Modern crocodylian systematics has been dominated by investigations of higher-level relationships aimed at resolving the disparity between morphological and molecular data, especially regarding the true gharial (Gavialis). Consequently, no studies to date have provided adequate resolution of the interspecific relationships within the most broadly distributed, ecologically diverse, and species-rich crocodylian genus, Crocodylus. In this study, Bayesian and ML partitioned phylogenetic analyses were performed on a DNA sequence dataset of 7,282 base pairs representing four mitochondrial regions, nine nuclear loci, and all 23 crocodylian species. The analyses were performed on a suite of partitioning strategies to investigate the modeling effects of partition choice in phylogenetic analyses. Bayesian lognormal relaxed-clock dating analyses also were performed on the dataset, calibrated from the rich crocodylian fossil record. A robust interspecific phylogeny of Crocodylus is reconstructed, and subsequently used in ML and Bayesian ancestral character-state reconstructions to test hypotheses about the biogeographic history and evolutionary ecology of the genus. The results demonstrate that the genus originated from an ancestor in the tropics of the Late Miocene Indo-Pacific, and rapidly radiated and dispersed around the globe during a period marked by mass extinctions of fellow crocodylians. The results also prove paraphyly of Crocodylus, and reveal more diversity within the genus than recognized by current taxonomy. This study also establishes a baseline for assessing the utility of various model selection criteria for objectively selecting the optimal partitioning strategy within ML and Bayesian frameworks. The results indicate that gene identity is a poor method of partition choice. Furthermore, the results of the ancestral character-state reconstructions suggest ML and Bayesian methods produce more realistic and reliable results than parsimony.

Because of a dearth of modern quantitative studies of the ecology and evolution of birds, our understanding of the biogeography of the Bornean avifauna is rudimentary. Although paleogeographic evidence provides a well known mechanism for speciation in the lowlands of Borneo (i.e., repeated colonization and isolation of populations on Sunda islands caused by sea level changes), the process of speciation in montane areas is less easily explained. Knowledge about the processes responsible for montane speciation in Borneo is desirable because mountains clearly have played a key role in the evolution of the islands avifauna, as evidenced by the relatively large number of montane endemics (25 of Borneos 39 endemic bird species are montane). To provide insight into the dynamics of montane speciation in Borneo, I examined the phylogeographic relationships of selected populations of four species across five sites in the mountains of Sabah, Malaysian Borneo. To determine the connectivity of bird populations among these sites, I compared DNA sequences of the second and third subunits of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene of four species that have populations at all the sites: Streaky-breasted Spiderhunter (Arachnothera affinis), Temmincks Babbler (Pellorneum pyrrogenys), Grey-throated Babbler (Stachyris nigriceps), and Ochraceous Bulbul (Alophoixus ochraceus). Divergence among populations at the five sites was very low, but interesting genetic patterns were still evident. The spiderhunter, which is distributed in both lowland and highland areas, showed no phylogenetic structure among its populations, as would be expected of a vagile species that is not restricted montane areas. For P. pyrrogenys and S. nigriceps, the most geographically distant population was phylogenetically distinct from all others. This isolation-by-distance pattern is reasonable for these small babblers, which are restricted to high elevations and assumed to be poor dispersers. The bulbul displayed the most interesting pattern. Its population in western Sabah near the Sarawak border was distinct from all others. Because this bulbul is presumed to be a good disperser and thus capable of moving among all the localities, this result suggests that the Lumaku birds represents a group with closer genetic ties to Sarawakian mountain populations than to Sabahan populations.

