Mammalian nucleostemin (NS) is a nucleolar GTP-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). Nucleostemin 1 (NS1) is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity, and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed GFP-NS1 enrich within the nucleolar granular regions as expected, while over-expressed GFP-NS1 localizes throughout the nucleolus and nucleoplasm, and to several transcriptionally active inter-bands of polytene chromosomes. Over-expression caused melanotic tumors and larval and pupal lethality. RNAi depletion of NS1 caused a loss of imaginal (precursor) cells in the larval midgut, and an apparent block in the nucleolar release of large ribosomal subunits in the terminally differentiated larval midgut polyploid cells. Depletion of 60% of NS1 transcripts lead to larval and pupal lethality. Ultra-structural examination of highly differentiated larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes with a concomitant appearance of cytoplasmic pre-autophagosomes. We interpret the appearance of these structures as indicators of cell stress response.

Geographic range differences among species may result from differences in their physiological tolerances. In the intertidal zone, marine and terrestrial environments intersect to create a unique habitat, across which physiological tolerance strongly influences range. The snail Melampus bidentatus occurs in coastal salt marshes in the western Atlantic and Gulf of Mexico. I have used sequence data from one mitochondrial (CO1) and two nuclear markers (histone H3 and a mitochondrial carrier protein, MCP) to identify three cryptic species within Melampus bidentatus, and to infer their relationships to other Melampus. To identify microhabitat differences between two cryptic species, I modeled their distributions using both marine and terrestrial environmental data. Temperature largely explained their range differences, but other environmental components (precipitation, salinity, and tidal height) explained facets of the range that temperature cannot. To test for phylogenetic conservation in freeze tolerance, I tested the mean lower lethal temperature (LT50) of three temperate and three tropical species. Cryptic species of M. bidentatus are significantly more freeze tolerant than their tropical relatives, although there was variation among locales within species, most likely due to microhabitat variation. The temperate species M. floridanus was also freeze tolerant, but without testing the LT50 of its closest relatives in the Pacific, I cannot determine this represents an independent evolution of freeze tolerance, or if this trait is more widely shared among Melampus. Nonetheless, the lack of freeze tolerance in the most basal species that I have tested (M. bullaoides), and the predominantly tropical distributions of most ellobiids, suggests that the evolution of freeze tolerance has allowed for the invasion of the temperate zone by Melampus. Using massively parallel sequencing, I have isolated > 500,000 expressed sequence tags and assembled these into ~20, 000 seasonally expressed transcripts. A comparison of these transcripts has revealed 2 candidate markers to test for their association with freeze tolerance in M. bidentatus, and many more markers that can be used for further phylogenetic analyses in Melampus. Further work to examine variation in both the sequence and expression of these proteins is needed to determine if they underlie adaptive differences among species.

Although birds of the forest canopy are an important component of tropical forest ecosystems, difficulty accessing the forest canopy has limited the advancement of knowledge pertaining to this group of species. Here I test methods for the study of canopy birds in lowland Neotropical rainforests, and identify recurring patterns of community structure in canopy bird assemblages as well as processes potentially responsible for these patterns. I used three methods to assess differences in ground-based and canopy-based methods for detecting forest birds in a 100-ha plot of lowland rainforest in northern Honduras: (1) point counts from the ground; (2) repeat censuses from two canopy trees; and (3) single censuses from multiple canopy trees. Ground methods significantly underestimated species and familial richness as well as abundances of individuals in the canopy stratum, and I predict that ground methods miss 25 to 50% of the species richness for some migrant and resident families and underestimates the density of some species by as much as 25%. I compared two distant canopy bird assemblages based on >11,000 detections at lowland rainforest sites in Honduras and Amazonian Brazil. Richness of canopy birds was similar between sites, despite overall higher forest bird richness in Brazil. Honduras and Brazil differed significantly in abundance distributions, with greater evenness characterizing the Brazil assemblage. Long-distance migrants and species of forest edges and open habitats were underrepresented at both sites when compared to null expectations drawn from regional species pools. Long-distance migrants were relatively more important in Honduras, where they constituted a third of canopy birds. Species richness of omnivores amongst core canopy species was greater than expected at both sites, and omnivores dominated the canopy in terms of species richness and individual abundance. Functional morphology analyses indicated that core canopy birds are more similar phenotypically than would be expected by chance. Similarity in functional morphology may result from environmental filtering selecting for phenotypes optimally suited for survival in the harsh canopy environment. Phylogeny seems to be an important underlying component of morphological similarity, however, and may exert a structuring force on the canopy bird assemblage through phylogenetic niche conservatism.

