Academic literature on the topic 'Orchid, pollination'

Orchids and their pollinators have developed fascinating co-adaptations that promote orchid pollination. This 6-page fact sheet details the various and often strange ways that orchids attract pollinators. The kinds of insects and animals that pollinate orchids and orchids’ reproductive anatomies and processes are also covered. Written by Haleigh Ray and Wagner Vendrame, and published by the UF Department of Environmental Horticulture, June 2015. ENH1260/EP521: Orchid Pollination Biology (ufl.edu)

A single plant might be visited by many flower visitors but not all might act as pollinators. Legitimate pollinators might also differ considerably in their efficiency, limiting pollination success. Unsuitable climatic conditions such as rain also affect pollinator activity. However, in the evergreen rainforest there is no prolonged dry season and flowering occurs usually under rain. Here, we explore the dependence on pollinators and the efficiency of flower visitors for the fruiting success of 10 Andean rainforest orchids. All species were self-compatible but strictly pollinator-dependent. Overall, we found low levels of fruit set in control flowers while experimental geitonogamous and cross-pollinations increased fruit set, revealing extensive pollination limitation in all populations. Seed viability dropped considerably after self and geitonogamous pollinations suggesting the possibility of early-acting inbreeding depression. Even though we monitored flower visitors on an extensive survey, few visitors were seen in these species and even fewer acted as legitimate pollinators. Thus, even though orchid pollination might be extremely diversified, these results show that few visitors are pollinating these species, explaining the low levels of fruit set recorded in the area studied.

The lack of appropriate pollinators is believed to be an impediment to naturalization of orchids. We studied the self-compatibility and pollination of an invasive, showy, rewardless, terrestrial orchid ( Cyrtopodium polyphyllum (Vell.) Pabst ex F. Barrios) in southern Florida. Pollination treatments indicated that the orchid is self-compatible but not autogamous. Visitor observations indicated that the orchid is pollinated by an invasive specialist oil-collecting bee ( Centris nitida Smith). The presence of the invasive Centris nitida has probably contributed to the naturalization of this probable oil reward mimicking orchid in Florida. We contrasted our results in the orchid’s invasive range with those reported in two studies in the orchid’s native range. In the invasive range in Florida, the orchid experiences a lower inbreeding depression. In both areas, the orchid is pollinated by Centris species and the pollination of the orchid is apparently enhanced by the presence of oil-rewarding species of the Malpighiaceae.

Many orchids achieve pollination by deceptive means. Sexually deceptive orchids are pollinated by male insects, which are lured to flowers that mimic the sex pheromones and/or appearance of their female conspecifics. This specialised pollination strategy was recently confirmed for the first time in a Pterostylis species in Australia. We investigated whether this pollination strategy may also be operating in Pterostylis species in New Zealand where generalised plant–insect pollination strategies are most commonly documented. The breeding systems of Pterostylis oliveri Petrie and Pterostylis irsoniana Hatch were investigated in the field with pollination treatments. Sticky traps were set up over flowering P. oliveri, P. irsoniana and Pterostylis venosa Colenso to catch potential pollinators of the flowers. Insects caught carrying orchid pollinia were identified, and the pollinia were identified to plant species with nuclear rDNA internal transcribed spacer (nrDNA ITS) sequences. Both P. oliveri and P. irsoniana were found to be self-compatible, but dependent on insects for pollination. Pollinia from each of the three Pterostylis spp. were found to be carried species-specifically by male fungus gnats (Diptera: Mycetophilidae): only Mycetophila latifascia fungus gnats carried the pollinia of P. oliveri, Morganiella fusca gnats carried the pollinia of P. irsoniana, and Tetragoneura sp. carried the pollinia of P. venosa. The pollinator specificity indicates that each of the male fungus gnat species was attracted to the flowers of a specific Pterostylis orchid. This strongly suggests that each of the orchid species emit a specific floral volatile, most probably resembling the sex pheromones of the female conspecifics, to lure their male pollinators. These are the first documented cases of highly specialised sexually deceptive pollination in New Zealand orchids, which were thought to be predominantly self-pollinating.

