Rho GTPases are molecular switches that mediate the formation of dendritic spines actin-enriched protrusions that make excitatory synapses with nearby neurons. Once activated by guanine nucleotide exchange factors (GEFs), GTPases regulate actin dynamics within spines. The roles of several Rho GTPases are known in spines; the functions of many GEFs, however, are unclear. Here, we show that the Rac GEF Asef2 is important for the development of dendritic spines. Endogenous Asef2 localizes to spines, and spine and synapse density can be increased or decreased by expression or knockdown of Asef2, respectively. This effect is GEF activity-dependent, since mutation of Asef2s GEF domain blocks spine formation. Also, knockdown of Rac blocks Asef2-mediated spine formation, suggesting that Asef2 activates Rac to promote spine development. Finally, the actin-binding protein spinophilin also regulates Asef2 function by targeting Asef2 to spines. In a related project, we investigated the regulation of Asef2 activity by identifying putative phosphorylation sites. Six phosphorylation sites were identified by mass spectrometry. Most of these sites cluster around the autoinhibitory region of Asef2, suggesting that they may regulate Asef2 activity. To test this, one of the residues serine 106 was mutated to alanine (S106A, non-phosphorylatable analogue) or to aspartic acid (S106E, phosphomimetic analogue), and the effect of these mutations on cell migration (another actin-dependent process) was assessed. The S106A mutant inhibited Asef2-mediated Rac activation, cell migration, and adhesion turnover (a component of cell migration), while the S106E mutant promoted Rac activation but did not influence cell migration compared to wild-type Asef2. These results suggest that phosphorylation is an important mechanism for regulating Asef2 activity and function.

Since Darwin, biologists wonder how organisms cope with environmental variation, why there are so many species, and how species form. In my dissertation, I explore how species of long-lived, clonal reef organisms originate across depth gradients. In Chapter 2, I evaluate the strength of depth to isolate populations by comparing the genes and morphologies of pairs of depth-segregated populations of the candelabrum coral Eunicea flexuosa across the Caribbean. Eunicea flexuosa is a long-lived clonal cnidarian that associates with an alga of the genus Symbiodinium. Genetic analysis revealed two depth-segregated lineages, each genetically well-mixed across the Caribbean. Survivorship data, combined with estimates of selection coefficients based on transplant experiments, suggest that selection is strong enough to segregate these two lineages. Limited recruitment to reproductive age, even under weak annual selection advantage, is sufficient to generate habitat segregation because of the cumulative selection accrued during prolonged pre-reproductive growth. I then studied in detail the genetic diversity of Symbiodinium in each Shallow and Deep E. flexuosa lineage (Chapter 3). I sampled colonies of the two ecotypes across depths at three Caribbean locations. I find that each host lineage is associated with a unique Symbiodinium variant. This relationship between host and alga is maintained when host colonies are reciprocally transplanted. Even when the clades of both partners are present at intermediate depths, the specificity between host and algal lineages remained. I then test whether the Shallow/Deep adaptive divergence occurs in natural populations, by examining the frequencies of juvenile and adults. The habitat distributions of the two lineages are more distinct when inferred from adults than from juveniles. Selection coefficients from cohort data correlates with that from transplant experiments (Chapter 4). The two lineages form a narrow hybrid zone (100 m), with coincident clines of both the host coral and its algal symbiont. Effective dispersal estimates derived from the hybrid zone are small (20 m) for a broadcast spawner with a large dispersing potential (50 km). Ecological factors associated with depth act as filters generating strong barriers to gene flow despite extensive dispersal, altering morphologies, and contributing to the potential for speciation in the sea.