Transcription factors are important for many aspects of gene regulation in eukaryotes. YY1 (Yin-Yang 1) is a particularly interesting example of a highly conserved zinc-finger transcription factor, involved in transcriptional activation, repression, initiation, and in chromatin modification. YY1 is ubiquitously expressed in mammals, and its binding sites are found in ~10% of human genes as well as in repetitive elements. It is a targeting protein of the Polycomb complex and is involved in mammalian genomic imprinting. First, we explored the evolutionary history of YY1 using 62 species and formation of its paralogs, YY2 and REX1, which are found in mammals, and Pho and Phol, which are found in Drosophila. We confirmed the specificity of the consensus YY1 binding site and the differences of the target binding motifs of YY2 and REX1 which are reflected in their amino acid sequences. We found that the core motif, CCAT, is conserved for all three homologs and that YY2 and REX1 were produced via retrotransposition events early in the mammalian lineage. Second, we identified unusual clusters of YY1-binding motifs found in the coding regions of olfactory receptor genes (OLFRs) in mammals but not in fish. Olfactory genes provide scent detection and are the largest class of genes in mammals. Statistical analysis indicates that the core of the YY1-binding motifs cannot be acounted for by conserved amino acid motifs or overall protein homology. Thus selection has acted at the DNA level rather than at the protein level in preserving these YY1-binding sites within coding regions. Therefore, YY1 is likely to play a crucial role in regulating the expression of OLFRs. Third, we produced a new method of microarray data analysis predicated on the positions of genes along a chromosome as well as their expression levels. This technique is supplementary to traditional microarray data analysis and adds a new dimension to finding target genes of interest by looking for co-regulation. Overall, this work provides a coherent background to the evolution of YY1 and its homologs. It provides strong evidence that coding sequences of genes can encode information both at the DNA level and the protein level.

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. In addition, integrin TM homomeric association was also observed. But the role of this interaction remains elusive. In this work, the platelet integrin, αIIbβ3, has been used to elucidate the roles of integrin lower leg and TM homomeric association in integrin signalling. We first addressed whether the separation of integrin αβ lower leg is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain is critical for integrin bidirectional signaling. Formation of the TM homooligomers was observed in micelles and bacterial membranes previously, and it has been proposed that this homomeric association is important for integrin activation and clustering. We then addressed whether integrin TM domains form homooligomers in mammalian cell membranes using cysteine mutagenesis scanning method. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding, or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homooligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, the integrin TM homooligomerization is not required for integrin activation, ligand binding and signaling.

Ecological mechanisms proposed to explain community assembly and the maintenance of biodiversity are hypothesized to fall along a theoretical continuum bounded at one extreme by deterministic processes (niche assembly) and at the other extreme by stochastic processes (dispersal assembly). In this dissertation, I explore the idea that the position of ecological communities along the niche-dispersal assembly continuum is dynamic in space and time. Using field experiments in a high-diversity longleaf pine savanna, I test the general hypothesis that ecological filters (competition, disturbance, and resource availability) contribute to niche assembly through their effects on established plant species and recruitment from the species pool. Consistent with dispersal-assembly theory, I found that dispersal from the species pool strongly limited local species diversity regardless of the presence of these three niche-based ecological filters. Importantly, however, some ecological filters (e.g., space limitation in communities with low-intensity fire disturbance and establishment limitation imposed by drought and high-rainfall conditions) limited the extent to which community assembly was influenced by dispersal, suggesting ecological conditions that reduce stochastic community assembly in high-diversity communities. I examined the generality of these patterns by conducting a meta-analysis of >60 published experiments. I found that dispersal strongly limited species richness in a wide range of plant communities, but that dispersal had a stronger positive effect on species richness in more disturbed communities and when the species pool contained high species diversity and functional-trait diversity, supporting the hypothesis that community assembly reflects a dynamic interplay between species-pool diversity and local environmental heterogeneity. My results suggest a conceptual model for community assembly in high-diversity pine savannas, with implications for other species-rich plant communities. I propose that characteristics of high-diversity communities (large species pools and pervasive recruitment limitation in populations of many rare species) generally contribute to stochastic community assembly, but that niche-based ecological filtering of resident species and immigrating species can shift high-diversity communities towards more deterministic community assembly. This conceptual framework has broader implications for understanding the maintenance of biodiversity and species coexistence in communities of contrasting diversity and for conserving biodiversity in longleaf pine communities threatened by habitat loss, fragmentation, and environmental change.

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature-sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA. yghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature-sensitive phenotype: targeted deletion of both genes in a wild-type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca2+, Ba2+, Sr2+, or Mg2+ or 300 to 500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. Lud135 is non-motile and overproduces outer membrane vesicles that contain FliC. Preliminary results from the topological analysis of YqjA suggest it contains 4 transmembrane domains with a large cytoplasmic domain that is enriched in positive amino acids. yghB and yqjA belong to the conserved and widely distributed dedA gene family, for which no function has been reported. The two open reading frames encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli.

