Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite. / Doctor of Philosophy / Field dodder (Cuscuta campestris) is a parasitic plant that lacks leaves and roots and attacks a wide range of plants, such as tomato and beets. Dodders are not able to carry out full photosynthesis and thus are incapable of producing enough food or obtaining water to survive on their own, so they parasitize other plants. Dodders have developed specialized structures called haustoria that allow them to take resources directly from their hosts. Studying dodder is challenging due to the dependence of the parasite on its host, such that effects of one plant on the other are hard to disentangle. We developed new tools and methodologies that allow us to explore the biology of dodder in unprecedent ways. We developed an Artificial Host System (AHS) that allows the growth and study of dodder without involving a living host plant. Thanks to this new tool, we were able to improve understanding of the function of the haustorium, discover nutrients and growth factors that are indispensable for dodder development, and prove that dodder growth benefits from light. Using the AHS, we compared haustorial regions and shoot tips of dodder to identify genes specific to haustorial function. Additionally, we studied the responses of dodder to bacteria to understand how it reacts to microbial colonization. Our studies contribute with the development of novel methodologies that allow unprecedent discoveries into the biology of dodder. We expect that this work will promote the study of parasitic plant biology.

A conexão hidráulica entre plantas parasitas e suas hospedeiras representa a formação de um sistema hidráulicos híbrido. Além disso, plantas parasitas apresentam condutâncias estomáticas muito elevadas e potenciais hídricos mais extremos do que suas hospedeiras. Por estes motivos, estas associações representam ao mesmo tempo um desafio técnico de fusão funcional de dois sistemas complexos, e uma fonte de stress e sobrecarga para as hospedeiras, que tem sua copa competindo com plantas em regime de balanço hídrico muito díspar. Os objetivos deste trabalho foram analisar forma e função da conexão entre Psittacanthus robustus e Vochysia thyrsoidea, de maneira interdisciplinar entre a anatomia da madeira e arquitetura hidráulica, para testar hipóteses relacionadas a estrutura da conexão, mudanças associadas a formação do sistema híbrido e influências da sobrecarga sobre as propriedades das hospedeiras. A estrutura da conexão foi avaliada revelando novidades. A interpretação dos tecidos que formam os haustórios foi revisada, foram descritas modificações nas duas espécies relacionadas à conexão e descobriu-se uma nova estrutura nos haustórios, para a qual se propôs uma hipótese funcional. Os galhos parasitados apresentaram potenciais hídricos mais baixos, no entanto são mais eficientes no transporte de seiva. Isto se relaciona a alterações morfoanatômicas que condicionam redução da resistência ao fluxo: maior contato entre vasos, paredes intervasculares e membranas de pontoações mais delgadas. Discute-se a reação das hospedeiras no contexto de respostas ao stress hídrico / The hydraulic connection between parasitic plants and their hosts represents the formation of a hybrid hydraulic system. Besides, parasitic plants show high stomata conductance and more extreme water potentials. Because of it, these associations represent both a technical challenge for the fusion of two complex systems in a function unit, and a source of stress and overload for the hosts. The aim of this work was to analyze form and function of the connection between Psittacanthus robustus and Vochysia thyrsoidea, to test hypothesis related to connection structure, changes associated to the formation of the hybrid system and influence of the overload on the host´s properties. The interpretation of haustoria tissues was reviewed, modifications on both the species were described and a new structure was discovered in the haustoria, to which we proposed a functional hypothesis. Parasitized branches have lower water potentials, but are more efficient in sap conduction. This correlates to morphoanatomical modifications related to reduction in xylem resistance: more intervessel contacts and thinner intervascular walls and pit membranes. The reactions observed on the hosts are discussed in light of water stress responses

