In this dissertation I describe a number of patterns and interesting aspects associated with the evolution of snake mitochondrial genomes (mtDNA). I also attempt to resolve the phylogeny of squamates, focusing on the relationship between the snakes and lizards. The results of this study indicate that snakes and worm lizards (amphisbaenians) appear to share an exclusive common ancestor, and snakes appear to have undergone strong selective pressure that shaped snake mtDNAs. Snake mtDNAs have several unique features, including a compact size, duplicated control regions, and an elevated evolutionary rate. Based on the correlation resulting from the asymmetric replication of mtDNA, the usage of control regions was inferred to be species specific. In snake mtDNAs, the magnitude of the rate acceleration varied considerably among genes and over time, and it appears that these changes at the nucleotide and protein level co-occurred with snake mtDNAs incurring a reduction in size and a duplication of the control region. In snake mtDNA, many unique amino acid substitutions were identified in all protein-coding genes. In the Cytochrome C Oxidase subunit I (COX1) protein, one of three proposed proton transfer channels was enhanced by several unique substitutions. Additionally, strong positive selection was detected on the COX1 gene of alethinophidian snakes. These may be causally related to the energetic demands imposed by the radical energy requirement in the early digestion period of alethinophidian snakes. Observations of change in COX1 gene suggest that, due to the relaxation of selective pressure or a population bottleneck, numerous deleterious substitutions accumulated on snake ancestral lineages. Then the impaired functions were recovered, or even enhanced by adaptation. During this period, the evolutionary rate of snakes was accelerated as well. In this research, the phylogenetic placement of snakes was inferred using the complete mtDNA of 65 vertebrates by maximum likelihood (ML) and partitioned-Bayesian inference. Snakes were placed as the sister taxon to worm lizards, and this branching pattern is strongly supported by Bayesian inference-derived posterior probability. The jackknife simulation also supports the sister relationship between snakes and worm lizards, cumulatively rejecting the hypothesis of marine origins of snakes.

SVA is a composite repetitive element named after its main components, SINE, VNTR and Alu. There are ~3000 SVA elements in the human genome. A genomic distribution analysis indicates that SVA elements are enriched in G+C-rich regions but have no preferences for inter- or intra-genic regions. A phylogenetic analysis of these elements resulted in the recovery of six subfamilies that were named SVA_A to SVA_F. The composition, age and genomic distribution of the different subfamilies have been examined. Subfamily age estimates indicate that the expansion of four SVA subfamilies (SVA_A, SVA_B, SVA_C and SVA_D) began before the divergence of human, chimpanzee and gorilla, while subfamilies SVA_E and SVA_F are restricted to the human lineage. Furthermore, I examined the amplification dynamics of SVA elements throughout the primate order and traced their origin back to the beginnings of hominid primate evolution, approximately 18 to 25 million years ago, which makes SVA elements the youngest family of retrotransposons in the primate order. Gene duplication is one of the most important mechanisms for creating new genes and generating genomic novelty. Retrotransposon-mediated sequence transduction (i.e. the process by which a retrotransposon carries flanking sequence during its own mobilization) has been proposed as a gene duplication mechanism. SVA elements are capable transducing 3 flanking sequence during retrotransposition. I examined all the full-length SVA elements in the human genome to assess the frequency and impact of SVA-mediated 3 sequence transduction. The results showed that ~53 kb of genomic sequence has been duplicated by 143 different SVA-mediated transduction events. In particular, I identified one group of SVA elements that has duplicated the entire AMAC gene three times in the human genome via SVA-mediated transduction events, which happened before the divergence of humans and African great apes. In addition to the original AMAC gene, the three transduced AMAC copies contain intact open reading frames (ORFs) in the human genome and at least two are actively transcribed in different human tissues. Thus, duplication of entire genes and creation of new gene families via retrotransposon-mediated sequence transduction represent an important mechanism by which mobile elements impact their host genomes.

