Ethylene and carbohydrate deprivation were investigated as possible causes of bud abortion in Lilium longiflorum Thunb. Silver thiosulfate (STS) was investigated as an inhibitor of ethylene-induced abortion. Fourteen days of 92.5% irradiance reduction increased bud abortion when plants were exposed to 2.07 mM ethephon. Percent bud abortion was 39% and 60% for plants grown in full irradiance and reduced irradiance, respectively. Ethephon resulted in 54% abortion, regardless of irradiance at 4.15 mM. A 70% irradiance reduction for 14 days did not increase bud abortion when plants were treated with ethephon. STS was applied to plants at visible bud +2 weeks followed by ethephon application 2 days later. Bud abortion was reduced from 69 to 13% with 2 mM STS; the STS x ethephon interaction was significant. STS inhibited ethephon-induced bud abortion when applied at visible bud, 4 weeks prior to ethephon application; However, STS application at flower bud initiation did not prevent/reduce ethephon-induced bud abortion.

Arabidopsis thaliana HYL1 is a nuclear doublestranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 primiRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3’ and 5’ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1- dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5’ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced primiRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

Although polyploidy is believed to be a major source of evolutionary novelty, few studies have examined the genetic basis of phenotypic variation in wild polyploids. In this thesis I have studied the genetic basis of flowering time variation in the wild tetraploid crucifer Capsella bursa-pastoris, as well as the evolutionary history of this species. First, phylogenetic methods were employed to test hypotheses on the origin of C. bursa-pastoris. Based on DNA sequences from two chloroplast DNA loci and three independent nuclear genes, we found no support for the notion of C. bursa-pastoris as an autopolyploid of the diploid C. grandiflora, or an allopolyploid of C. grandiflora and C. rubella, even though some C. bursa-pastoris accessions shared alleles with C. rubella at nuclear loci. Using divergence population genetic methods, a larger sample of accessions and data for six duplicated nuclear genes, we found that allele sharing in sympatry was better explained by introgressive hybridization than by multiple origins of the tetraploid. The genetic basis of flowering time variation was examined using three approaches. A gene expression microarray study revealed that early- and late-flowering accessions differ in circadian rhythm, as well as in the gibberellin pathway affecting flowering time. Second, two QTL (Quantitative Trait Loci) for flowering time map to duplicated linkage groups. Third, polymorphisms at the candidate genes CRYPTOCHROME1 (CRY1), in one of the QTL regions, and FLOWERING LOCUS C (FLC) are associated with natural flowering time variation. Different FLC splice site polymorphisms are associated with flowering time in samples from Western Eurasia and China. The CRY1 association is only found in Europe, where alleles introgressed from C. rubella have an effect on flowering time. In conclusion, duplicated genes, introgressive hybridization and splicing variation may all have played a role in the evolution of flowering time variation in C. bursa-pastoris.

The transition to flowering is a major developmental switch in angiosperms, the timing of which is expected to be important for fitness. In this thesis the focus has been on identification of genes affecting natural variation in flowering time in Brassica nigra. The background for this thesis is an earlier QTL-mapping study in B. nigra. The genomic area with the greatest effect on flowering time in that study contained a homolog to the CONSTANS gene, which is known to affect flowering time in A. thaliana. When studied more closely this gene did not seem to affect flowering time variation in B. nigra. Near the B.nigra CO gene (BniCOa), however, we identified a homolog to the related CONSTANS LIKE 1 (COL1) gene. In A. thaliana COL1 has not been shown to be associated with induction of flowering but since the B. nigra homolog (BniCOL1) in the QTL area showed surprising amounts of variation between early and late flowering plants we set out to test if this variation was associated with flowering time variation. In the first paper we found a significant association between flowering time and one indel (Ind2) in the coding region. Motivated by the results in paper one, we searched for evidence of selection at the BniCOL1(paper two). In paper three the aim was to validate the results from the first paper in a larger sample of populations, and to check whether the association found in paper I could reflect linkage disequilibrium with areas outside of the gene. Finally, in paper four we investigate the general evolution of three CONSTANS-LIKE genes in B. nigra, namely BniCOL1, BniCOa and BniCOb.