Coral reefs are the most productive and species rich ecosystems in the ocean yet we lack knowledge about the distribution of genetic variation the within and among reef species, particularly for the sponges (Porifera). My dissertation describes how genetic variation at mitochondrial and nuclear genes is partitioned among and within species in the sponge genus Callyspongia. I compared patterns of genetic diversity and population subdivision in the mitochondrial and nuclear genomes of one species, C. vaginalis, in Florida (Chapter 2). Previous work revealed three divergent mitochondrial lineages, but nuclear alleles did not correspond to either mitochondrial clade or geography. Coalescent simulations showed mito-nuclear discordance was not the result of incomplete lineage sorting. Instead, patterns in mitochondrial and nuclear DNA were consistent with changes in population size and sperm-mediated gene flow. Across the Caribbean, I found subdivision in C. vaginalis for mitochondrial and nuclear DNA was concordant, suggesting geographic features and habitat discontinuity are important for structuring populations at large spatial scales (Chapter 3). Clustering analyses found C. vaginalis populations were divided west-east and model-based tests of species boundaries supported a cryptic lineage in Central America. Phylogeographic patterns for three invertebrate sponge commensals also showed a west-east split between Florida and the Bahamas. I tested for mismatches between morphological and molecular species boundaries for seven Caribbean Callyspongia species (Chapter 4). Genetic distances calculated within and among species support C. fallax, C. tenerrima, and C. plicifera as distinct species. However, C. armigera, C. longissima, C. ?eschrichtii and C. vaginalis shared alleles across loci and genetic distances among these taxa overlapped distances within them. Model-based species delimitation supported the hypothesis that these latter four taxa represent one evolutionarily significant unit. This dissertation demonstrates that in the common reef sponge Callyspongia vaginalis, demographic processes and geography influence population structure at small and large spatial scales, respectively, and genetic markers from different genomes can show contrasting patterns. My work also shows the relationship between morphology and evolutionary history is not straightforward in sponges and points to the importance of inter- and intraspecific genetic data for a thorough documentation of biodiversity in marine invertebrates.

Barataria Bay, in southeast Louisiana is home to productive oyster reefs that are both ecologically and economically important, but was threatened by the Deepwater Horizon oil spill in April 2010. This study was designed to determine how the oil spill affected long- and short-term oyster recruitment, and spat (juvenile oysters) cellular health. I also investigated how predators in the bay affect spat survival. Four study sites were selected in Barataria Bay: two control and two oiled, each with a low and high salinity location. To determine whether there were long-term effects of the oil spill on oyster recruitment, tiles were placed at each site in 2012 and 2013 and spat recruitment quantified monthly. Results indicate that in 2012, recruitment varied more with salinity, but in 2013 when early summer salinity was lower, recruitment only occurred at the control sites. Tiles with 5 mL of light crude oil absorbed were used to study the short-term effects of oil on oyster and barnacle recruitment in 2013. Spat recruitment was lower on oiled tiles but there was no difference in spat size between treatments; alternately, there was a slight tendency for oil to increase barnacle recruitment. The effect of No (0 ppm), Low (500 ppm) or High (25,000 ppm) oil concentrations in 10 and 20 PSU seawater on spat cellular function was determined. Lysosomal stability was lower in low and high oil treatments suggesting oiling can affect spat health after just 10 days of exposure. The effect of predators on spat survival at each site was determined by quantifying predation on spat with no cage or in predator exclusion cages with 0.5, 1.0, or 3.0 cm mesh openings. The presence of a cage reduced predation of oyster spat. Predation rates were greatest on spat without cages suggesting larger predators such as blue crabs and oyster drills, with access to exposed spat, may play greater roles in post-settlement spat mortality than other predators such as mud crabs. Oyster recruitment and spat survival seems to depend more on salinity and predation than long-term hydrocarbon contamination. However, the short-term effect of hydrocarbon contamination can detrimentally impact spat recruitment and health; thus, clean-up efforts immediately following an oil spill and continuous monitoring efforts are necessary to maintain healthy oyster populations.

