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.

Mechanisms responsible for the diversity of plant defenses in natural populations are poorly understood. This dissertation examines how environmental heterogeneity and succession influence the distribution, expression, and adaptive value of resistance and tolerance of goldenrod (Solidago altissima). Within replicate early- and late-successional fields, I quantified the spatial structure of herbivore damage and goldenrod resistance, tolerance, and fitness traits, and assessed whether the spatial structure of defense was correlated with edaphic conditions and neighboring plant community. I then conducted a common garden experiment to empirically measure genetic variability of resistance and tolerance and quantify their associated fitness costs and selection gradients. Lastly, I conducted a field experiment to test the prediction that successional changes in the environment alter the adaptive value of resistance and tolerance. To this end, I transplanted goldenrod genets into early- and late-successional fields and measured defense levels, fitness costs, and selection gradients. Field surveys revealed that defense traits were strongly spatially structured (i.e., autocorrelated), and late stages were more strongly structured than early stages. There were stage-specific relationships between defense and neighbor variables. Damage was correlated with vegetative cover in early stages, and tolerance traits were positively correlated with canopy cover in late stages. In the common garden, I found genetic variability in resistance and tolerance, but low heritability. Herbivores imposed strong selection for increased tolerance but not resistance. Results from the field experiment revealed an apparent succession-induced shift in the expression and adaptive value of resistance and tolerance. Resistance was 41% greater and tolerance was 97% lower for goldenrods planted in early than in late stages. Resistance was more adaptive in early stages and tolerance may be beneficial in mitigating the effects of both herbivory and low light availability in late stages. This represents the first study to examine plant resistance and tolerance to herbivory in a spatiotemporal context. I conclude that environmental heterogeneity and succession may be important mechanisms promoting the spatial and temporal variability of plant defense expression in nature. Fluctuating adaptive landscapes may have important implications for trait evolution and should be incorporated into future studies of plant defense.

A confluence of factors determines the biological diversity we observe today. Here, I conducted three investigations of the historic, geographic and ecological factors that shaped the morphological and genetic diversity of rainforest birds in Southeast Asia. (1) In the Oriental Dwarf Kingfisher (Ceyx erithaca) complex, the birds are highly polymorphic in plumage. Quantitative analysis of this variation indicated that a large proportion of birds in Sumatra, the Malay Peninsula and Borneo have plumages intermediate between the northerly black form from mainland Asia and the southerly rufous form from Java. Phylogenetic analyses indicated that birds from continental Southeast Asia (north of the Malay Peninsula) were well differentiated from those from insular Southeast Asia. This genetic distinction correlates well with a fixed plumage difference (mantle coloration). Coalescent analyses showed that the plumage polymorphism was caused by past genetic introgression between the two parental forms. (2) I sampled 16 lowland rainforest bird species primarily from the Malay Peninsula and Borneo to test the long-standing hypothesis that animals on different Sundaic landmasses intermixed extensively when low sea-levels during the Last Glacial Maximum (LGM) exposed land-bridges. This hypothesis was rejected in all but five species through coalescent simulations. Environmental niche modeling showed that the presence of unsuitable habitats between western and eastern Sundaland during the LGM coincided with deeper inter-population genetic divergences. The distinctiveness of the northeastern Borneo populations of some species may be underlain by a combination of factors that included riverine barriers, LGM expansion of montane forests and regional physiography. (3) I further investigated the population divergence and demographic histories of three bird species that possessed disparate ecological characteristics. Multilocus analyses revealed changes in effective population sizes that were driven by long-term changes in the environment, instead of high-frequency glacial cycles. Populations from Borneo exhibited stronger demographic growth than those from mainland Southeast Asia, suggesting regional differences in environmental changes or directional colonization. The species with the widest habitat breadth also showed the greatest amount of inter-landmass gene flow. This adds to the growing body of empirical work indicating an association between a species ecological characteristics and its population connectivity over evolutionary time-scales.