Arthropods may enter diapause to escape environmental insult. Diapause is an endogenously controlled dormant state defined by developmental arrest and species-specific physiological changes (e.g., metabolic depression and upregulation of compounds that protect cell structure and function). Although physiological changes have been documented for a number of species in diapause, biochemical and molecular regulation of diapause remains largely unexplained. Aerobic metabolism in diapause, Artemia franciscana, embryos is reduced up to 92 % compared with post-diapause embryos. Differences in isolated mitochondria are insufficient to account for respiratory depression because mitochondria in diapause embryos are structurally similar to mitochondria in post-diapause embryos. Respiratory control ratios and P:O flux ratios of mitochondria from diapause embryos are equal to or higher than those of mitochondria from post-diapause embryos. State 3 and state 4 respiration rates on pyruvate are equivalent in the two stages, and mitochondria isolated from diapause embryos show a moderate, 15-27 % reduction with succinate. Cytochrome c oxidase activity is 53 % lower in diapause embryos, but the minimal impact on mitochondrial respiration appears to be due to the 31 % excess of COX capacity in these embryos. Allonemobius socius embryos enter diapause 3-4 d post-oviposition as indicated by their morphology and DNA embryo-1. There is not an acute downregulation of metabolism during diapause in this species. Diapause embryos consume O2 at the same rate as morphologically similar non-diapause embryos. Diapause and non-diapause embryos exhibit unusually high [AMP]/[ATP] and low [ATP]/[ADP] during early embryogenesis, suggesting that these embryos may be hypoxic early in development. However, superfusing 3 d embryos with O2 enriched air only partially relieves the hypoxic state, which indicates the unusual energy status is an ontogenetic feature not fully explained by oxygen limitation. Subtractive hybridization and qPCR identified 6 genes predicted to regulate diapause entry in A. socius. Reptin, TFDp2, CYP450, AKR are significantly upregulated in pre-diapause embryos, and ACLY and Capthesin B-like protease are downregulated compared to non-diapause embryos. The need for genes upregulated in pre-diapause embryos appears to be transient as these genes are substantially downregulated 10 d after diapause entry. Taken together, these studies provide an integrative examination of mechanisms underlying diapause entry in arthropods.

In the vertebrate retina, multiple cell types express g protein-coupled receptors linked to phospholipase C. The signaling pathway engendered by activation of this enzyme can involve Ca²⁺-permeable transient receptor potential (TRP) channels. To begin to understand the role of these channels in the retina, we undertake an immunocytochemical localization of two TRPC channel subunits, TRPC1 and TRPC4. TRPC1 expression was observed in amacrine cells and their process in the chicken retina. TRPC4 expression was much more widespread with some degree of labeling found in all layers of the retina, and was shown to be expressed in Müller glial cells. Thus, the distributions of these two subunits indicate that different retinal cell types express TRPC channels containing different subunits. Recently, several sphingolipids have been demonstrated to play key roles in Ca²⁺ mobilization in neurons. Sphingosine-1-phosphate is a sphingolipid metabolite that has been shown to activate a class of g protein-coupled receptors (S1PRs) in other cell types. In the present study, we examine the signaling properties of S1P in retinal amacrine cells. S1P produced a noisy, inward cation current in amacrine cells that occurred through activation of S1P1R and S1P3R. The S1P-induced current was PLC-sensitive and was eliminated with La³⁺ and Gd²⁺, suggesting activation of TRPCs. S1P also elicited cytosolic Ca²⁺ elevations. The S1P-induced Ca²⁺ increase was mediated by S1P1R and S1P3R and was a result of both release of Ca²⁺ from internal stores and Ca²⁺ influx. Single-cell PCR amplification of TRPC channel subunits 1, 4, and 5 confirmed expression of these subunits in amacrine cells, suggesting that S1P is capable of activating TRPC-mediated Ca²⁺ entry in retinal amacrine cells through a novel lipid signaling pathway.

