published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Identifying sites under evolutionary pressure and predicting the effects of substitutions at those sites are among the greatest standing problems in bioinformatics and computational biology. Moreover, the two problems have traditionally been separated by the enormous chasm that exists between molecular evolutionary biologists interested in the evolutionary process and theoretical chemists interested in free energy changes. As a result, identifying sites under selective pressure has most often left out any semblance of structural biology and biochemistry; likewise, theoretical chemistry tends to rely strictly on first principles calculations rather than thinking first about biologically simple and interpretable results. Here, I have tried to integrate these two intuitions with regard to protein function and evolution. First, I developed a model that implements structural measurements into a traditional structure-blind molecular evolutionary model. This structure-aware model performs significantly better at identifying sites under both purifying and diversifying selection than its structure-blind counter part. Second, I go further to understand the extent to which structural features of any kind can predict the evolutionary process. By comparing site-wise evolution between human and avian influenza, I find that structural features can account for 24% to 36% of the evolutionary pressure on influenza hemagglutinin. Third, I developed a computational method based on first principles molecular dynamics simulations to predict the biological effect of substitutions in the Machupo virus--Human receptor protein--protein interface. I found that relatively simple energetic proxies offer a reasonable substitute for rigorous free energy calculations; such simple proxies could allow non-experts to naively implement first principles methods without being forced to consider all possible degrees of freedom for post hoc calculations. / text

Plant microRNAs have been shown to have important roles in regulating diverse processes ranging from reproductive development to stress response. In the first two chapters, I focus on miRNA diversity in Aquilegia studying both anciently evolved broadly conserved and rapidly evolving species specific miRNAs. In chapter one, I utilize Aquilegia's critical phylogenetic position between the well developed models Arabidopsis thaliana and Oryza sativa to study the evolution of ancient miRNAs across the angiosperms. In chapter two, I utilize smallRNA high-throughput sequencing to annotate Aquilegia specific miRNAs and, in the process, uncover the novel regulation of a floral homeotic gene by an Aquilegia-specific miRNA. In chapter three, I look at the tissue specific development of miRNA regulation in the bioenergetically relevant model organism Populus trichocarpa. High-throughput smallRNA sequencing from four diverse tissue sets including leaves, xylem, mechanically treated xylem, and pooled vegetative and reproductive tissues were analyzed, revealing a total of 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Expanding on my work with the petal identity pathway, I turned a broader analysis of Aquilegia petal spurs. Petal spurs are the distinguishing characteristic of Aquilegia and are argued to be a key innovation in the adaptive radiation of the genus. In the fourth chapter, I explore the cellular basis of extreme spur length diversity in the genus and find that a single parameter, cell shape, can explain this morphological range. Next, I seek to describe the cellular patterns that give rise to a spur primoridia from an initially flat laminar petal and find that spur initiation is characterized by concentrated, prolonged, and oriented cell divisions. Inspired by this quantitative analysis of growth, chapter five looks at the mechanisms of shape change in cucumber tendrils. I find that anisotropic contraction of a multi-layered gelatinous fiber ribbon explains coiling in cucumbers. Surprisingly, we discover that tendrils display twistless-overwinding when pulled and exhibit an unforeseen force-extension response as a result. These results provide the design basis for twistless springs with tunable mechanical responses and serve as a clear example of how the biological systems can inspire applied mechanical designs.

Symbiosis is one of the most widespread evolutionary strategies on Earth. In the deep-sea, symbioses between chemosynthetic bacteria and invertebrates are abundant at hydrothermal vents and cold seeps. These mutualisms, in which symbiont carbon fixation provides for host nutrition, are analogous to the ancient endosymbioses that resulted in the chloroplast and the eukaryotic mitochondrion. However, the evolutionary processes that led to the widespread dispersal of deep-sea organisms and the mechanisms by which symbioses are initiated and maintained are poorly characterized. This thesis examined the evolution of deep-sea mussels (Bathymodiolinae) and their chemosynthetic symbionts. Bathymodioline mussel taxonomy is in need of a comprehensive systematic revision because the majority of named genera do not constitute monophyletic groups. First, this thesis demonstrated that mussels found on the Northeast Pacific Ridges are members of Adipicola, a paraphyletic genus within Bathymodiolinae, refining the evolutionary history of this poorly characterized group. Second, an updated multi-locus phylogeny of bathymodiolines was presented and used to evaluate the statistical evidence for previously proposed hypotheses describing the directional evolution of bathymodioline traits. The results indicated that patterns of directional evolution in this group are not well supported and instead suggests that trait evolution has proceeded in a non-directional manner. Third, this thesis presented the first evidence of detection and abundance of bathymodioline symbionts in the deep-sea environment, providing direct evidence that these symbionts are environmentally acquired. Fourth, this thesis presented the first multi-locus phylogenies of bathymodioline symbionts and tested the hypothesis of environmental acquisition of symbionts in this group. The results demonstrated that symbiont and host lineages are decoupled, which is consistent with the environmental acquisition hypothesis. Finally, environmental acquisition implies that symbionts have opportunities to exchange genetic information with other bacterial strains and evidence for recombination in bathymodioline symbionts is also presented. This thesis advances our understanding of the evolutionary history of bathymodioline symbioses by clarifying host and symbiont evolutionary history and symbiont transmission strategy. In aggregate, these results suggest that bathymodiolines are more flexible with regard to the habitats they inhabit and the symbionts they harbor than previously understood.