Vibrio vulnificus is a Gram-negative organism that is found in estuarine environments. The bacterium is capable of causing gastroenteritis, wound infections, and septicemia, often resulting in death for susceptible individuals. Monoclonal antibodies (MAbs) to five lipopolysaccharide (LPS) O-serovars have previously been used as a serotyping system in epidemiological studies. Here is the first report of two additional MAbs capable of recognizing two new LPS serovars (O6 and O7), further increasing the number of V. vulnificus that can be typed. Analysis of V. vulnificus isolates from the United States, Europe, and Asia demonstrated that the O1/O5 serovar is dominate in clinical isolates, serovars O3 and O4 are dominate in the environmental isolates of Europe, and that most environmental isolates from United States are nontypeable. Additionally, MAb, RS1, was chosen for further analysis by having DNA coding for its light and heavy chains cloned into a phage-display system. The published primers, designed to be used with the pComb3X display system, produced primer dimers and were unsuccessful in generating the single chain variable fragment (scFv) to be cloned into the phage vector. Modification of the existing primers inhibited the formation of primer dimers and allowed for the successful generation of the scFv. The resulting phage-displayed antibody, PJ1, displayed similar binding characteristics as RS1 showing the usefulness of phage-display as an alternative method of maintaining pre-existing MAbs. Lastly, it was proposed that as V. vulnificus undergoes capsular switching it is possible that LPS switching occurs as well. Analysis of the data suggests that capsule and LPS switching are not directly related. Interestingly, LPS switching seems to occur in a unidirectional manner on two separate branches that converge on serovar O5 suggesting that O5 may be a group of sugars that are used as a building block for other serovars. This allowed for the formation of a speculative LPS model. The stability of serovars O6 and O7 in the LPS switching assay make them difficult to place in relationship to serovar O5 and further analysis will be needed to more accurately place them on the LPS model.

Central America has two howling monkey species: the widespread mantled howling monkey (Alouatta palliata) and the endemic and endangered black howling monkey (A. pigra) limited to southeastern Mexico, northern Guatemala and Belize. Studies that verify the distribution of these species are needed, especially in their contact zones where sympatry is reported. Their evolutionary history remains controversial. My study examines their distribution at a local scale in a potential contact zone in eastern Guatemala through direct observations and interviews and at a regional scale across the entire isthmus using data from museum specimen localities, study sites, historic records and field surveys. Using GIS I analyzed the distributions against geographic and ecological features to infer current barriers between both species and explore the possibility of their role in the initial speciation. I found no evidence for current sympatry in eastern Guatemala; instead parapatry is maintained by a riverine barrier and by ecological adaptation, as only A. pigra occurs in the cold montane habitats further inland. My study reveals broader elevational and vegetational tolerances by A. pigra than previously reported. My results suggest differences in elevation and cold tolerances by the two species which I consider an important ecological barrier separating them at present. I identified the highland massif of northern Central America and their associated coniferous and subalpine vegetation as a geographic barrier. In contrast to other studies, I propose that both species ranges are not adjacent throughout, but separated by these mountains and only coming into contact in a broad sympatry zone in the lowlands north of the highland massif in Mexico and in the narrow parapatry zone in Guatemala. I present an alternative biogeographic hypothesis that proposes an initial split by the northern Central American highland massif during cold periods that resulted in the isolation of the A. pigra lineage in the Yucatan peninsula and in the further divergence in cold tolerance.

This dissertation explores effects of windstorm and fire disturbances on the clonal and reproductive biology of cane (Arundinaria gigantea Muhl., Poaceae). In this collection of studies, multiple disturbances interact in complex ways, and some interactions appear only after a substantial lag. One implication of this research is that multiple, interacting disturbances might strongly influence the boundary between monodominant and species-rich plant communities. Cane is the only bamboo native to the United States, and once covered vast areas of bottomlands in the southeastern U.S. in monodominant stands called canebrakes. The study took place on the Buckhorn Wildlife Management Area in Tensas Parish of northeastern Louisiana, in the Mississippi alluvial valley. Here in 2000 a tornado blew down a large swath of bottomland hardwood forest. The four-year experimental study focused on the effects of windstorm and prescribed fire on cane ramet dynamics, reproduction and regeneration from seed. The effect on cane of a large windstorm-generated gap is accelerated new ramet production and increased ramet density. Cane spreads continually, albeit irregularly. This suggests that cane stands might shift location over time as small forest gaps open and close. The effects of fire on cane are complex, and some may be lasting. In the large wind-generated forest gap, populations of cane ramets grow faster for having been burned. Forest-grown ramet populations decline in the year of fires, but growth rates rebound the next year. Unburned populations decline during the studys final year, but previously burned populations do not, suggesting that fire one year might impart resistance to shocks one or more years later. The dissertation proposes how a sequence of windstorm and fire disturbances might promote natural canebrake formation. The counterpoint is that without periodic disturbances, cane ramet populations decline. The dissertation explores cane reproductive ecology and regeneration, and discusses three critical stages: seed production, germination and seedling establishment. Cane seeds and seedlings appear to germinate and survive most frequently on sites receiving partial sunlight with a layer of leaf litter. The final chapter describes how these experimental results inform cane restoration, and suggests three pathways by which cane restoration might be achieved.

