Browsing by Subject "Plant biology"

The Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.

Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.

Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.

Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.

This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.

One of the most important questions in evolutionary biology is how new genes and new functions arise and evolve. Among the theories addressing this question, gene duplication is one of the most popular. Previous study has shown that two threonine deaminase (TD) gene copies exist in Solanum lycopersicum, and these two copies have very different functions and low sequence similarities. The primary objective of this study was to widen our understanding of this gene duplication and the subsequent evolutionary processes affecting the duplicate copies by first collecting additional TD sequences from related species, building a gene tree, and inferring the point of gene duplication. The evolutionary processes acting on this gene were then analyzed using the program PAML. Results indicate that 1) The TD duplication probably occurred in before the split of the Solanoideae from the Nicotianoidea; and 2) there is strong evidence for positive selection on one of the TD copies after gene duplication, while for the other TD copy, only weak evidence for positive selection was found; and 3) adaptive improvement of the copy with new function probably spanned a period of at least 25 million years.

In this project I expand the current model of hierarchical transition to include transition by individuation in addition to transition by integration and I apply my model of transition by individuation to the evolution and development of the filamentous fungi. I accomplish this in two parts. In the first section, I defend a general Hierarchy Thesis: A differentiated hierarchical whole can arise not only through the integration of individuated parts but also by the individuation of parts within an integrated whole. I elaborate an expanded model of hierarchical transition and discuss the relevance of part-level selection and part-hood regulation for different modes of transition.

In the second section, I defend a Mycology Thesis: The filamentous fungi have evolved a developmental cellularization process to meet ecological and reproductive demands for coenocytic growth in early development and cellularization in later development. I elaborate the origins and evolution of the filamentous fungi and argue that this history provides cases of hierarchical transition via individuation in both phylogeny and ontogeny.

The project provides an expanded evolutionary-developmental framework for hierarchical transition and a framing narrative for the evolutionary development of filamentous fungi, an evolutionarily significant and ecologically ubiquitous group, and has implications for the study of similar organisms outside Fungi and of hierarchical transition in general.

This dissertation examines issues concerning sap flux scaled estimates of the canopy-averaged transpiration rate of trees per unit leaf area (E_L) and stomatal conductance (G_S), as well as their implications in the water and carbon balance of individuals and stands, with the final goal of an integrated assessment of 11 years of such data from two species (Pinus taeda and Liquidambar styraciflua) at the Duke Free Air Carbon dioxide Enrichment (Duke FACE) facility. These issues include (1) the effects of allometric relationships and xylem characteristics on the gas phase transport of water from leaves and the hydraulic supply of it, (2) consideration of the hydraulic capacitance in the inference of stomatal behavior from sap flux data and (3) the dynamic modeling of stomatal conductance to environmental drivers using Bayesian techniques. It is shown that a) for resolution of sap flux in conifers at the scale of minutes under dynamic conditions, time constants for both stomatal responses and hydraulic capacitance of sapwood must be considered, (b) nighttime conductance can lead to large errors in rates of sap flux measured under some conditions, (c) variation in allometry between P. taeda individuals can lead to different rates of transpiration and carbon assimilation per unit leaf area and that (d) hydraulic time constants for the stems of mature P. taeda at Duke FACE trees varied by the stem length considered and were on the order of 30-45 minutes for a 10-m segment. An analysis incorporating all these elements leads to the conclusions that (e) both elevated CO₂ (eCO₂) and fertilization (FR) resulted in proportionally larger reductions in the E_L and G_S of P. taeda as soil moisture decreased with (f) eCO₂ having little to no effect in months of high soil moisture and (g) FR leading to ~14% reduction of GS under high soil moisture in absence of eCO₂, while (h) both eCO₂ and FR led to reduced E_L and G_S of L. styraciflua across soil moisture conditions.

The fern family Cystopteridaceae has been a thorn in the side of fern phylogeneticists, on many levels. Until this thesis, its basic existence (as a deeply isolated clade) and composition were unrecognized, hypotheses as to the relationships of its constituents within the broader fern tree-of-life were wildly inconsistent, the relationships of its genera to each other were contested, the species limits within those genera weakly understood, and the relationships among those species unknown. This thesis first establishes the broad evolutionary context for the family, which is that it is the first-diverging branch in Eupolypods II (it is sister to the rest of the eupolypod II clade). Eupolypods II is a large clade, containing nearly a third of extant fern species, making the Cystopteridaceae's position pivotal to a full understanding of fern evolution.