In this dissertation I explored the processes that generated high species diversity in a continental adaptive radiation of passerine birds: the family Furnariidae. Although there is a vast amount of information on the microevolutionary mechanisms that generate speciation and adaptation, much less is known about the processes underlying large-scale patterns of diversity. I used new techniques that exploit information contained in phylogenetic trees to investigate the diversification process in Furnariidae at a macroevolutionary scale. First, I conducted the first quantitative study that demonstrated that the Furnariidae constitute a case of rapid diversification in both species numbers and morphology as compared to six closely related families. To evaluate phenotypic diversity, I developed a new multivariate extension of the variance, which I called 'proper variance', and a new simulation procedure to test for differences in phenotypic diversity when complete phylogenies are not available. I found that the Furnariidae, although not particularly diverse in body size, is unusually diverse in shape. These results support the idea that the Furnariidae constitute a continental adaptive radiation. To explore processes responsible for high species diversity in the Furnariidae, I first investigated the effect of dispersal ability on species proliferation. Assuming that dispersal in birds is highly influenced by flying ability, I used an index of wing shape as a surrogate for flying ability. Using a nearly complete species-level phylogeny of the Furnariidae and a recently developed method, I found a negative correlation between flying ability and speciation rates, suggesting that low dispersal has stimulated species proliferation in Furnariidae. Finally, I investigated the effect of three key innovations on furnariid diversification: adaptations for trunk climbing, spiny-tail morphologies, and skull kinesis. I found that increases in diversification rates are not associated with major morphological changes. In addition, I found that climbing specialization had a negative effect on speciation rates, but that the spiny-tail morphology was positively correlated with speciation rates. These results provide mixed support for a role of key innovations promoting speciation. My results suggest a stronger role of geographic isolation (low dispersal) than ecological opportunities in species diversification in the continental radiation of the Furnariidae.

High stress environments can reduce species diversity. How such stress-induced reduction in predator diversity impacts prey survival is less well studied. Brackish waters in estuaries are stressful, species depauperate areas, but also prime oyster habitat in Louisiana. Surveys revealed reduced bivalve predator diversity at the low salinity (high stress) site. Exclosure experiments indicated highest prey mortality at the high salinity (low stress) site. Predator enclosures corroborated the field study results, with reduced consumption rates at the low salinity site for both stone crabs, Menippe adina and oyster drills Stramonita haemastoma, but not blue crabs, Callinectes sapidus. Blue crab mortality in enclosures was relatively low at all sites, while stone crab and oyster drill mortality were higher at the medium and low salinity sites. As predator diversity increases, interactions between predators can impact prey mortality. Therefore I studied how the bivalve predators interact, how interactions impact prey survival, and how interactions change with stress. Interactions between blue crabs and stone crabs had an additive effect on bivalve mortality. Videotaping suggested blue crabs fed longer than stone crabs, and that interactions did not impede feeding. Bivalve mortality was however lower than predicted in blue crab-oyster drill combinations, suggesting interference reduced feeding by oyster drills. Salinity did not affect multiple predator interactions or feeding times. Prey preference by predators also affects prey mortality. Hooked mussels, Ischadium recurvum, had higher mortality than oysters, Crassostrea virginica, in field and laboratory experiments, possibly because the thinner-shelled mussels were easier to consume. Chapter 4 examined prey preference in two important predator species. Blue crabs preferred small hooked mussels. Because profit did not differ with mussel size, stone crabs because their stronger claws were less prone to damage showed no size preference, and large mussels required force generation near levels that can damage claws, I concluded blue crabs consumed small mussels to reduce risk of claw damage, or to minimize handling times to limit their own predation risk.

Mutualisms are reciprocally exploitative interactions providing net benefits to both partners. These interactions can be exploited, in turn, by individuals that take advantage of benefits offered by one or both partners in a mutualism, while offering no benefits in return. For many mutualist-exploiter interactions the mechanisms allowing exploitation, and the maintenance of mutualisms in the face of exploitation, are still poorly understood. Here I describe manipulative field and laboratory experiments to investigate the mechanisms used by an exploiter to invade an ant-plant mutualism. I tested two non-mutually exclusive hypotheses for how a coreid (Mozena sp., Hemiptera: Coreidae) feeds on mymecophytic acacia trees (Vachellia spp.) while avoiding attack by resident ants: chemical defense and chemical mimicry. I found that chemical compounds produced by Mozena sp. in both the metathoracic gland and the cuticle reduced the number of ant attacks and cuticular compounds appeared to be essential in escaping recognition on ant-occupied Vachellia spp. trees. The compounds were effective on multiple colonies and for multiple ant species, thus they are not strictly host- or species-specific. In addition, gas chromatography and mass spectrometry analyses of cuticular compounds revealed a close match between chemical profiles of Mozena sp. and Pseudomyrmex spinicola ants, suggesting chemical mimicry is the primary mechanism by which Mozena sp. exploits the ant-acacia mutualism. To examine the prevalence of a cost of exploitation for plant partners in exploited mutualisms, I conducted a meta-analysis of studies from the published literature. I found that exploitation has a weak, negative, but insignificant impact on the reproductive success of mutualistic plants. Collectively, these analyses illuminated methods by which exploiters may succeed in infiltrating mutualisms and suggested that the relatively low costs of exploitation may account for the lack of destabilization and degradation by exploiters of some mutualistic interactions.