There are approximately 30,000 species in the family Orchidaceae, with some species growing terrestrially and others growing as epiphytes. Though the pollination biology of many of these species is not well known, there has been a diversity of taxa recorded as orchid pollinators. Insects, birds, and even a record of a mammal species have been documented as successful pollinators, while some orchids are able to reproduce without the use of a pollen vector. The goal of this review is to provide an overview of orchid pollination tactics, with references to more specific studies of each, and to analyze a large subset of publications to determine differences in pollinator taxa and specificity between epiphytic and terrestrial orchid genera. This review examines pollination data from over 400 orchid species, including 74 epiphytic and 83 terrestrial orchid genera. Two pollinator taxa, Coleoptera and Hymenoptera (Class: Insecta), were found to pollinate significantly more terrestrial than epiphytic orchid genera, while other taxa showed no significant differences. Hymenoptera were the dominant taxa of pollinator in regards to the overall number of species recorded; however, based on species interaction webs that were built, the Lepidoptera (Class: Insecta) have stronger interactions with the orchid species they pollinate, suggesting a more specific relationship between the two.

This review summarises scientific knowledge concerning the mycorrhizal associations, pollination, demographics, genetics and evolution of Australian terrestrial orchids relevant to conservation. The orchid family is highly diverse in Western Australia (WA), with over 400 recognised taxa of which 76 are Declared Rare or Priority Flora. Major threats to rare orchids in WA include habitat loss, salinity, feral animals and drought. These threats require science-based recovery actions resulting from collaborations between universities, government agencies and community groups. Fungal identification by DNA-based methods in combination with compatibility testing by germination assays has revealed a complex picture of orchid–fungus diversity and specificity. The majority of rare and common WA orchids studied have highly specific mycorrhizal associations with fungi in the Rhizoctonia alliance, but some associate with a wider diversity of fungi. These fungi may be a key factor influencing the distribution of orchids and their presence can be tested by orchid seed bait bioassays. These bioassays show that mycorrhizal fungi are concentrated in coarse organic matter that may be depleted in some habitats (e.g. by frequent fire). Mycorrhizal fungi also allow efficient propagation of terrestrial orchids for reintroduction into natural habitats and for bioassays to test habitat quality. Four categories of WA orchids are defined by the following pollination strategies: (i) nectar-producing flowers with diverse pollinators, (ii) non-rewarding flowers that mimic other plants, (iii) winter-flowering orchids that attract fungus-feeding insects and (iv) sexually deceptive orchids with relatively specific pollinators. An exceptionally high proportion of WA orchids have specific insect pollinators. Bioassays testing orchid-pollinator specificity can define habitats and separate closely related species. Other research has revealed the chemical basis for insect attraction to orchids and the ecological consequences of deceptive pollination. Genetic studies have revealed that the structure of orchid populations is influenced by pollination, seed dispersal, reproductive isolation and hybridisation. Long-term demographic studies determine the viability of orchid populations, estimate rates of transition between seedling, flowering, non-flowering and dormant states and reveal factors, such as grazing and competition, that result in declining populations. It is difficult to define potential new habitats for rare orchids because of their specific relationships with fungi and insects. An understanding of all three dimensions of orchid habitat requirements can be provided by bioassays with seed baits for fungi, flowers for insects and transplanted seedlings for orchid demography. The majority of both rare and common WA orchids have highly specific associations with pollinating insects and mycorrhizal fungi, suggesting that evolution has favoured increasing specificity in these relationships in the ancient landscapes of WA.

Floral deception has been observed in several genera in angiosperms, but is most common in the Orchidaceae. Pollination mechanisms in food deceptive plants are often difficult to assess, as visitation frequency by insects requires numerous hours of field observations to ascertain. Here, for the first time, we describe in detail and validate a simple and effective method that extends previous approaches to increase the effectiveness of pollination studies of food deceptive orchids. We used an orchid of southwest Australia, Diuris brumalis (Orchidaceae), that visually mimics model plants belonging to the genus Daviesia (Faboideae). Arrays of orchid flowers were placed and moved systematically in proximity to model plants, resulting in rapid attraction of the pollinators of D. brumalis. We compared pollinaria removal (as an indicator of pollination success) in naturally growing orchids with pollinaria removal in arrays of orchid flowers in the same sites. We showed that the proposed method greatly enhances pollinator attractiveness in food deceptive systems with very low pollination rates, and we compared its efficiency with other similar methods. The approach can be used for observing pollinator behavioural patterns and confirming effective pollinators for food deceptive species with low insect visitation rates.