A remarkable property of developing organisms is the consistency and robustness within the formation of the body plan. In many animals, morphological pattern formation is orchestrated by conserved signaling pathways, through a process of strict spatio-temporal regulation of cell fate specification. Although morphological patterns have been the focus of both classical and recent studies, little is known about how this robust process is modified throughout evolution to accomodate different morphological adaptations.

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<p>In an ever-changing world, evolution is an essential process that may allow organisms to adapt to their environment through natural selection. All evolutionary processes act through a single fundamental medium: genetic variation. Polyploidy, or whole genome duplication, is a major mechanism for evolutionary change because it is both widespread across taxa and results in a proliferation of genetic material that evolution can act upon. The key questions addressed here are: (1) How does chromosome pairing during meiosis in allopolyploids affect the magnitude of genetic variation?, (2) How does the genome of polyploids evolve following formation, and what genetic mechanisms govern this evolution?, and (3) How does genetic and genomic evolution in polyploids affect phenotypic evolution? I use the shy monkeyflower, <italic>Mimulus sookensis</italic>, a tetraploid of hybrid origin between <italic>Mimulus guttatus</italic> and <italic>Mimulus nasutus</italic>, to address these focal questions. In order to develop a foundation to aid in interpretation of my findings, I first investigate the evolutionary history of <italic>M. sookensis</italic>. Chromosome counts establish that <italic>M. sookensis</italic> is indeed an allotetraploid, and a review of taxonomic literature reveals that this species is heretofore undescribed. By analysing the patterns of genetic variation at chloroplast and nuclear loci in <italic>M. guttatus</italic>, <italic>M. nasutus</italic>, and <italic>M. sookensis</italic>, I show that <italic>M. sookensis</italic> has recurrently formed from <italic>M. guttatus</italic> and <italic>M. nasutus</italic>. Crossing experiments within <italic>M. sookensis</italic> indicate that recurrent origins can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages.</p><p>To address my focal questions, I take advantage of an intriguing and striking difference in flower size among <italic>M. sookensis</italic>, <italic>M. guttatus</italic>, and <italic>M. nasutus</italic>. The flowers of <italic>M. sookensis</italic> and <italic>M. nasutus</italic> are small and remarkably similar to one another, while the flowers of <italic>M. guttatus</italic> and diploid and tetraploid F1 hybrids between <italic>M. guttatus</italic> and <italic>M. nasutus</italic> are large and showy. This phenotypic divergence in flower size between <italic>M. sookensis</italic> and <italic>M. guttatus</italic>-like hybrids indicates that small flower size has evolved in <italic>M. sookensis</italic>. Using genetic marker data and phenotypic measurements from synthetic neoallotetraploid <italic>Mimulus</italic>, I demonstrate that there are low levels of fragment loss and phenotypic variation in neoallotetraploids; this suggests that homeologous pairing and recombination following polyploidization is not a major source of genetic variation or phenotypic evolution in <italic>M. sookensis</italic>. Analysis of the whole genome sequence of two <italic>M. sookensis</italic> lines reveals that <italic>M. sookensis</italic> is a fixed heterozygote throughout its entire genome, in that it has retained both a <italic>M. guttatus</italic>-like and <italic>M. nasutus</italic>-like subgenome, neither of which have been removed through homeologous recombination. These subgenomes have been homogenized by widespread gene conversion, and do not appear to have been differentially affected by deletions or deleterious mutations. Finally, to directly characterize the genetic architecture of flower size in <italic>M. sookensis</italic>, I cross a large-flowered synthetic neoallotetraploid <italic>Mimulus</italic> to small-flowered <italic>M. sookensis</italic>. I then employ a novel genotyping-by-sequencing approach to identify quantitative trait loci (QTL) associated with flower size. I find that there is one locus that accounts for a large proportion of phenotypic variation, and four other loci also contribute to flower size variation between the parental lines. Some of these loci co-localize with previously identified loci for flower size in diploid <italic>Mimulus</italic>, while others do not. Altogether, genetic marker data, phenotypic analysis of neoallotetraploids, whole genome sequence data, and QTL mapping data suggest that the genetic variation necessary for flower size evolution was likely caused by both gene conversion and new mutations, but not homeologous recombination. These results suggest that trait evolution in polyploids may be affected by the unique attributes of polyploids, but that new mutations are always an important source of genetic variation, regardless of ploidy level.</p> / Dissertation

Emancipating the forelimbs from locomotion for use in other activities, such as food manipulation, is a major evolutionary milestone. A variety of selective forces and evolutionary correlates may influence the evolution of various degrees of skill with which the forelimbs are used. Using the order Carnivora as a test group, I assesed the relative influence of six factors: relative brain size, neocortical volume, manus proportions, body size, phylogenetic relatedness, type of locomotion and diet. I developed a rating system to describe the dexterity of individual species and compared the scores to the six factors using modern comparative methods. Only phylogeny and diet were significanly correlated with forelimb dexterity. More specifically, forelimb dexterity tends to be higher in caniform than in feliform carnivorans and decreases with increasing specialisation on vertebrate prey. I conclude that food handling and feeding niche breath have a significant effect upon the evolution of skilled forelimb movements. / xii, 151 leaves : ill. ; 28 cm.