Understanding the factors that influence the formation and location of distribution boundaries is important for the study of evolutionary processes. These factors can be studied effectively at suture zones, regions containing disproportionally high numbers of contact zones (CZs) and phylogeographic breaks (PBs). Together, CZs and PBs offer complementary views of current and historical factors that separate or bring together populations of closely related taxa. For my dissertation, I studied a suture zone in northern Amazonia, where ~100 pairs of taxa replace one another geographically. I analyzed the Guiana Shield avifauna, using bird distributions to redefine the boundaries of the Guianan area of endemism. I showed that the Rio Branco is an important biogeographical barrier and a natural western limit for this area, although smaller rivers, savannas, and mountains also play a significant role. A multivariate approach revealed that the Branco/Negro interfluvium represents a transitional zone for birds, suggesting that the longstanding view of Amazonia as a mosaic of parapatric areas of endemism likely represents an oversimplification of current patterns. I investigated the role of rivers in maintaining and generating biodiversity by testing predictions of the riverine barrier hypothesis. Using a comparative phylogeographic approach, I found that phenotypically differentiated populations across rivers are reciprocally monophyletic. The lower Rio Negro represents a stronger barrier to gene flow than does the upper Rio Negro, but no genetic homogenization occurs towards the headwaters. Most riverine barrier hypothesis predictions were not supported, suggesting that rivers are key to maintaining biodiversity, but not for its generation. Finally, I explored the role of physical and ecological factors in the location of CZs and PBs. PBs cluster along physical barriers, whereas CZs aggregate at the headwaters of large rivers. Nearly half of the pairs that come into contact hybridize, and show significantly lower genetic distances than pairs that come into contact and do not interbreed, suggesting that time of isolation as inferred from genetic data may predict their likelihood of hybridization. Ecological niche models showed significant levels of niche divergence between pairs, suggesting that environmental variables cannot be ruled out as factors influencing the location of suture zones.

Microsporidia are ubiquitous, obligate intracellular eukaryotes that cause chronic diarrhea and disseminated diseases in humans, especially in immunocompromised individuals. Macrophages, cellular components of the innate immune system, are believed to be the source of dissemination of this pathogen throughout the body. Little is known about the innate immune response to microsporidia. Macrophages are a source of interleukin (IL)-12 and IL-23 and play an essential role in the link between innate and adaptive immunity. The focus of this thesis is the investigation of the p38 mitogen-activated protein kinase (MAPK) signaling mechanisms and IL-12 and IL-23 production regulated by Toll-like receptor (TLR) 2 and TLR4 engagement with pathogenic and nonpathogenic species of microsporidia. IL-12 and IL-23 production by primary human macrophage were induced in response to challenge with avirulent but not virulent species from 12 to 24 hour time points. Using western blot, we found that activated p38á MAPK is continuous from three to 24 hours post infection of human macrophages with avirulent species. Activation of p38á MAPK is transient when infected with pathogenic species as we only detected phosphorylation at three hours and six hours post infection. These data suggest that activation of p38 MAPK may be necessary for the proper innate immune response to microsporidia to control infection. Using small interfering RNA, p38á, ã, and ä MAPK were knocked down in primary human macrophages and resulted in a decrease in IL-12/IL-23 p40 production when infected with nonpathogenic species. Thus, additional isoforms of p38 MAPK may regulate the production of IL-12 and/or IL-23 which is a novel finding to the field of microsporidian research and immunology as a whole. MAPK phosphatases (MKP) 1 and/or MKP5 may be negative regulators of this IL-12 and/or IL-23 response. Increased expression of MKP5, but not MKP1, was observed in MDMs challenged with pathogenic species for six hours. The deactivation of p38 MAPK by MKPs may result in the diminished levels of IL-12 and IL-23 observed in virulent infections and thus leading to host susceptibility to microsporidian infection.