The translocation of orchids (Orchidaceae) cannot be successful if one is unaware of their effective pollinators and plant–pollinator interactions. Cypripedium guttatum is a generalized food-deceptive orchid, which is highly threatened in the Republic of Korea, thus, requiring immediate translocation actions. Although effective pollinators of the orchid are well known in China, little is known about the pollinators in the Republic of Korea and the ecological context in which the orchid can be successfully pollinated. To briefly assess the pollination of C. guttatum prior to translocation, we conducted a one-month survey of general pollination and the community-wide plant–pollinator network properties. Over 21 h of observation, we found that an effective pollinator of the orchid was the sweat bee Lasioglossum virideglaucum. The network was significantly specialized and modular, but not significantly nested. L. virideglaucum (pollinator) and Arabis gemmifera (plant) were determined to be keystone species, based on network metrics. A total of six network modules were identified and the flower colors of the plant species belonging to the C. guttatum module were purple, white, and yellow. After comparing the daily network patterns, we found that pollination of the orchid was accomplished when various flowering plant species bloom, and the nestedness value was high. This study revealed that high plant and pollinator richness could increase the chance that the deceptive orchid would be pollinated. Our study suggests that the network properties of this food-deceptive orchid community could provide useful insight into understanding the ecologically suitable habitat for the translocation of the highly threatened orchid species C. guttatum.

Food deception as a pollination strategy has inspired many studies over the last few decades. Pollinator deception has evolved in many orchids possibly to enhance outcrossing. Food-deceptive orchids usually have low pollinator visitation rates as compared to rewarding species. They may benefit in visitations from the presence (magnet-species hypothesis) or, alternatively, absence of coflowering rewarding species (competition hypothesis). We present data on pollinator visitations on a deceptive, terrestrial orchidCalypso bulbosa, a species with a single flower per plant and whose flowering period partly overlaps with rewarding, early flowering willows (Salixsp.) and later-flowering bilberry (Vaccinium myrtillus). When surveying inactive bumblebee queens on willows in cool weather, about 7% of them carriedCalypsopollinia. Most common bumblebee species appeared to visit and thus pollinateCalypso. Bumblebees typically visited one to threeCalypsoflowers before flying away, providing some support for the outcrossing hypothesis. We conclude that, regarding the pollinations strategy, both magnet-species and competition hypotheses have a role in the pollination ofCalypso, but on different spatial scales. On a large scale rewarding species are important for attracting pollinators to a given region, but on a small scale absence of competition ensures sufficient pollination rate for the deceptive orchid.

Prior to undertaking conservation translocations of plants with specialised pollination systems, it is important to ensure the presence of pollinators at recipient sites. Here, for two threatened species, Caladenia concolor Fitzg. and Caladenia arenaria Fitzg. (Orchidaceae), we determine (i) the pollination strategy used, (ii) which floral visitors are involved in pollination, and (iii) whether the pollinator species are present at potential translocation sites. For both orchid species, pollination was primarily achieved by nectar-foraging thynnine wasps, with a single species responsible for pollination in C. concolor, whereas C. arenaria utilised at least two species to achieve pollination. Both orchid species secreted meagre quantities of sucrose on the upper surface of the labellum. Visits to C. concolor occurred primarily in the late afternoon, with some wasps perching on the flowers overnight. Surveys revealed that pollinators were present at all extant populations and most potential translocation sites for both orchids. The specialisation on one pollinator species in C. concolor means that the distribution of the pollinator needs to be considered for conservation translocations. With C. arenaria, the risk of hybridisation with other Caladenia that are known to share one of its pollinator species needs to be taken into account when selecting translocation sites.

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

Abstract This chapter focuses on designing the orchard, site preparation, orchard structures, nursery tree source selection, successful pollination, planting and the use of plant growth regulators in cherry production.

Abstract With an ever-changing climate, the decline in the bee population is forcing humans to find supplemental ways to pollinate. A mechanical substitute for a bee needs to be as efficient or better than bee pollination since it will require one person to do the work of many bees. Pollinating one flower at a time would be too time consuming to be effective, so a drone mounted pollen distribution device is the best direction to go to solve this problem. This is a game changing design to prevent a lack of food-producing plant growth as the bee population decreases over time. The design is a low-cost alternative that can be adjusted to meet the consumer needs based on the type of pollination. This design is easy to manufacture en masse due to its simplicity. The design and operation have been optimized using stress, torque, and safety factor analyses to ensure that the product can withstand the loads applied during operation. This drone pollinator will have the ability to work for various drone types and plant pollens. With the use of commercial drones, the ability to distribute pollen to a large orchard or forested area will allow humans to supplement bee pollination on a large scale in the farming and forest conservation industries.