As plantas holoparasitas dependem inteiramente de recursos retirados do xilema e floema hospedeiros e são representadas atualmente por pelo menos sete linhagens, cujo posicionamento filogenético continua sendo revisto. A família Apodanthaceae inclui plantas aclorofiladas, holoparasitas de caules, cujo corpo vegetativo apresenta extrema redução. Por serem endoparasitas, tornam-se evidentes apenas na época da floração, quando as diminutas flores rompem a periderme do hospedeiro. Incluem-se nessa família três gêneros, sendo Pilostyles o de mais ampla distribuição. Apesar de existirem estudos minuciosos sobre o sistema endofítico no gênero, sua fenologia, ciclo de vida e ontogênese floral continuam desconhecidos. O presente estudo objetiva elucidar essas questões, de modo a proporcionar uma compreensão do sistema reprodutivo e do processo evolutivo das flores dessas intrigantes angiospermas, servindo de base para estudos futuros que visem a estabelecer homologias com outros clados. Primeiramente foi realizado um levantamento dos hospedeiros parasitados, analisando-se padrão de distribuição das flores e a razão sexual das populações de Pilostyles existentes nas áreas de estudo no Brasil e México. Indivíduos foram selecionados aleatoriamente para a realização dos testes de polinização, observação e coleta de visitantes, coleta de néctar e de sementes parasitas, e acompanhamento fenológico. Em laboratório realizaram-se os experimentos de germinação e as técnicas de preparo e análise histológicas das sementes e flores de Pilostyles. Com relação ao ciclo de vida, observou-se que: 1) as vespas aparecem como os visitantes mais freqüentes; 2) a dispersão provavelmente seja resultado de endozoocoria por aves; 3) a germinação provavelmente ocorre diretamente no caule hospedeiro; 4) as fenologias da hospedeira e da parasita estão diretamente relacionadas, havendo inclusive possibilidade de sincronismo. Com relação à razão sexual das populações, concluímos que: 1) a determinação do sexo das flores responde a estímulos específicos de cada espécie hospedeira; 2) a distribuição espacial das flores esta diretamente relacionada à razão sexual das populações; 3) existem flores monoclinas em número constante em todas as populações, sendo tais flores funcionalmente estaminadas. A partir do estudo da anatomia floral e embriologia, pudemos observar que: 1) as flores são envolvidas por quatro verticilos estéreis (de fora para dentro: 2 + 2 + 4 + 4); 2) a região estigmática é restrita à área das papilas; 3) a deiscência dos microsporângios é resultado da expansão e ressecamento do órgão; 4) os tricomas vesiculares secretam uma substância pegajosa, provavelmente responsável por unir os grãos de pólen e grudá-los ao corpo do polinizador; 5) a coluna das flores estaminadas é formada através de fusão pós-genital de uma coluna estilar central e de uma coluna externa, que traz no bordo superior os sacos polínicos. Concluindo, acreditamos que tais dados merecem destaque pelo ineditismo para a literatura atual da família Apodanthaceae e, principalmente, na sua capacidade de ancorar futuros estudos comparativos com outros táxons, servindo de base na avaliação das propostas taxonômicas vigentes. / Holoparasitic plants depend entirely on resources from the host xylem and phloem and are currently represented by at least seven lineages, whose phylogenetic position is still being reviewed. The Apodanthaceae family includes stem holoparasitic plants, achlorophyllous, whose vegetative body is extremely reduced. Because they are endoparasites, they become evident only at the time of flowering, when the tiny flowers emerge from the hosts bark. There are three genera in this family, being Pilostyles the one with broader distribution. There are detailed studies on the endophytic system of this genus, but its phenology, life cycle and floral ontogeny remain unknown. This study aims to elucidate these issues, in order to provide an understanding of the reproductive system and the evolutionary process of the flowers of these intriguing angiosperms. We hope these could be a basis for future studies that aim to establish homologies with other groups of angiosperms. First it was made a survey of parasitized hosts in Brazil and Mexico areas, and then the distribution pattern of flowers and sex ratio of the Pilostyles populations were analyzed. Individuals were randomly selected for the experiments of pollination, observation and collection of floral visitors, collecting nectar and phenological monitoring. Meanwhile, in the laboratory, were performed germination experiments and the histological analysis of Pilostyles seeds and flowers. Regarding the life cycle, we observed that 1) the wasps are the most frequent