<p> Differential gene expression in response to internal and external stimuli is studied in detail to understand the intricate mechanisms underlying response to various environmental stressors in microorganisms. MarR family transcriptional regulators have been studied for their involvement in such mechanisms. This work elucidates the mechanism of urate-induced attenuation of DNA binding of HucR, a MarR homolog, and extends this mechanism to describe a novel subfamily of MarR homologs responsive to urate, proposing a physiological relevance of utilizing urate as a signaling molecule.</p> <p> HucR (hypothetical urate regulator) binds to the shared promoter region between uricase and <em>hucR</em> genes. It has high specificity for urate in attenuation of DNA binding. The ligand-binding site in HucR was identified using molecular-dynamics guided mutational analysis, leading to a proposed mechanism for the attenuation of DNA binding upon interaction of urate. According to this model, urate is anchored in the binding pocket by W20 and R80 while a charge-repulsion displaces D73, which propagates the conformational change to the DNA recognition helix. </p> <p> A possible extension of this mechanism to other MarR homologs was examined through homology search where a number of MarR homologs were identified as conserving the residues involved in urate binding. Further, they show high sequence identity in helix-3, which includes the conserved aspartic acid residue and in the DNA recognition helix, a sequence conservation that correlates to the conservation of bases in their proposed 18 bp consensus dyadic-binding site. To further investigate this phenomenon, <em>Agrobacterium tumefaciens</em>-encoded PecS, which conserves these residues, was studied in detail. PecS binds to the shared promoter region between the genes <em>pecS </em>and<em> pecM </em>while urate attenuates DNA binding <em>in vitro </em>and elevates the transcript levels<em> in vivo.</em> This study thus identifies a novel subfamily of MarR family transcription factors that bind urate and proposes a novel signalling function of urate, wherein invading bacteria utilize urate produced by the host to promote successful host colonization. </p>

Scheffersomyces stipitis (=Pichia stipitis) is a xylose-fermenting and assimilating yeast (Ascomycota: Saccharomycotina) consistently isolated from the gut of Odontotaenius disjunctus (Coleoptera: Passalidae). This filamentous yeast is often attached to the wall of the posterior hindgut of the beetle by a holdfast. Gallery walls of white-rotted wood inhabited by the beetle are lined with macerated wood and frass that includes the yeast and other microbes. Previous studies have mentioned the relationship between the passalid and this yeast; however, passalid beetles also harbor a community of prokaryotes in their guts that have not been characterized. Experiments using culturing and cloning techniques and whole gut isolations have been performed in this work to characterize gut microbes. Introduction of yeast-free diets as well as diets lacking hemicellulose backbone components xylose and xylan served to test the hypothesis that S. stipitis is associated with the beetle host due to continuous environmental transmission. Treatments with antibiotics were attempted to selectively remove the yeast that is attached to the gut. Because attempts to cure the beetle of the yeast failed, introduction of foreign yeasts was performed to determine if the hindgut is saturated with microbes and to determine if the native yeast can be removed or replaced. Microsatellite and species-specific probes were used to confirm that the identity of the yeasts recovered were the same as those introduced. Results showed six taxa of three major groups, γ-Proteobacteria, β-Proteobacteria, and Firmicutes, were represented in easily isolated, culturable prokaryotes of passalid beetle guts. Diet manipulation without removing the native yeast suggested that continued recruitment from the environment might not be necessary for adults for up to 11 days. All treatments with antibiotics failed to selectively remove the yeast from the beetle surface and gut. Yeasts introduced in feeding experiments showed that non-native yeasts could be maintained for up to ten days after feeding, evidenced by microsatellite primers; however, the native yeast was never displaced. Production of a holdfast by the non-native yeasts was not observed when gut tissues were examined with light microscopy. Additionally, killer factors of the native and foreign yeasts were not discovered.

Acetyl-CoA carboxylase is the first and committed step of de novo fatty acid synthesis in all organisms. In Escherichia coli, the enzyme is expressed as separate proteins for the three functional components: a biotin carboxylase, a biotin carboxyl carrier protein, and a carboxyltransferase. The carboxyltransferase enzyme has an α2β2 heterotetrameric quaternary arrangement. The crystal structure of the β subunit revealed a zinc-binding domain, a feature common among nucleic acid-binding proteins. Carboxyltransferase preferentially binds mRNA coding for its two subunits over other nucleic acids, suggesting a means by which the enzyme can regulate its own expression. In the first study, the role played by the zinc-binding motif in carboxyltransferase is revealed through site-directed mutagenesis of the four coordinating cysteinyl residues. Results indicate that the zinc-binding domain is involved in both enzymatic activity of the enzyme as well as mediating binding of the enzyme to its own subunit mRNA. In this utility, the zinc-binding domain as a structural feature physically links the two functional aspects of the enzyme, possibly as a means to evolutionally conserve the capacity to regulate its own translation. In the second study, the individual interactions of carboxyltransferase with substrate and carboxyltransferase with mRNA are representated by mathematical modeling in an effort to validate these interactions function as a single system in regulating the activity and expression of carboxyltransferase in response to the metabolic state of the cell. Comparison of experimental and simulation results validate the model while also suggesting a more complex mechanism of carboxyltransferase translational regulation not captured by the current model.