The evolution of the Eupolypods II is marked by an "ancient, rapid radiation" at the base of the clade, which helps to explain the difficulty that this broad group has historically posed to evolutionary biologists. Molecular data from five plastid loci show that Eupolypods II is comprised of 10 deeply divergent lineages, each worthy of recognition at the rank of family: Cystopteridaceae, Rhachidosoraceae, Diplaziopsidaceae, Hemidictyaceae, Aspleniaceae, Thelypteridaceae, Woodsiaceae, Onocleaceae, Blechnaceae, and Athyriaceae. The ancestors of Cystopteridaceae diverged from those of the rest of the clade approximately 100 million years ago, and the family is now comprised of five extant genera: Acystopteris, Cystoathyrium (the only genus for which we lack molecular data--it may be extinct), Cystopteris, Gymnocarpium, and ×Cystocarpium.

Within the family, the relationships of Cystoathyrium are unknown. Acystopteris is sister to Cystopteris, and those two genera, together, are sister to Gymnocarpium. Gymnocarpium is the maternal parent of ×Cystocarpium, so that genus falls within Gymnocarpium in phylogenetic trees based on maternally transmitted loci (i.e., plastid or mitochondrial loci). Plastid data resolve a basal trichotomy in Gymnocarpium, among the G. disjunctum clade, the G. robertianum clade, and core Gymnocarpium. The earliest diverging branch of core Gymnocarpium is the morphologically anomalous G. oyamense, followed by a split that separates G. appalachianum and G. jessoense parvulum (on one side) from G. remotepinnatum and G. jessoense jessoense, on the other. In Acystopteris, the first division surprisingly separates A. taiwaniana (which is frequently treated as a variety of A. japonica) from A. japonica + A. tenuisecta (which are morphologically very distinct from each other).

The evolution of Cystopteris is, as expected, more complex. The first lineage to diverge from the rest of the genus is the one that gave rise to C. montana. The next division, however, is unclear; molecular data infer a trichotomy among the sudetica clade (containing C. sudetica, C. moupinensis, and C. pellucida), the bulbifera clade (containing C. bulbifera and its related allopolyploids C. tennesseensis and C. utahensis), and the C. fragilis complex. Within the C. fragilis complex relationships (and species limits) get particular messy. The diploid species of eastern North America--C. protrusa--is sister to the rest of the complex, but after that point the major named species (including C. fragilis and C. tenuis) cease to be monophyletic, being found on both sides of a major split, alongside such taxa as the Australian/New Zealand C. tasmanica, the Hawaiian C. douglasii, and the Mexican C. membranifolia and C. millefolia.

In the context of the deep divergence of Gymnocarpium from Cystopteris, and the complicated species-level patterns of relationship within each genus, it is particularly surprising that molecular data confirm that ×Cystocarpium is a hybrid between Gymnocarpium dryopteris and a European tetraploid member of the Cystopteris fragilis complex. The ancestors of Cystopteris diverged from those of Gymnocarpium approximately 58 million years ago, meaning that the ×Cystocarpium hybridization event (which happened very recently) united genomes that contain, between them, over 100 million years of independent evolution. This breadth of divergence makes ×Cystocarpium the most extreme example of wide hybridization currently documented, with important implications for the pace of evolution of reproductive isolation, and thus for species formation.

This thesis ends with a tentative synopsis of the Cystopteridaceae (Appendix E). The family, as construed here, contains five genera and approximately 36 species (three in Acystopteris, one in Cystoathyrium, ~25 in Cystopteris, seven in Gymnocarpium, and one in ×Cystocarpium), plus two named subspecies (one each in Cystopteris and Gymnocarpium), and eight named sterile hybrids (three in Cystopteris and five in Gymnocarpium). Each of these tallies is highly subjective--much further research, with an emphasis on cytological and low-copy nuclear data, is necessary before we can hope to have any confidence in the species limits and finer-scale evolutionary patterns in this family.