Patterns of community structure may be examined using phylogenetic and morphological data; these patterns can then be used to infer the processes that gave rise to these patterns. Communities made up of similar species may be structured by habitat filtering, wherein only species with traits necessary to survive in a particular location are found there. Communities made up of dissimilar species may have been structured by competition, which reduces overlap in resource use. I examined the sensitivity of phylogenetic community structure (PCS) metrics to changes in phylogeny and community delimitation method, investigated patterns of PCS and correlation to environmental variables at multiple spatial and taxonomic scales, and assessed whether morphological data gave results similar to phylogenetic data using North American desert bats as a model system. I found that PCS metrics were robust to moderate changes to phylogeny and that these metrics also trend in the same direction regardless of delimitation method. Bat communities tended to be made up of species that were significantly more closely related than expected by chance, or phylogenetically clustered, at large spatial and taxonomic scales; this tendency towards clustering decreases with decreasing scale. Phylogenetically clustered communities also tended to occur in harsher environmental conditions than more overdispersed communities, or those made up of species not closely related. From a morphological perspective, communities were made up of species that were morphologically clustered or not significantly different from random. Morphological community structure was positively correlated with PCS, indicating that these different datasets gave similar results. These results indicate that North American desert bat communities are made up of phylogenetically and morphologically similar species and that environmental variables such as temperature and seasonality may influence community structure. This suggests that habitat filtering is playing a predominant role in structuring these communities.

Circadian clocks are the endogenous timing mechanisms that facilitate organisms to adapt to the daily cycles on the earth. Previous studies have demonstrated that cyanobacterial circadian clock enhances fitness under cyclic environment. In this dissertation, the adaptive value of circadian clocks were further confirmed in cyanobacteria under various culture conditions, and the potential mechanism of the clock-mediated fitness enhancement was explored. By establishing the global metabolic profiles of cyanobacterial strains, I found that the metabolism of cyanobacteria can be affected by the disruption of circadian clocks, which could be the potential mechanism of the clock-mediated fitness enhancement. In addition, a novel timing mechanism driven by kaiCRp, which is a homolog of cyanobacterial central clock gene kaiC, was discovered in a purple non-sulfur bacterium, and this timing mechanism also enhances fitness under light-dark cycles.

The zebrafish epithalamus, which includes the dorsal habenular nuclei and pineal complex, displays robust molecular and anatomical asymmetries. The full elaboration of molecular and anatomical differences between the left and right dorsal habenular nuclei depends on the formation of a left-sided parapineal organ. This work has yielded significant insights into the role of Tbx2b during parapineal development using both the candidate gene approach as well as the non-biased, transcriptome analysis approach. Through the candidate approach, it was determined that Tbx2b acts in a permissive manner during parapineal specification, but it is necessary for proper parapineal migration. Further, the inhibitory roles of Flh and Nr2e3 during formation of parapineal neurons have been characterized. Using RNA-sequencing to perform differential expression analysis between wild-type and tbx2b-deficient pineal complex cells has yielded many targets that are downstream of Tbx2b. Together, these studies also provided a novel insight into a potential genetic interaction system between Tbx2b and Nr2e3 during cone versus rod photoreceptor differentiation.

Transcription by RNA polymerase III (Pol III) requires sequential assembly of Pol III-specific transcription factors. At the tRNA gene, the TFIIIC complex recognizes and specifically binds at intragenic promoter elements A-box and B-box and aids the assembly of TFIIIB to upstream of the transcriptional start site. Upon binding, Pol III is recruited near start sites and transcription of tRNA genes is initiated. Apart from transcription of a gene, these bound Pol III complexes influence transcription, chromatin state and genome organization of neighboring RNA polymerase II (Pol II)-transcribed genes. Such effects are known as extra-transcriptional effects of Pol III complex. Our study provides evidence of a unique extra-transcriptional activity of assembled Pol III transcription complexes at the tRNA gene that blocks progression of intergenic RNA polymerase II transcription. We demonstrated that the Pol III transcription complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream non-coding intergenic SUT467. The protection is predominately mediated by binding of the TFIIIB complex. Failure to block this readthrough resulted in compromised ATG31 translation. Given the recent discovery of widespread pervasive transcription in yeast, protection of neighboring genes from intergenic transcriptional interference may be a key extra-transcriptional function of assembled RNA polymerase III complexes. Our data from RNA-seq analysis demonstrated genome-wide effects of DNA bound Pol III complexes on neighboring chromosomal loci, by comparing expression profiles from tfc6 under-expressing mutants and wild-type S. cerevisiae strains. Reduced TFIIIC occupancy in mutant strains altered Pol II derived transcripts and displayed 5 or 3 extension of protein-coding genes, readthrough from non-coding transcripts and increase in the transcription of genes near the potential TFIIIC binding sites, including tRNA genes and putative ETC sites. Interestingly, not all genes in the vicinity of TFIIIC binding sites were transcriptionally mis-regulated, suggesting variable strength of influence on Pol II transcripts by TFIIIC bound sites. Finally, as observed in SUT467-ATG31 readthrough, we anticipated translation defects in 5 or 3 extended transcripts in mutants. Overall these genome-wide results suggest much complex regulatory role of Pol III transcription factors bound sites than previously anticipated.