visitors; 2) the dispersion is probably a result of endozoochory by birds; 3) germination probably occurs directly in hosts stem; 4) the host and parasite phenologies are directly related; there is a possibility of timing. Regarding the sex ratio of the populations, we conclude that: 1) sex determination of Pilostyles flowers seems to respond to specific stimuli of each host species; 2) the spatial distribution of staminate and pistillate flowers is directly related to the sex ratio of the populations; 3) there are monoclinous flowers in constant number in all populations, and these flowers are functionally staminate. From the study of floral anatomy and embryology, we observed that: 1) the flowers are surrounded by four whorls of scales, thereby arranged from outside to inside: 2 + 2 + 4 + 4; 2) the stigmatic surface is restricted to the area with papillae, 3) the dehiscence of the pollen sacs is a result of synandrium expansion and dryness, occurring initially by individual transverse slit; 4) the vesicular trichomes secrete a sticky substance, probably responsible for uniting the pollen grains and stick it to the pollinators body; 5) the column of staminate flower is formed by post-genital fusion of a central column, the sterile gynoecium, and an external column, which brings the pollen sacs in the upper board. In conclusion, we believe that such data should be highlighted by the novelty for the Apodanthaceae family literature, and especially in its ability to underpin comparative studies with other taxa, being a basis to evaluate current taxonomic proposals.

A demographic census and disease assessment was conducted in 2007 and 2015 in three Tennessee populations of the dioecious shrub,;Buckleya distichophylla (Santalaceae). Population sizes were relatively stable over time and plant heights and numbers of stems per clump were similar among populations. Seedlings were present in all populations, where they represented 14–19% of individuals. Two populations had an equal male:female sex ratio, but a third population was male-biased. Nonflowering individuals comprised 33–41% of individuals in a population. The majority of plants in all populations had high vigor. Spatial analyses revealed clusters of seedlings in two populations and a cluster of low-vigor plants in one population. Cronartium appalachianum, a rust fungus dependent upon;Pinus virginiana and B. distichophylla as primary and alternate hosts, respectively, was present in all populations with prevalences on B. distichophylla of 19–29%, but there was no spatial clustering of disease in populations. The tree species nearest to B. distichophyllavaried among populations with Tsuga canadensis predominant in one population, T. caroliniana in another, and P. virginiana in the third. Buckleya distichophylla in proximity to P. virginiana had a higher than expected prevalence of C. appalachianum infection.

As plantas holoparasitas dependem inteiramente de recursos retirados do xilema e floema hospedeiros e são representadas atualmente por pelo menos sete linhagens, cujo posicionamento filogenético continua sendo revisto. A família Apodanthaceae inclui plantas aclorofiladas, holoparasitas de caules, cujo corpo vegetativo apresenta extrema redução. Por serem endoparasitas, tornam-se evidentes apenas na época da floração, quando as diminutas flores rompem a periderme do hospedeiro. Incluem-se nessa família três gêneros, sendo Pilostyles o de mais ampla distribuição. Apesar de existirem estudos minuciosos sobre o sistema endofítico no gênero, sua fenologia, ciclo de vida e ontogênese floral continuam desconhecidos. O presente estudo objetiva elucidar essas questões, de modo a proporcionar uma compreensão do sistema reprodutivo e do processo evolutivo das flores dessas intrigantes angiospermas, servindo de base para estudos futuros que visem a estabelecer homologias com outros clados. Primeiramente foi realizado um levantamento dos hospedeiros parasitados, analisando-se padrão de distribuição das flores e a razão sexual das populações de Pilostyles existentes nas áreas de estudo no Brasil e México. Indivíduos foram selecionados aleatoriamente para a realização dos testes de polinização, observação e coleta de visitantes, coleta de néctar e de sementes parasitas, e acompanhamento fenológico. Em laboratório realizaram-se os experimentos de germinação e as técnicas de preparo e análise histológicas das sementes e flores de Pilostyles. Com relação ao ciclo de vida, observou-se que: 1) as vespas aparecem como os visitantes mais freqüentes; 2) a dispersão