Morphological characters from the gametophyte and sporophyte generations have been used in land plants to infer relationships and construct classifications, but sporophytes provide the vast majority of data for the systematics of vascular plants. In bryophytes both generations are well developed and characters from both are commonly used to classify these organisms. However, because morphological traits of gametophytes and sporophytes can have different genetic bases and experience different selective pressures, taxonomic emphasis on one generation or the other may yield incongruent classifications. The moss order Hookeriales has a controversial taxonomic history because previous classifications have focused almost exclusively on either gametophytes or sporophytes. The Hookeriales provide a model for comparing morphological evolution in gametophytes and sporophytes, and its impact on alternative classification systems. Sometimes, placement of certain groups within Hookeriales remains challenging even at the molecular level. That is the case of the genus Calyptrochaeta. We study diversification dynamics in this genus to elucidate possible mechanisms obscuring its placement and we address biogeographic questions using the Tropical Conservatism scenario as our null hypothesis. Furthermore, to better understand biogeographic patterns in the Southern Hemisphere, infraspecific molecular patterns are compared in two species of the genus Calyptrochaeta (i.e., C. apiculata and C. asplenioides) and vicariance and recent long distance dispersal are tested to explain the disjunct distributions observed in these species.

In this study we reconstruct relationships among pleurocarpous mosses in or associated to the Hookeriales, in Calyptrochaeta, and within Calyptrochaeta. Six molecular markers are explored in total from all three genome compartments to reconstruct the evolution of morphological characters and habitat preferences in our phylogenies. Divergence times are estimated in a Bayesian framework using a relaxed molecular clock, and diversification rates are calculated on the chronograms resulting from these estimations.

As a result, we found that the Hookeriales, as currently circumscribed, are monophyletic and that both sporophyte and gametophyte characters are labile. We documented parallel changes and reversals in traits from both generations. We show that diversification rates in Calyptrochaeta have changed through its history. Also, though we lack support to clearly reject the tropical conservatism hypothesis, our data point to a more complex scenario where both temperate and tropical species can be ancient and give rise to one another, since shifts between tropical and temperate regions seem to be possible in any direction. Finally, we have show that recent long distance dispersal best explains the distribution of both C. apiculata and C. asplenioides in the Southern Hemisphere.

Root system architecture (RSA) is the spatial distribution of roots of individual plants. As part of a collaborative effort I adapted a gellan gum based system for imaging and phenotyping of root systems in maize. This system was first used to perform a survey of 26 distinct maize varieties of the Nested Association Mapping (NAM) population. The analysis of these data showed a large amount of variation between different RSA, in particular demonstrating tradeoffs between architectures favoring sparse, but far reaching, root networks versus those favoring small but dense root networks. To study this further I imaged and phenotyped the B73 (compact) x Ki3 (exploratory) mapping population. These data were used to map 102 quantitative trait loci (QTL). A large portion of these QTL had large, ranging from 5.48% to 23.8%. Majority of these QTLs were grouped into 9 clusters across the genome, with each cluster favoring either the compact of exploratory RSA. In summary, our study demonstrates the power of the gellan based system to locate loci controlling root system architecture of maize, by combining rapid and highly detailed imaging techniques with semi-automated computation phenotyping.

Plants use an array of photoreceptors to measure the quality, quantity, and direction of light in order to respond to ever-changing light environments. Photoreceptors not only determine how and when individual plants complete their life cycles, but they also have a profound and long-term macroevolutionary influence on species diversification. Despite their significances, very little is known about photoreceptors across plants as whole, and we lack a comprehensive view of photoreceptor evolution.

In my dissertation, I investigate the origin and evolution of three of the most prominent photoreceptor gene families in plants: phytochromes, phototropins and neochromes. Using newly available transcriptomic and genomic data, I completed the first in-depth survey of these photoreceptor families across land plants, green algae, red algae, glaucophytes, cryptophytes, haptophytes, and stramenopiles.

Phytochromes are red/far-red photoreceptors that play essential roles in seed germination, seedling photomorphogenesis, shade-avoidance, dormancy, circadian rhythm, phototropism, and flowering. Here, I show that the canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte green algae plus land plants), and I identify the most likely sequence whereby the plant phytochrome structure evolved from its ancestral phytochrome. Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts, and Selaginella apparently possess a single phytochrome gene copy, whereas independent gene duplications occurred within mosses, lycopods, ferns, and seed plants, leading to diverse phytochrome families in these clades. My detailed phylogeny encompasses all of green plants and enables me to not only uncover new phytochrome lineages, but also to make links to our current understanding of phytochrome function in Arabidopsis and Physcomitrella (the major model organism outside of flowering plants). Based on this robust evolutionary framework, I propose new hypotheses and discuss future directions to study phytochrome mechanisms.

