<p>Closely related populations or species often occupy ecologically disparate habitats. Adaptation to new habitats can maintain genetic variation within a species or eventually lead to speciation. Local adaptation to different environments has been repeatedly demonstrated in plants and animals, however the traits and genes that underlie this adaptation are poorly understood. This is because many traits differ between divergent populations and species. One way to solve this problem is to separate a trait from its genetic background through genetic manipulation and look for differences in fitness between genetically manipulated individuals. </p><p>My dissertation focuses on investigating the traits and genes that allow two species of Monkey flower, Mimulus laciniatus and Mimulus filicifolius, to survive in a unique habitat. Most closely related Mimulus species, such as M. guttatus, occur in streams and seeps, but M. laciniatus and M. filicifolius have each colonized a harsh granite outcrop environment. Another unique characteristic that both these species share is a lobed leaf shape. Because of the physiological properties of lobed leaves they should be adaptive in a dry, exposed granite outcrop. M. laciniatus also flowers earlier than nearby M. guttatus and is a small flowered self-fertilizing species while M. guttatus has large flowers and is highly outcrossing. Early flowering allows plants to escape the onset of seasonal drought while a self-fertilizing mating system and small flower size is often correlated with the occupation of harsh habitats. </p><p>In chapter one I describe a new granite outcrop endemic species of Mimulus, M. filicifolius based on morphological divergence from M. laciniatus. M. filicifolius was previously categorized as M. laciniatus but it is geographically disjunct and its leaves are more finely dissected (Sexton, Ferris, and Schoenig 2013). In the second chapter I explore whether M. filicifolius is genetically divergent and reproductively isolated from M. laciniatus using genetic sequence, microsattelite, and hybrid fertility data from four members of the M. guttatus species complex with highly overlapping geographic ranges: M. guttatus, M. nasutus, M. lacinaitus, and M. filicifolius. In the third chapter I investigate the genetic basis of leaf shape differences in three members of the M. guttatus species complex, M. laciniatus, M. nudatus, and M. guttatus using bulk segregant analysis to map quantitative trait loci. In the fourth and final chapter I examine the genetic basis of flowering time, floral size, and leaf shape divergence between sympatric M. guttatus and M. laciniatus populations in a common garden using quantitative trait locus (QTL) mapping, phenotypic selection on flowering time, flower size, and leaf shape in M. laciniatus x M. guttatus hybrids in a reciprocal transplant experiment in the field, and whether QTL's from my common garden experiment overlap fitness QTL's in the field by genotyping hybrid individuals that survived to flower in the field.</p> / Dissertation

Many soybean [Glycine max (L.) Merrill] cultivars have narrow leaflet shape but it is not known if all of these lines derive this trait from the ln gene or another locus. This project was conducted to determine the inheritance of the narrow leaflet trait in several soybean genotypes and wild [Glycine soja Sieb. et Zucc.] accessions, and also to determine the allelism of the genes for this trait in the selected lines. The parents, F1, F2 and F2:3 generations were grown at Kentland Research Farm near Blacksburg, VA or in the greenhouse. The F2 and F2:3 generations (where available) were observed for segregation in leaflet shape. The populations were scored as having either broad or narrow leaflets using visual classification and leaf measurements when necessary. 'Camp' was crossed with broad leaflet parent 'Essex' to study the inheritance of the narrow leaflet trait in Camp. Observation of the F2 and F2:3 generations lead to the conclusion that a single recessive gene controls leaflet shape in Camp. Narrow leaf parents 'SRF 400' and Camp were crossed with lines having the ln gene (T41, S56, and D64-4731). None of the crosses among Camp, T41, SRF 400, S56 and D64-4731 segregated for leaflet shape in the F2 generation leading to the conclusion that they all have the ln allele at the same locus controlling lanceolate leaflet shape. T313, a line containing a gene for narrow rugose leaflets (lnr), was crossed with Camp to study allelism between the lnr and ln genes. Segregation for leaflet shape was observed in the F2 and F2:3 generations allowing the conclusion that the lnr gene controlling the narrow rugose leaflet trait in T313 is at a locus independent from the ln gene. A deficiency of narrow rugose plants was observed in all of the populations with T313 as a parent, and was theorized as being caused by selection against lnr gametes. After adjustment for the lnr deficiency, the F2 data appeared to fit a 9 broad : 3 narrow : 4 narrow rugose ratio. Three G. soja lines were crossed to broad and narrow leaflet parents and the F2 generations were examined to determine the inheritance of the very narrow leaf phenotype. The results indicate that there are one or two recessive genes controlling narrow leaflet shape in the G. soja accessions, which are not allelic to the ln gene. Since these populations were not advanced to the F3 generation, definite conclusions cannot be drawn about the genetics of the very narrow leaf phenotype. / Master of Science

