co-flowering rewarding plants seems to be important for maintaining pollinators in orchid populations. Comparisons with orchid fruit set indicated that individual co-flowering species may be facilitators or competitors for pollinator attention, affecting orchid reproductive success.

To effectively manage an endangered plant species, managers must have an adequate knowledge of the habitat of the species and its reproductive ecology. This study describes the plant community associated with the endangered western prairie fringed orchid (WPFO) (Platanthera praeclara) in Manitoba and determine whether the plant community differed between sites and between six distances from the centre of orchid beds. In addition, this study exarnines factors which may affect the pollination ecology, specifically pollinator visitation. Assessment of orchid habitat occurred at four sites, separated by at least 800 m, in the Tall Grass Prairie Preserve (TGPP) in southeastern Manitoba in 2005 and 2006. The four sites were broadly similar in that each was dominated by graminoids (sedges, rushes, and grasses) and shared a moderate number of forb and graminoid species. The most abundant forbs included Galium boreale, Fragaria virginiana and Hypoxis hirsuta. The measures of species richness (species richness and rarified richness) varied significantly between distances from the centre of the orchid beds, but not between sites. The diversity measures (the Shannon-Weiner and Simpson indices) and the evenness measures (Shannon-Weiner evenness and rank log abundance slope) were significantly different between sites but not distances from the centre of the orchid beds. Ordination methods (Principal Components Analysis and Multiple Discriminant Analysis) showed the plant community at one site differed from that of the other sites, but the `plant community was not significantly different between distances from orchid bed centre. Pollinator (ie: sphinx moth) visitation rates, measured using seed capsule production, the presence of moth scales, and pollinaria removal, varied considerably between sites and between years. The abundance of nectar sources and larval hosts for the pollinators at the sites was not related to visitation rates. The visibility and proximity of anthropogenic light sources to orchids did not correspond with rates of pollinator visitation. Sphinx moth activity at the TGPP was not significantly correlated with weather conditions. The plant community associated with the WPFO was broadly similar at different sites, though differences in composition and diversity were evident. Controlled experiments would be useful in determining the effect of various factors, including those examined in this study, on the pollinator visitation rates to the WPFO in Manitoba.