Seed dispersal and seedling establishment the two stages in seedling recruitment set the spatiotemporal distribution of new individuals in plant communities. Diversity often increases at the seed to seedling transition, making it critical for species coexistence. Debate continues regarding the effects of each stage on the community structure of diverse forests. Neutral theories postulate a strong role of dispersal, whereas niche-differentiation theories suggest that environmental conditions may be more important. This dissertation tested the effects of dispersal, competition and predation on the structure of the seedling layer in a pristine Amazonian rainforest. Seed-addition experiments broadly tested the relative importance of dispersal and environmental conditions on seedling community structure. Dispersal treatments explained more variance in community structure than did environmental conditions. This was the first variance- partitioning study to show that dispersal affects not only seedling density, but also diversity and species composition. Two more narrowly focused studies tested the intensity of competition among seedlings, and examined the effects of various mammalian predators on seedling recruitment. Evidence for inter-seedling competition was weak: individual growth and survival rates were generally unrelated to stem density, and seedlings zones of influence rarely overlapped substantially. As predators, small and medium-sized mammals reduced seedling density, whereas large mammals had no detectable effects. Furthermore, small mammals generated a rare-species advantage, the fundamental element of frequency dependence. Integrating the three studies, we suggest that dispersal is more important for seedling community structure than are environmental conditions. Given the low density of seedlings in xii Neotropical forests, we infer that competition among tree seedlings is largely irrelevant to their recruitment. Seed predators operated in a distinctly non-neutral manner, preferentially removing seeds of common and large-seeded species. Despite the powerful effects of predation, dispersal explained more variance in seedling recruitment than did all aspects of environmental variation (including predation). Taken together, the results of these three experiments support a view that, at least for young plants, and at small scales, dispersal may more strongly influence the species composition of tropical trees than environmental conditions, consistent with predictions from neutral models.

Obesity and Type 2 diabetes are associated with high fat diet (HFD), reduced mitochondrial mass and function and insulin resistance as characterized by glucose disposal and relative to body fatness. We hypothesized that (a) HFD affects expression of genes involved in mitochondrial biogenesis and function, (b) carbohydrate metabolism and storage is under transcriptional control and (c) both overall fatness and characteristics of adipose tissue influence the interplay between free fatty acids (FFAs) and insulin-stimulated glucose disposal. To test hypotheses a and b, we fed 10 insulin-sensitive males an isoenergetic HFD for 3 days with muscle biopsies before and after intervention. Oligonucleotide microarrays revealed 370 genes differentially regulated in response to HFD (Bonferonni adjusted p < 0.001). Expression of six genes involved in oxidative phosphorylation decreased. PGC1α and PGC1β mRNAs decreased by ~22%. Seven genes in the carbohydrate metabolism pathway changed in response to HFD, and three genes confirmed by qRT-PCR: PFKFB3, PDK4 and GYS1. In a separate experiment, C57Bl/6J mice were fed HFD for three weeks and the same OXPHOS and PGC1 mRNAs decreased by ~90%, Cytochrome C and PGC1α protein by ~40%, while the same glucose metabolism genes changed by ~70%. These results suggest a mechanism whereby HFD downregulates genes necessary for oxidative phosphorylation and mitochondrial biogenesis, as well as glucose utilization and storage. These changes mimic those observed in diabetes and insulin resistance. To test hypothesis c, we measured changes in respiratory quotient (ΔRQ; metabolic flexibility) before and during euglycemic-hyperinsulinemic clamps in healthy young males. Anthropometric, laboratory measurements, fat biopsies and fat cell size (FCS) were measured after overnight fast. Adipose tissue gene expression (qRT-PCR) was measured. Metabolic inflexibility (lower ΔRQ) was associated with higher body fat, larger FCS and higher insulin-suppressed FFAs. ΔRQ was not related to fasting FFAs, but lower ΔRQ was associated with lower serum adiponectin levels. Higher adipose tissue inflammatory gene expression was associated with higher insulin-suppressed FFAs and lower ΔRQ. These results indicate fatness, adipocyte hypertrophy, blunted insulin suppression of FFAs, decreased adiponectin levels and inflammation, are associated with decreased insulin-stimulated glucose uptake and oxidation, an important component of metabolic inflexibility.