The Atacama Desert in Chile extends from latitudes 17º S to 28º S between longitudes 69º W and 71º W. It has been reported that surface soils in the hyper-arid region contain low numbers of culturable heterotrophic bacteria. These soils are considered Mars-like and offer an ideal setting to investigate the application of life detection systems. Thirty-three surface sites were sampled to examine further the extent of the hyper-arid region. We also excavated four soil pits, 40 cm to 90 cm in depth, to explore subsurface microbial communities. One pit was dug in a southern region of the desert compared to the location of the pits in the hyper-arid region. Samples were examined using culture-dependent techniques, including serial dilution plating methods on five media for the cultivation of heterotrophic bacteria. Using 16S rRNA gene sequence comparisons, 1,260 organisms have been recovered and identified. Fifty-four percent of the samples obtained from within the hyper-arid region show numbers of culturable bacteria above the detection limit, yet there is evidence of microbial patchiness in surface and subsurface soils. In some samples, no bacterial colonies were retrieved; in the majority, less than ten were recovered. One pit showed an increase in CFUs/g at 40 cm then dropped back to levels near and below the detection limit. A second pit showed an increase at 10 cm with numbers near and below the detection limit at further depths. The southern soil pit had CFUs/g up to four orders of magnitude greater than those in the hyper-arid region. Direct count methods employing DAPI epifluorescence microscopy were applied to samples, but proved suitable only for a sample having the highest CFUs/g of soil (7.4 x 105) due to the determined detection limit for the technique utilized on the minimal-life containing soils. A chemical composition analysis was performed on all soil samples and showed that elevated ion concentrations may correlate with low numbers of culturable bacteria. The data obtained for the desert samples point to the importance of developing surface and subsurface sampling protocols for future missions to Mars searching for evidence of past or present life.

LINE-1 (Long Interspersed Element-1/L1) and Alu are two active retrotransposon families in the human genome that have the potential to create genomic instability either during the insertion of new elements or through ectopic recombination. However, recent in vitro analyses have demonstrated that these elements also repair DNA double-strand breaks, hence contributing to the maintenance of genomic integrity. As such, the comprehensive role of mobile elements in either creating or mitigating instability in primate genomes remains unclear. The recent sequencing of the chimpanzee and rhesus macaque genomes uniquely facilitates the accurate resolution of this question, as three-way computational alignment of the human genome with two other hominoid genomes allows human lineage-specific changes (i.e., those younger than 5-6 million years) to be accurately dissected out. Here, using a combined computational and experimental approach, we have attempted to provide an unbiased picture of the contribution of the Alu and L1 families to human genomic stability. In the first analysis described herein, we assessed levels of genomic deletion associated with L1 retrotransposition and reported 50 deletions resulting in the loss of ~18 kb of human genomic sequence and ~15 kb of chimpanzee genomic sequence. We developed models to explain the observed bimodality of the deletion size distribution, and showed that overall, in vivo deletions are smaller than those observed in cell culture analyses. Next, we quantified Alu recombination-mediated deletion in the human genome subsequent to the human-chimpanzee divergence and described 492 deletions (totaling ~400 kb of human genomic sequence) attributable to this process. Interestingly, the majority of these deletions are located within known or predicted genes, opening the possibility that a portion of the phenotypic differences between humans and chimpanzees may be attributed to this mechanism. In the third analysis, we reported the in vivo existence of an endonuclease-independent insertion pathway for L1 elements and characterized twenty-one loci where L1 elements appear to have bridged genomic lesions. We show that these insertions are structurally distinguishable from classical L1 elements and suggest that this pathway may escape the purifying selection thought to be acting on endonuclease-dependent L1 loci in the genome.

Amacrine cells mediate complex lateral signaling in the inner plexiform layer of the vertebrate retina. Glutamate is the primary excitatory neurotransmitter that shapes the retinal circuitry by activating ionotropic glutamate receptors (iGluRs)and metabotropic glutamate receptors (mGluRs). One step in understanding the signaling in the inner retina is to understand the role and expression patterns of these glutamate receptors. The expression of iGluRs in amacrine cells is well documented in the literature. This study addresses the localization of Group I mGluRs in the retina. Pre-embedding immunocytochemistry combined with electron microscopy was used to study the expression of Group I mGluRs in the chicken retina. Results indicate that Group I mGluRs are expressed in the synaptic sites of the outer plexiform (OPL) and inner plexiform (IPL) layers and specifically at amacrine cell synapses. In order to understand the intricacies of amacrine cell signaling mechanisms it is important to dissect out the signaling mediated by Ca2+ in these cells. In this dissertation, Ca2+ dependent local signaling in amacrine cells, more specifically the Ca2+ transport mechanisms involving mitochondria and ER, are explored. Calcium imaging experiments were performed on cultured chick amacrine cells. The results presented here suggest that there is physiological interplay between mitochondria and ER. In addition to this, it is demonstrated that ryanodine receptors are specifically involved in Ca2+ transport, probably via calcium-induced calcium release (CICR). Blocking the mitochondrial uniporter with FCCP or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP 37157 revealed that mitochondria also influence the duration of glutamate-dependent cytosolic Ca2+ elevations. The effects of FCCP and CGP were detectable only in amacrine cell bodies and also in regions of processes next to mitochondria, thus revealing a spatial limit to the effects of mitochondria on cytosolic Ca2+. Together, these results contribute to our understanding of how local signaling is achieved in the vertebrate retina.