Many eukaryotes use the ribonucleoprotein telomerase to replenish telomeric sequences lost during conventional DNA replication. In Saccharomyces cerevisiae, telomerase contains the catalytic reverse transcriptase, Est2p, the templating RNA, TLC1, and two accessory proteins, Est1p and Est3p. Little is known about how the components of telomerase assemble away from the telomere. However, studies monitoring the localization of TLC1 RNA have led to the hypothesis that the telomerase complex assembles in the cytoplasm and is imported through a pathway previously shown to transport TLC1. Because Est1p abundance is uniquely cell-cycle regulated, we reasoned that the model for telomerase assembly may involve additional complexity. Here we show that the nuclear localization of Est1p requires three cis-acting nuclear localization sequences (NLSs). Mutation of an NLS within Est1p perturbs telomere maintenance in a manner that is rescued by fusion with an exogenous NLS. Nuclear import of Est1p requires a pathway different from that utilized by TLC1 RNA, indicating that assembly of TLC1 and Est1p in the cytoplasm is unlikely. These investigations reveal that one key step of telomerase biogenesis occurs in the nucleus and have implications for how the cell might regulate other such complexes whose activity is restricted in the cell cycle.

<p>DNA sequencing costs have decreased dramatically over recent decades, resulting in a flood of phylogenetic information available to researchers. While it is often assumed that additional data will lead to more accurate conclusions, it also raises a number of problems for phylogeneticists, including mundane computational issues such as data management and complex statistical problems such as obtaining a single species tree from multiple conflicting gene trees. Developing new methods to make better use of existing data and probe the causes of conflicting signal will be necessary to confidently resolve phylogenies in the genomic era.</p> <p>Here, we examine two current problems in statistical phylogenetics and attempt to address them in a Bayesian framework. The first problem involves inflated tree lengths in Bayesian phylogenies, which can be an order of magnitude longer than maximum likelihood estimates. We developed EmpPrior, a program which queries TreeBASE for datasets similar to the focal data, then estimates parameters from each dataset to inform priors on the focal data. This approach greatly improves the tree length credible intervals in four exemplar datasets and, when combined with other approaches such as the use of a compound Dirichlet prior on tree length, can nearly eliminate the problem of inflated trees.</p> <p>The second problem involves incongruence between morphological and molecular phylogenies in squamates. Here, we use posterior prediction with inferential test statistics to investigate whether systematic error may be biasing inference in the molecular data. While we detected some model violation in most of the 44 genes, the genes with the most model violation were more distant from the molecular phylogeny. This suggests that model violation is not a major source of error in the molecular data. Hence, the source of incongruence between the molecular and morphological squamate topologies remains unknown.</p> <p>In both problems, we found that incorporating tools such as informed priors and posterior prediction from Bayesian statistical literature into phylogenetic analyses can improve results and help uncover why different datasets lead to conflicting topologies. As phylogenetic datasets continue to grow, using methodological best practices will only become more important if we want to have confidence in our conclusions.</p>