The relationship between leaf and its environment has resulted in a tremendous diversification of leaf shape within and between plants species, which is important to cope with the differing environmental conditions. Arabidopsis lyrata is a prime model plant that shows leaf shape variation within species and between related species. In Cardamine and Capsella, the RCO genes (homeodomain leucine zipper family transcription factors) are involved in shaping leaves, yielding more complex shaped leaves (leaflets). In A. thaliana, over the course of evolution, the RCO-A and RCO-B paralogous genes have been deleted that led to the loss of lobes (leaf simplification). Based on previous quantitative trait locus (QTL) mapping results, these gene family members are thought to control leaf shape variation also in A. lyrata. Since the paralog involved in leaf shape variation is unknown, both copies of the RCO genes (AL6G13350 and AL6G13360) were sequenced. The study aimed to identify whether DNA sequence variation exists in the two paralogous RCO genes, which could explain the phenotype variation both within population and between A. lyrata populations, along with related species A. arenosa. The results showed limited variation in the coding regions in the form of indels, single nucleotide polymorphisms (SNPs) and amino acid substitutions resulting in no significant difference in phenotype between genotype (p&gt;0.139). The most variants were rare and increasing the number of individuals within the populations, to cover the full phenotypic spectrum, may lead to different results. Not being able to obtain the nucleotide sequence of the promotor region, further analysis is required since it is an important region for gene expression, which could explain phenotype variation for leaf shape in Arabidopsis lyrata.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22287号 / 理博第4601号 / 新制||理||1660(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 酒井 章子, 教授 髙林 純示, 教授 田村 実 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Une grande diversité de formes foliaires caractérise le monde végétal. Cette diversité s'étend des feuilles simples avec des marges lisses aux feuilles composées, avec des marges disséquées. Cependant, les dentelures des marges de ces feuilles simples ou composées se développent en suivant un mécanisme similaire. Ce mécanisme repose sur l'action des gènes NO APICAUX MERISTEM/ CUP-SHAPED COTYLEDONS (NAM/CUC) ainsi que sur la voie auxinique. Chez Arabidopsis, qui possède des feuilles simples, un équilibre entre les expressions de CUC2 et de son répresseur, miR164, est nécessaire au bon développement des dents. Nous avons montré qu'un autre membre de la famille CUC, CUC3, contribue également au développement de ces dents chez Arabidopsis. Bien que son action soit principalement dépendante de CUC2, il agit également plus tard au cours du développement foliaire. En outre, nous avons démontré qu'une boucle de rétro-contrôle entre CUC2 et la voie auxinique permet le développement de dents avec plus ou moins marquées. Nous avons également montré qu'un modèle d'expression temporelle existe entre l'auxine et le module CUC2-miR164. En outre, la production de plantes transgéniques de Cardamine hirsuta, un proche parent d' Arabidopsis, qui possède des feuilles composées, a mis en évidence l'importance des éléments cis-régulateurs dans le promoteur de CUC1 de Cardamine hirsuta. En effet, la divergence de ces éléments cis-régulateurs entre les promoteurs de CUC1 de Cardamine hirsuta et d' Arabidopsis pourrait expliquer que CUC1 soit fortement exprimé dans les feuilles de Cardamine hirsuta alors qu'il est faiblement exprimé dans celles d' Arabidopsis. / A wide diversity of leaf shapes characterises the plant world. This diversity ranges from simple leaves with smooth margins to compound leaves with dissected margins. However, all serrations of simple or compound leaf margins are developed using a similar mechanism. This mechanism includes the action of the NO APICAL MERISTEM/CUP-SHAPED COTYLEDON (NAM/CUC) genes as well as the auxin pathway. In Arabidopsis simple leaves, a balanced expression of CUC2 and its repressor miR164 is controlling the serrations development. We have shown that another member of the CUC family, CUC3, also contributes to the serration development in Arabidopsis simple leaves. While its action is mainly dependent of the one of CUC2, it also acts later during leaf development. Additionally, we have demonstrated that a feed-back loop was regulating the CUC2 and auxin pathways, in order to form leaves with more or less incisions. We also shown that a temporal expression pattern was established between the auxin and the CUC2-miR164 module. Moreover, generation of transgenic Cardamine hirsuta plants, a close relative of Arabidopsis, that possesses compound leaves, has enlighten the importance of cis-regulatory elements in the promoter of CUC1 from Cardamine hirsuta. Indeed, the divergence of cis-regulatory elements between promoters of CUC1 from Cardamine hirsuta and Arabidopsis could explain that CUC1 is expressed strongly in Cardamine hirsuta leaves whereas it is weakly expressed in Arabidopsis leaves.

Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1&deg;C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea&rsquo;s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.

Leaf shape is remarkably variable among plants, and hence likely has major consequence for ecological function and fitness. My thesis addresses the ecological significance of clinal variation for a leaf shape polymorphism in Ipomoea hederacea (lobed leaves dominate the north, entire-shaped leaves restricted to the south), and investigates the role of adaptation and demography in shaping its evolutionary history in its eastern North American range. To evaluate the adaptive value of the cline, I surveyed leaf shape genotypes from 77 populations , and found a steep latitudinal leaf shape cline that was not reflected in 173 neutral genetic markers. Furthermore, the leaf shape locus was a genomic outlier, implicating divergent selection in generating or maintaining the cline. I investigated the thermoregulatory and freezing tolerance properties of the leaf shape genotypes, and discovered that lobed leaves remain marginally warmer at night, and a 1&deg;C decrease separated mildly damaged and severely frost damaged tissue, potentially suggesting that a critical ambient temperature could drive differential leaf shape damage. I further explored three additional hypothesized selective agents (insect herbivores, flowering phenology and growth), and showed that these putative agents impose selection on I. hederacea, but do not differentiate between leaf shapes. These studies highlighted the challenge of identifying selective agents, even for a polymorphic trait with hypothesized selective mechanisms. To understand the contribution of adaptation and demography in shaping I. hederacea&rsquo;s evolutionary history, I sequenced 7 nuclear loci from 192 individuals sampled from 24 populations and characterized patterns of nucleotide diversity. I demonstrated that I. hederacea is genetically structured in patches consistent with long-distance dispersal, genetically depauperate, and undergoing range expansion, suggesting a recent founder event or metapopulation dynamics. My thesis represents a comprehensive evaluation of the key processes affecting a polymorphism that influences plant morphology, geographical distribution, and population history.