The project investigated the molecular genetic and biochemical basis of pollination-induced senescence of Phalaenopsis flowers. This experimental system offered unique advantages in that senescence is strictly regulated by pollination, providing the basis to experimentally initiate and synchronize senescence in populations of flowers. The postpollination syndrome in the Phalaenopsis orchid system was dissected by investigating the temporal and spatial regulation of ACC synthase gene expression. In the stigma, pollen-borne auxin induces the expression of the auxin-regulated ACC synthase (PS-ACS2) gene, resulting in ACC synthesis within 1 h following pollination. Newly formed ACC is oxidized by basal constitutive ACC oxidase to ethylene, which then induces the expression of the ethylene-regulated ACC synthase(PS-ACS1) and oxidase (ACO1) genes for further autocatalytic production of ethylene. It is speculated that during the 6-h period following pollination, emasculation leads to the production or release of a sensitivity factor that sensitizes the cells of the stigma to ethylene. ACC and ethylene molecules are translocated from the stigma to the labellum and perianth where ethylene induces the expression of PS-ACS1 and ACO1 resulting in an increased production of ACC and ethylene. Organ-localized ethylene is responsible for inrolling and senescence of the labellum and perianth. The regulation of ethylene sensitivity and signal transduction events in pollinated flowers was also investigated. The increase in ethylene sensitivity appeared in both the flower column and the perianth, and was detected as early as 4 h after pollination. The increase in ethylene sensitivity following pollination was not dependent on endogenous ethylene production. Application of linoleic and linoleic acids to Phalaenopsis and Dendrobium flowers enhanced their senescence and promoted ethylene production. Several major lipoxygenase pathway products including JA-ME, traumatic acid, trans-2-hexenal and cis-3-hexenol, also enhanced flower senescence. However, lipoxygenase appears to not be directly involved in the endogenous regulation of pollination-induced Phalaenopsis and Dendrobium flower senescence. The data suggest that short-chain saturated fatty acids may be the ethylene "sensitivity factors" produced following pollination, and that their mode of action involves a decrease in the order of specific regions i the membrane lipid bilayer, consequently altering ethylene action. Examination of potential signal transduction intermediates indicate a direct involvement of GTP-binding proteins, calcium ions and protein phosphorylation in the cellular signal transduction response to ethylene following pollination. Modulations of cytosolic calcium levels allowed us to modify the flowers responsiveness to ethylene.

The project objective was to more fully understand how the motion of pollen grains may be controlled by electrostatic forces, and to develop a reliable mechanized pollination system based upon sound electrostatic and aerodynamic principles. Theoretical and experimental analyses and computer simulation methods which investigated electrostatic aspects of natural pollen transfer by insects found that: a) actively flying honeybees accumulate ~ 23 pC average charge (93 pC max.) which elevates their bodies to ~ 47 V likely by triboelectrification, inducing ~ 10 fC of opposite charge onto nearby pollen grains, and overcoming their typically 0.3-3.9 nN detachment force resulting in non-contact electrostatic pollen transfer across a 5 mm or greater air gap from anther-to-bee, thus providing a theoretical basis for earlier experimental observations and "buzz pollination" events; b) charge-relaxation characteristics measured for flower structural components (viz., 3 ns and 25 ns time constants, respectively, for the stigma-style vs. waxy petal surfaces) ensure them to be electrically appropriate targets for electrodeposition of charged pollen grains but not differing sufficiently to facilitate electrodynamic focusing onto the stigma; c) conventional electrostatic focusing beneficially concentrates pollen-deposition electric fields onto the pistill tip by 3-fold as compared to that onto underlying flower structures; and d) pollen viability is adequately maintained following exposure to particulate charging/management fields exceeding 2 MV/m. Laboratory- and field-scale processes/prototype machines for electrostatic application of pollen were successfully developed to dispense pollen in both a dry-powder phase and in a liquid-carried phase utilizing corona, triboelectric, and induction particulate-charging methods; pollen-charge levels attained (~ 1-10 mC/kg) provide pollen-deposition forces 10-, 77-, and 100-fold greater than gravity, respectively, for such charged pollen grains subjected to a 1 kV/cm electric field. Lab and field evaluations have documented charged vs. ukncharged pollen deposition to be significantly (a = 0.01-0.05) increased by 3.9-5.6 times. Orchard trials showed initial fruit set on branches individually treated with electrostatically applied pollen to typically increase up to ~ 2-fold vs. uncharged pollen applications; however, whole-tree applications have not significantly shown similar levels of benefit and corrective measures continue. Project results thus contribute important basic knowledge and applied electrostatics technology which will provide agriculture with alternative/supplemental mechanized pollination systems as tranditional pollen-transfer vectors are further endangered by natural and man-fade factors.