provavelmente seja resultado de endozoocoria por aves; 3) a germinação provavelmente ocorre diretamente no caule hospedeiro; 4) as fenologias da hospedeira e da parasita estão diretamente relacionadas, havendo inclusive possibilidade de sincronismo. Com relação à razão sexual das populações, concluímos que: 1) a determinação do sexo das flores responde a estímulos específicos de cada espécie hospedeira; 2) a distribuição espacial das flores esta diretamente relacionada à razão sexual das populações; 3) existem flores monoclinas em número constante em todas as populações, sendo tais flores funcionalmente estaminadas. A partir do estudo da anatomia floral e embriologia, pudemos observar que: 1) as flores são envolvidas por quatro verticilos estéreis (de fora para dentro: 2 + 2 + 4 + 4); 2) a região estigmática é restrita à área das papilas; 3) a deiscência dos microsporângios é resultado da expansão e ressecamento do órgão; 4) os tricomas vesiculares secretam uma substância pegajosa, provavelmente responsável por unir os grãos de pólen e grudá-los ao corpo do polinizador; 5) a coluna das flores estaminadas é formada através de fusão pós-genital de uma coluna estilar central e de uma coluna externa, que traz no bordo superior os sacos polínicos. Concluindo, acreditamos que tais dados merecem destaque pelo ineditismo para a literatura atual da família Apodanthaceae e, principalmente, na sua capacidade de ancorar futuros estudos comparativos com outros táxons, servindo de base na avaliação das propostas taxonômicas vigentes. / Holoparasitic plants depend entirely on resources from the host xylem and phloem and are currently represented by at least seven lineages, whose phylogenetic position is still being reviewed. The Apodanthaceae family includes stem holoparasitic plants, achlorophyllous, whose vegetative body is extremely reduced. Because they are endoparasites, they become evident only at the time of flowering, when the tiny flowers emerge from the hosts bark. There are three genera in this family, being Pilostyles the one with broader distribution. There are detailed studies on the endophytic system of this genus, but its phenology, life cycle and floral ontogeny remain unknown. This study aims to elucidate these issues, in order to provide an understanding of the reproductive system and the evolutionary process of the flowers of these intriguing angiosperms. We hope these could be a basis for future studies that aim to establish homologies with other groups of angiosperms. First it was made a survey of parasitized hosts in Brazil and Mexico areas, and then the distribution pattern of flowers and sex ratio of the Pilostyles populations were analyzed. Individuals were randomly selected for the experiments of pollination, observation and collection of floral visitors, collecting nectar and phenological monitoring. Meanwhile, in the laboratory, were performed germination experiments and the histological analysis of Pilostyles seeds and flowers. Regarding the life cycle, we observed that 1) the wasps are the most frequent visitors; 2) the dispersion is probably a result of endozoochory by birds; 3) germination probably occurs directly in hosts stem; 4) the host and parasite phenologies are directly related; there is a possibility of timing. Regarding the sex ratio of the populations, we conclude that: 1) sex determination of Pilostyles flowers seems to respond to specific stimuli of each host species; 2) the spatial distribution of staminate and pistillate flowers is directly related to the sex ratio of the populations; 3) there are monoclinous flowers in constant number in all populations, and these flowers are functionally staminate. From the study of floral anatomy and embryology, we observed that: 1) the flowers are surrounded by four whorls of scales, thereby arranged from outside to inside: 2 + 2 + 4 + 4; 2) the stigmatic surface is restricted to the area with papillae, 3) the dehiscence of the pollen sacs is a result of synandrium expansion and dryness, occurring initially by individual transverse slit; 4) the vesicular trichomes secrete a sticky substance, probably responsible for uniting the pollen grains and stick it to the pollinators body; 5) the column of staminate flower is formed by post-genital fusion of a central column, the sterile gynoecium, and an external column, which brings the pollen sacs in the upper board. In conclusion, we believe that such data should be highlighted by the novelty for the Apodanthaceae family literature, and especially in its ability to underpin comparative studies with other taxa, being a basis to evaluate current taxonomic proposals.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第18605号 / 人博第701号 / 新制||人||167(附属図書館) / 26||人博||701(吉田南総合図書館) / 31505 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 加藤 眞, 教授 市岡 孝朗, 教授 瀬戸口 浩彰, 教授 宮本 嘉久, 教授 新宮 一成 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