Photoperiodism refers to the organism’s ability to detect and respond to seasonal changes in the daily duration of light and dark and thus constitutes one of the most significant and complex examples of the interaction between the organism and its environment. This thesis attempts to describe the prevalence of variation in a photoperiodic response, its adaptive value, and its putative genetic basis in a common cruciferous weed, Capsella bursa-pastoris (Brassicaceae). Furthermore, the thesis presents a first comprehensive comparative overview of the circadian clock mechanism in an early land plant, Physcomitrella patens (Bryophyta), thus providing insights into the evolution of the plant circadian system. In an introductory survey of global gene expression changes among early- and late flowering accessions of C. bursa-pastoris we found an enrichment of genes involved in photoperiodic response and regulation of the circadian clock. Secondly, by phenotyping circadian rhythm variation in a worldwide sample of accessions with known flowering time, we detected robust latitudinal clines in flowering time and circadian period length, which constitute strong indications of local adaptation to photoperiod in the shaping of flowering time variation in this species. In an attempt to elucidate putative genetic causes for the correlated variation between circadian rhythm and flowering time, we found that sequence variation and diverged expression in components regulating light input to the clock, PHYTOCHROME B (PHYB) and DE-ETIOLATED 1 (DET1) make them strong candidate genes. Finally, we present a comparative study of circadian network topology in the moss P. patens. Phylogenetic analyses and time series expression studies of putative clock homologues indicated that several core clock genes present in vascular plants appeared to be lacking in the moss. Consequently, while the clock mechanism in higher plants constitutes at least a three-loop system of interacting components, the moss clock appears to comprise only a single loop. We conclude that C. bursa-pastoris is a highly suitable model system for the further elucidation of the molecular variation that influences adaptive change in natural plant populations. Furthermore, we believe that the continuing study of the seemingly less complex circadian network of P. patens not only can provide insights into the evolution of the plant circadian system, but also may help to clarify some of the remaining issues of the circadian clock mechanism in higher plants.

<p>Speciation is a constantly ongoing process whereby reproductive isolating baririer build up over time until groups of organisms can no longer exchange genes with each other. Adaptation is thought to play a major role in the formation of these barriers, although the genetic mechanisms and geographic mode underlying the spread of barriers due to adaptive evolution is poorly understood. Critically, speciation may occur in stages through the formation of intermediate partially reproductively isolated groups. The idea of such widespread ecotypes has been the subject of great controversy over the last century. Even so, we have relatively little understanding about whether widespread ecotypes exist, wheather they are reproductively isolated, and how adaptive alleles are distributed among partially isolated groups. In this dissertation, I examined these issues in widespread coastal perennial and inland annual ecotypes of the yellow monkeyflower, Mimulus guttatus. First, I determined that coastal and inland populations comprise distinct ecotypic groups. I then determined that these ecotypes are adapted to their respective habitats through genetically based flowering time and salt tolerance differences. I assessed the genetic architecture of these adaptations through quantitative trait loci (QTL) analysis and determined the geographic distribution of the underlying alleles through latitudinally replicated mapping populations. I quantified the contribution of these loci to adaptation in the field through the incorporation of advance generation hybrids in reciprocal transplant experiments. In the process, I discovered a widespread chromosomal inversion to be involved in the adaptive flowering time and annual/perennial life-history shift among the ecotypes. Overall, the results of this study suggest that widespread reproductively isolated ecotypes can form through the spread adaptive standing genetic variation between habitats and that chromosomal rearrangements can integral to this process.</p> / Dissertation

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19051号 / 農博第2129号 / 新制||農||1033 / 32002 / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 奥本 裕, 准教授 中﨑 鉄也 / 学位規則第4条第1項該当