Phototropins are blue-light photoreceptors that regulate key adaptive physiological responses, including shoot-positive phototropism, root-negative phototropism, chloroplast accumulation/avoidance, stomatal opening, circadian rhythm, leaf expansion, and seedling elongation I show that phototropins originated in the common ancestor of Viridiplantae (all green algae [charophytes, chlorophytes, prasinophytes] plus land plants). Phototropins repeatedly underwent independent duplications in all major plant lineages (mosses, lycopods, ferns and seed plants), except for liverworts and hornworts, where phototropin is a single-copy gene. Following each major duplication event, phototropins subsequently differentiated in parallel, resulting in two specialized (yet partially overlapping) functional forms that primarily mediate either low- or high-light responses. My gene phylogeny further suggests that phototropins have co-evolved with phytochromes, as is evident from their molecular interactions and strikingly similar gene duplication patterns. I hypothesize that the co-evolution of phototropins with phytochromes, together with their subsequent convergent functional divergences in phototropic responses, contributed to the success of plants in adapting to diverse and heterogeneous habitats.

Neochromes are chimeric photoreceptors that, by fusing phytochrome and phototropin modules into a single protein, are able to use both red/far-red and blue light to modulate phototropic responses. Neochromes were first discovered in ferns, and the evolution of neochromes was implicated as a key innovation that facilitated fern diversification under the low-light angiosperm canopies. Despite its significance from an evolutionary standpoint, the origin of neochromes has remained a mystery. Here I present the first evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in my large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 MYA, long after the split between the two plant lineages (at least 400 MYA). By analyzing the draft genome of the Anthoceros punctatus hornwort, I also discovered a novel phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was horizontally transferred to ferns, where it may have played a significant role in the diversification of modern ferns.

In summary, my studies identified the molecular origins of phytochromes, phototropins and neochromes, and reconstructed their respective evolutionary histories. This new framework for photoreceptor evolution will stimulate new research linking ecology, evolution, and photochemistry to understand how plants adapt to variable light environments.

Small noncoding RNAs (ncRNAs) are key regulators of plant development through modulation of the processing, stability and translation of larger RNAs. I generated small RNA datasets comprising over 200 million aligned Illumina sequence reads covering all major cell types of the root as well as four distinct developmental zones. These data were analyzed for three major types of small RNAs, namely microRNAs (miRNAs), repeat associated small interfering RNAs (ra-siRNAs), and trans-acting siRNAs (ta-siRNAs). 133 of the 243 known miRNAs were found to be expressed in the root, and most showed tissue- or zone-specific expression patterns. My collaborators and I identified 70 new high-confidence miRNAs, and knockdown of three of the newly identified miRNAs resulted in altered root growth phenotypes. Ra-siRNAs specify methylation by the RNA directed DNA methylation (RdRM) pathway, requiring the generation of additional methylation datasets. Preliminary analysis shows cell-type specific methylation patterns that correlate with small RNA and mRNA expression. Analysis of ta-siRNAs revealed new ta-siRNA generating loci, and a novel triggering miRNA for TAS1 loci. In summary, our study demonstrates the power of isolating individual cell types and developmental zones in combination with deep sequencing and computational analyses to obtain detailed profiles of ncRNAs, as well as to significantly extend the compendium of known functional RNAs.