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. The Dps homolog, Dps-1, from the radiation-resistant bacterium Deinococcus radiodurans has an extended N-terminal tail. In the crystal structure of Dps-1, the first ~30 N-terminal residues are invisible and the remaining 25 residues form a loop that harbors a novel metal binding site. The data presented here show that retention of this N-terminal metal site is necessary for formation of the dodecameric protein assembly. Previous studies have suggested that the lysine-rich N-terminus of Dps proteins participates in DNA binding. Accordingly, deletion of the N-terminal tail of Dps-1 obliterates DNA/Dps-1 interaction. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents show that Dps-1 interacts through the DNA major groove. Dodecameric Dps-1 can bind ≥ 22bp DNA duplexes with very high affinity (K_d ~0.4 nM); considering interactions in the DNA major grooves, the requirement for two complete helical turns implies optimal interactions involving two consecutive major grooves. The data further suggests that high-affinity DNA binding depends on occupancy of the N-terminal metal site. Stoichiometric titration of dodecameric Dps-1 with 22 bp DNA revealed the presence of 6 DNA binding sites in each dodecamer. DNA cyclization assays show that dodecameric Dps-1 inhibits DNA bending. Taken together, the mode of DNA interaction by Dps-1 is consistent with the previously proposed layered assembly of protein and DNA that leads to DNA compaction. Using Dps-1-promoter-lacZ fusion constructs, it is shown that Dps-1 expression in D. radiodurans is relatively constant throughout both exponential and stationary phase growth. As E. coli cells expressing Dps-1 feature significant nucleoid condensation, as shown by transmission electron microscopy and nucleoid staining, a role for Dps-1 in chromosomal DNA packaging is suggested. The presence of a novel iron exit channel is most likely responsible for the inability of Dps-1 to protect DNA from hydroxyl radical-mediated DNA degradation. The release of iron from the core upon DNA binding suggests that Dps-1 may be involved in the process of DNA degradation that contributes the first response to DNA damage.

In tropical forests, forest fragmentation that alters species interactions between plants and animals, especially mutualisms, will affect the regeneration of trees. Fragmentation reduced Duckeodendron cestroides seed dispersal quantity and quality. The percent and distance of dispersed seeds were twice as great in continuous forest (31%, 4.9 m) as in fragments (15%, 2.5 m). Differences were even more exaggerated for each tree's furthest dispersed seeds. Distributions of dispersed seeds across distance showed more seeds at all distances in continuous forest than in fragments. Dispersal differences were strongest in years when fruit production was high and resulted in a greater number of first-year seedlings at distances far from the parent tree in continuous forests. Fruit production and post-dispersal seed fate of Duckeodendron were also different between fragments and continuous forest. Fruit production, measured by fruit fall, was reduced in fragments, more dramatically in years when fruit production was high. In continuous forest, seeds protected from mammal seed predators or secondary dispersers had high rates of seedling establishment (55%), but the majority of unprotected seeds were rapidly removed (77%). Seed predation was 17 times greater in continuous forest (53%) than in fragments (3%). More seeds in fragments had delayed germination than in continuous forest, resulting in seedlings half as tall as their continuous forest counterparts. Despite these differences in the processes leading up to seedling establishment, there was no difference between the percent of seedlings that established in fragments or continuous forest. Although fragmentation had dramatic effects on D. cestroides, responses of species interactions to fragmentation can vary. Although the number, percentage, distance, and distributions of dispersed seeds were reduced in fragments for the large-seeded Duckeodendron, they were not for the small-seeded Bocageopsis multiflora. Seed dispersal of large-seeded species may be more susceptible to fragmentation than small-seeded species because large-seeded species rely on fewer, extinction prone dispersers. Three lines of evidence from other tropical fragmentation research support this hypothesis. A rapid survey failed to detect differences in fruit production between fragments and continuous forest, probably because small sample sizes and high intraspecific variability caused a Type II error.

The native stream fishes of the Hawaiian Islands are uniquely adapted to colonize and thrive in lotic habitats in this extremely isolated archipelago. Chapter 1 of this dissertation introduces the five native fishes of Hawaii's streams and describes the variety of lotic habitats they occupy. All five species have amphidromous life cycles, and, as such, their larvae must migrate to the ocean soon after hatching. Of the five species, Lentipes concolor is able to scale Hawaiis highest waterfalls during the migration back into fresh water, and therefore inhabits the widest variety of stream types. Adult L. concolor are found a few meters from the ocean in small streams that enter the ocean as terminal waterfalls. Lentipes concolor also inhabit inland stream reaches located at high elevations in large streams with low gradient terminal estuaries. This dissertation examined potential spatial and temporal patterns in the migration of newly hatched L. concolor larvae as they passively drifted from instream hatching sites to marine habitats. Results presented in Chapter 2 demonstrated that more larvae successfully completed seaward migration in terminal waterfall streams than did larvae spawned in the upper reaches of larger, higher-order streams. In Chapter 3, a significant diel drift pattern was observed during 24-hour sampling. Although larvae were captured in drift nets during both day and night, a preponderance of larvae during the first three hours following sunset was the most commonly observed trend. Results presented in Chapter 3 also suggest that in terminal-waterfall streams, spawning and therefore downstream movement of larvae occur more commonly during new-moon phases. A lack of distinct lunar periodicity in upland reaches of large streams is hypothesized to possibly be related to the high frequency and intensity of flash floods that may disrupt the sequence of courtship and spawning behavior of adult L. concolor. Chapter 4 discusses ways in which the new information contained in the previous two chapters may change current paradigms regarding the population ecology and conservation of native stream fishes in Hawaii.