Parasitism in plants is often accompanied by a suite of morphological and physiological changes resulting in a condition known as the ‘parasitic reduction syndrome’. With changes including extreme vegetative reduction, frequently beyond any resemblance to its photosynthetic relatives, accompanied by significant losses of genes linked to photosynthesis, the study of parasitic plants can be challenging. Conopholis (Orobanchaceae) is a small holoparasitic genus distributed across eastern and southwestern North America and Central America. This genus has never been the subject of a molecular phylogenetic or morphometric analyses. In addition, very little is known of the relationships among populations and of their post-glacial history. To investigate the species limits and phylogenetic relationships in Conopholis, we conducted a comprehensive molecular phylogenetic study of the genus as well as a fine-scale morphometric study. Based on plastid and nuclear sequences, Conopholis was found to contain three distinct and well-supported lineages which have varying degrees of overlap with previously proposed taxa. The clustering and ordination analyses of the morphometric study corroborated the molecular data, demonstrating the morphological differentiation between the three lineages detected within Conopholis. A taxonomic re-alignment is proposed for the genus that recognizes three species, C. americana, C. panamensis, and C. alpina. To address genetic diversity and phylogeographic structure of C. americana in eastern North America, microsatellite markers were developed and characterized for the first time in this species. Using these newly generated markers along with sequences from the plastid genome, the persistence of a minimum of two glacial refugia at the last glacial maximum were inferred, one in Florida and southern Alabama and another in the Appalachian Mountains near the southern tip of Blue Ridge Mountains. The diversity seen across the southern Appalachian Mountains supports the hypothesis that populations derived from the southern and northern refugia come together in this area.

Parasitism in plants is often accompanied by a suite of morphological and physiological changes resulting in a condition known as the ‘parasitic reduction syndrome’. With changes including extreme vegetative reduction, frequently beyond any resemblance to its photosynthetic relatives, accompanied by significant losses of genes linked to photosynthesis, the study of parasitic plants can be challenging. Conopholis (Orobanchaceae) is a small holoparasitic genus distributed across eastern and southwestern North America and Central America. This genus has never been the subject of a molecular phylogenetic or morphometric analyses. In addition, very little is known of the relationships among populations and of their post-glacial history. To investigate the species limits and phylogenetic relationships in Conopholis, we conducted a comprehensive molecular phylogenetic study of the genus as well as a fine-scale morphometric study. Based on plastid and nuclear sequences, Conopholis was found to contain three distinct and well-supported lineages which have varying degrees of overlap with previously proposed taxa. The clustering and ordination analyses of the morphometric study corroborated the molecular data, demonstrating the morphological differentiation between the three lineages detected within Conopholis. A taxonomic re-alignment is proposed for the genus that recognizes three species, C. americana, C. panamensis, and C. alpina. To address genetic diversity and phylogeographic structure of C. americana in eastern North America, microsatellite markers were developed and characterized for the first time in this species. Using these newly generated markers along with sequences from the plastid genome, the persistence of a minimum of two glacial refugia at the last glacial maximum were inferred, one in Florida and southern Alabama and another in the Appalachian Mountains near the southern tip of Blue Ridge Mountains. The diversity seen across the southern Appalachian Mountains supports the hypothesis that populations derived from the southern and northern refugia come together in this area.