Studies of demography and population structure give insight into important evolutionary processes such as speciation and diversification. In the present work I perform such studies in the genus Capsella, which has three species: C. grandiflora, an outcrossing diploid, C. rubella a selfing diploid, and C. bursa-pastoris a selfing tetraploid. These three species make a good model system for evolutionary studies because they encompass two major plant evolutionary processes: mating system shifts and polyploidization. To conduct my studies I have gathered a large number of samples across the distributions of each species and scored them both phenotypically and genotypically: more specifically we measured flowering time and collected DNA sequence data. In the tetraploid C. bursa-pastoris we applied an association mapping approach which takes population structure into account to search for genetic variation associated with variation in flowering time. Flowering time is an important and highly adaptive trait which is frequently subject to natural selection. We found evidence of association between flowering time and several single nucleotide polymorphisms (SNPs) within the flowering locus C (FLC) and cryptochrome 1 (CRY1). In the case of FLC these SNPs code for nonconsensus splice site variation in one of the two copies of the gene. The SNPs could potentially have functional consequences and our results imply that non-functionalization of duplicate genes could be an important source of phenotypic variation. Using a novel coalescent based approach, we investigated the polyploid origin of C. bursa-pastoris and find evidence supporting a recent autopolyploid origin of this species. In the two diploid species, I use sequence data to investigate population structure and demographic history and to assess the effects of selfing on C. rubella. Observed patterns of population structure and genetic diversity in C. rubella can be explained by a combination of both demographic history and mating system. Observed patterns in C. grandiflora suggest that the investigated populations do not deviate strongly from the SNM, which has rarely been found in modern demographic studies. Finally, we investigate the effect of sampling strategy on demographic inference. Extensive sampling both within and across our populations allow us to empirically test the effect of sampling strategy on demographic inference. We complement the empirical analysis with simulations and conclude that the effect of sampling strategy is in many cases weak compared with that of demographic events. Nevertheless, these effects are real and have the potential to lead to false inference and therefore sampling strategy should be carefully considered in demographic studies. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 725

In this thesis, I combine molecular analyses, common-garden and field experiments to examine how evolutionary and ecological processes influence patterns of genetic variation among and within populations of the declining, insect-pollinated, self-incompatible, perennial herb Primula farinosa. More specifically I examined 1) whether genetic diversity at neutral marker loci was related to habitat fragmentation and habitat stability, 2) whether floral display and flowering time were more strongly differentiated among populations than were putatively neutral marker loci, 3) whether adaptive population differentiation could be detected on a local spatial scale, and 4) whether floral display differentially affected male and female reproductive success. Genetic diversity at neutral marker loci was lower within fragmented populations on the Swedish mainland than within the more densely occurring populations on the island Öland, SE Sweden. On Öland, fluctuations in population size were more pronounced on thin than on deep soils, but genetic diversity was not related to soil depth. Among-population genetic differentiation in scape length and flowering time was stronger than that of neutral marker loci, which is consistent with divergent selection acting on these traits. Water availability should influence the length of the growing season and thus the time available for fruit maturation, but flowering time in a common-garden experiment was not related to estimates of water availability at sites of origin. In a reciprocal transplant experiment conducted among four populations separated by up to a few kilometres and growing in environment differing in water availability and grazing intensity, no evidence of local adaption was observed. Finally, in a field experiment, interactions with pollinators and antagonists differentially affected selection on floral display through male and female function. Taken together, the results indicate that habitat connectivity and environmental heterogeneity contribute to high neutral and adaptive genetic variation in Primula farinosa on the island Öland, SE Sweden, and illustrate that effects on both male and female reproductive success need to be considered to understand fully the evolution of floral display.

Flowering is one of the most important stages in determining the successful survival and spread in plants. The duration of the flowering period is closely associated with successful reproduction, making it essential to understand the importance and effects of the length of flowering on various macroecological variables across plant species. The effects of the length of flowering periods on the distribution range size of species have seldom been investigated. This project aims to identify any macroecological relationship that may exist between the length of flowering periods and the distribution ranges of plant species endemic to the eastern part of South Africa, a region well known for its floral diversity. Range size and flowering phenology data were collected for several genera that are centred in the region (Cussonia, Gymnosporia, Searsia, Streptocarpus, Pavetta, Plectranthus, Crinum, Eulophia, Gladiolus, Kniphofia, Satyrium, Watsonia and Zantedeschia). At genus level, the relationship varied considerably. While significant correlations between the two variables were retrieved in four genera, the meaning of these patterns differed. In some cases, these suggested that a larger range was achieved through successful pollination due to extended flowering periods, whereas in others, it is probably just an effect of different flowering seasons in different areas where the range is large enough to comprise diverse climates. When incorporating variables such as growth form (narrowly and broadly-defined) and genus identity in analyses of covariance between flowering durations and various measures of distribution, the association of genera was far greater than that of growth form. It can be concluded that both range size and the length of the flowering season are the result of numerous factors acting jointly, which differ across plant groups and are likely to be susceptible to changes in climate and biological invasions. This means that the relationship between range size and flowering period is driven by different factors in different genera, suggesting that the conservation of plant diversity in the face of global change will have to consider the complexity of flowering patterns, and it is likely that lineage-specific approaches for different plant groups will be necessary. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.