The delineation of closely related plant species is difficult, as finding a discrete and distinct set of characters can be problematic in taxa that exhibit extreme morphological variability. Such difficulties arise in the genus Sphagnum because of its diversity and variability, as well as its propensity to hybridize. This study asks (1) do the five Norwegian morphospecies of the Sphagnum recurvum complex, a group of closely related and taxonomically controversial boreal species, correspond to genetically distinct entities? And (2) are morphologically intermediate plants a result of phenotypic plasticity or interspecific hybridization? Using “next generation” RADseq-based phylogenetic analyses, three highly distinct clades emerged, corresponding to S. angustifolium, S. flexuosum, and a clade containing S. fallax, S. isoviitae and S. brevifolium. This result suggests the boundaries of S. fallax should be expanded. The lack of genetic differentiation among the members of the S. fallax clade and the clear separation of the three clades was supported by DAPC multivariate clustering and a novel analysis comparing the lineages revealed by individual loci. These analyses also tested for genetic admixture within plant samples. However, there were no intermediate samples between the genetic clusters discerned from any of the analyses, and the phylogenetic tree demonstrates strong clade cohesion. This lack of genetic intermediates suggests that the morphological variation observed in these populations is likely to be due to intraspecific genetic variation or phenotypic plasticity. A revised key and diagnoses for Norwegian species in the Sphagnum recurvum complex is provided to facilitate identification by collectors.

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

While local adaptation has been frequently demonstrated via reciprocal transplant experiments, our understanding of the genetic basis of it remains minimal. There is a notable lack of studies that identify naturally segregating variants, determine the traits controlled by these variants and characterize their fitness effects in the field. Such studies are critical for understanding how spatially varying selective pressures can drive population divergence and maintain genetic variation. The experiments presented here aim to characterize the genetic basis of local adaptation to serpentine soils in Mimulus guttatus. First, I show that serpentine and non-serpentine populations of M. guttatus are locally adapted to soil habitat wherein non-serpentine plants are unable to survive on serpentine soils. Serpentine tolerance appears to come at a cost as serpentine plants are smaller in the juvenile stage than non-serpentine plants when grown at non-serpentine field sites. These size differences may limit the competitive ability of serpentine tolerant plants in non-serpentine habitats which tend to be more heavily vegetated than serpentine habitats. Next I identify environmental variables that are important selective agents in the serpentine habitat. Using hydroponic assays to isolate an individual chemical variable of serpentine soils - low calcium levels to high magnesium levels (low Ca:Mg ratio) - I show that serpentine and non-serpentine populations of M. guttatus have significant differences in tolerance to low Ca:Mg. I then characterize the genetic basis of these ecotypic differences in survival and tolerance using quantitative trait locus (QTL) mapping. I identify a single, major QTL that controls both the ability to survive on serpentine soils and tolerance to low Ca:Mg ratio which suggests that M. guttatus populations have adapted to serpentine soils through an ability to tolerate the low levels of Ca while simultaneously not suffering from Mg toxicity. Furthermore, I show that this same QTL controls ability to survive on serpentine soils in a second, geographically distant population. However, preliminary work suggests that the two populations are not equally tolerant to each other's soils indicating that either other loci also contribute to serpentine tolerance and these are not shared between the two serpentine populations or that there are different serpentine tolerance alleles at the major QTL are not functionally equivalent. This work addresses long-standing questions in evolutionary biology regarding the number and effect size of loci that underlie adaptive traits by identifying a large effect locus that contributes to adaptive differences between M. guttatus populations.

This dissertation examines the processes that generate phenotypic variation in life cycles in seasonal environments. Collectively, a life cycle describes the stages an organism passes through during a generation. The timing, or phenology, of these transitions is often influenced by both environmental and allelic variation. Using the model organism Arabidopsis thaliana and both empirical and modeling approaches, I examine how correlations between life-cycle transitions, environment-dependent allelic effects, and epistasis generate patterns of life-cycle variation both within and between generations. In my first chapter, I use experiments to determine that many combinations of genetic, environmental, and developmental factors can create similar germination phenotypes, that maternal effects can influence phenotypes more than genetic differences, and that cross-generational effects can reduce variation in germination timing despite variation in flowering and dispersal time. In my second chapter, I use a modeling approach to consider the entire life cycle. I find that environmental variation is a major driver of phenotypic variation, and that considering the known geographic distribution of allelic variation across the range improves the match of model predictions to phenotypes expressed in natural populations. Specifically, variation in dormancy generated in the previous generation is predicted to cause life-cycle differences within a location, and the geographic distribution of allelic variation in dormancy interacts with local climatic environments to canalize an annual life history across the range. Finally, I test if allelic and environmental variation that affects early life stages can influence the environment experienced during reproduction. This environment determines both the time available for reproduction and the environment experienced during senescence. By implementing simple survival rules for flowering plants in the model, I show that time available for a plant to reproduce depends on earlier phenological traits and varies widely from year to year, location to location, and genotype to genotype. If reproductive trade-offs that underlie the evolution of senescence are environmentally sensitive, these results suggest that genetic variation in earlier life-stage transitions might shape senescence rates and whether they are environmentally responsive. In sum, my dissertation demonstrates the importance of pleiotropy, environment-dependent allelic expression, and epistasis in defining life-cycle variation, and proposes a novel way of predicting these relationships and complex life cycles under seasonal conditions.

Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.

I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.

Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.

In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.

Roots are developmentally plastic and highly dependent on the immediate environment. By studying root responses to abiotic stress, we have identified novel regulators of developmental modulation. When roots are deprived of phosphate (Pi), developmental programs are modulated to slow primary root growth and expand surface area through emergence of root hairs. By focusing on exposure time-periods of less than two days, we have described very early changes to root development in response to this condition that may reveal new mechanisms of root hair specification and emergence. Also, using transcriptomic analyses with high spatial resolution, we identified a kinase that is specifically induced in root vascular tissue within three hours of exposure and acts to modulate aspects of root development in response to deprivation of Pi. These data suggest that individual tissues play unique roles in whole organ development, and that interpretation of Pi -deprivation responses may change as we develop methods with resolution necessary to understand these roles. Beyond Pi, we compared transcriptomic data for four additional stresses and identified a novel stress-responsive transcription factor that modulates expression of a cell expansion protein. This putative network connection demonstrates the value of using high-dimensional data for inference of regulatory relationships. Overall, we have combined "-omics" approaches with reverse genetics to identify novel developmental regulators and described a phenotypic frame-work with resolution at which cellular mechanisms can be studied.

Recent changes in the timing of annual events are a sign that climate change is already impacting ecosystems. Carbon sequestration by forests increases with longer growing seasons. Biodiversity can be affected by mis-timing of events through shading interactions and frost damage. Projecting forests' ability to provide these ecosystem services in the future requires an understanding of trees' phenological responses to a new climate. I begin by proposing a first order definition of an `optimal' phenological response to warming: that the mean temperature following budburst should remain essentially constant. Analogously, the temperature preceding senescence can serve the same role.

To understand which environmental cues will drive future changes in phenology, I assimilate clues from observational and experimental literature. For budburst in woody plants, spring warmth, over-winter chilling and light drive nearly all behavior, but species' responses vary widely. Species using chilling or light as safety mechanisms against budburst during mid-winter thaws are thought to be less able to phenologically track a warming climate. However, I show that even species cued solely by spring warmth are likely to under-track temperature changes. Fall cues are more idiosyncratic, and a plant's driver of senescence is likely to vary from year to year.

Models are a tempting method to untangle species budburst cues and forecast phenology under warmer climate scenarios. I tested two models' ability to recover parameters used to simulate budburst data. The simpler model was cued only by spring warmth while the complex one modulated warmth requirements with chilling exposure. For the simple model, parameters could be recovered consistently from some, but not all, regions of parameter space. The complex model's parameters were largely unrecoverable. To understand the consequences of parameter uncertainty, I applied both models to an 18 year phenological record of 13 deciduous tree species. While a few species fell into identifiable regions of the simple model's parameter space, most did not, and projected budburst dates had wide parameter-derived uncertainty intervals. These bands were wider still under a 5°C warming scenario. Even greater uncertainty resulted from the complex model.

To better understand plants' potential for growing season extension I subjected seedlings to warmer climates in a series of open-topped chambers in sites at each end of the eastern deciduous biome. Soil and air were heated to 3 or 5°C above ambient, or left unheated. For nearly all species, warming hastened budburst and germination and delayed senescence. However, these events failed to track temperature changes, happening at warmer temperatures in hotter chambers. Individual species showed a remarkable variability of all events' dates within treatments, and even within chambers. Because phenological traits are heritable, this offers a potential for evolutionary response to climate change.

This research has shown that while individual trees extend their growing seasons under warmer temperatures, they typically under-respond to the magnitude of warming, suggesting forests' capacity for increased carbon sequestration may reach a limit. However, within populations, trees vary substantially in their phenological responses, forming a possibility for evolutionarily adaptation to changing cues.