Understanding the genetic basis of speciation is of fundamental importance to evolutionary biology and hybrid zones offer unique natural laboratories in which to investigate the ecological and evolutionary processes important in creating and maintaining biological diversity. By comparing introgression patterns of different loci, researchers can begin to identify genetic regions that contribute to reproductive isolation between hybridizing taxa. In taxa, like birds, with heterogametic females, Haldanes rule predicts that mtDNA and z-linked loci will introgress less than autosomal loci. I tested this prediction using the hybrid zone between Passerina cyanea (Indigo Bunting) and Passerina amoena (Lazuli Bunting), two species that hybridize where their breeding ranges overlap in the Great Plains of North America. Although a recent mtDNA-based phylogenetic hypothesis of the genus Passerina suggested these two species are not sister taxa, I found, using DNA sequence data from ten nuclear loci, that they are more closely related to each other than either is to P. caerulea (the mtDNA sister to P. ameona). Both cline-based and coalescent-based analyses of mtDNA (two genes), z-linked (two loci), and autosomal (four) loci indicated a reduction in introgression of both mtDNA and z-linked loci, relative to autosomal loci. These patterns, consistent with the predictions of Haldanes rule, suggested the sex-chromosomes may play a large role in reproductive isolation between P. cyanea and P. amoena. Using DNA sequence data from an additional eight z-linked loci, I explored patterns of differential introgression of ten z-linked loci. Introgression of one z-linked locus, VLDLR9, was significantly less than introgression of the other nine loci, pointing to a candidate region for reproductive isolation between P. cyanea and P. amoena. Interestingly, VLDLR9 is an intron of the very-low density lipoprotein receptor, which plays an active role in egg laying. Additionally, in a particular strain of chickens, a point mutation in the VLDLR gene produces females that do not lay eggs. While my data are insufficient to adequately address the role VLDLR may play in maintaining reproductive isolation between P. cyanea and P. amoena, the hypothesis that female buntings may have trouble laying eggs warrants further investigation.

There is mounting evidence of impacts of single base substitutions in both somatic and inherited genetic diseases. However, the occurrence of single base mutations in the germline and gametes has previously only been by conjecture and assumption. There is a need for understanding the risk of offspring inheriting small intragenic mutations, the frequencies at which these mutations are present in germ cells, and the impact that these inherited mutations may have son the predisposition to somatic disease during ones lifetime. The present study attempts to establish baselines of mutational frequencies in two well characterized genes in human tissues, and demonstrate the prevalence of mutations in two genes, Kristen-ras (KRAS) and TP53 in direct analysis of semen specimens. These two genes play important roles in human disease. Eleven normal human male volunteers semen specimens were tested for single base substitutions (SBS). A detection sensitivity of one mutant cell in 106 wild type cells was achieved with the aid of an ultra-sensitive detection method, which utilizes the combined power of the polymerase chain reaction, restriction endonuclease, and ligase chain reaction techniques. The first base of the TP53 gene codon 248, 282, 273 and the second base of codon 273 and the first base of the Kristen-Ras (KRAS) gene codon 12 were studied. The observed mutation prevalence was significantly higher in the first base of the codon 248 of the TP53 gene compared to the first base of codon 273 or the first base of codon 282, both (both P< 0.006). Similarly, there was a significant difference between the prevalence of the first base of codon 12 of the KRAS gene and the first base of codon 273, or the first base of codon 282 (both P< 0.04). Interestingly, the SBS mutation prevalence was nearly the same in the first base of codon 12 KRAS gene and the first base in codon 248 of the TP53 gene (P< 0.006). The spectra of base substitution mutations observed in this study may provide clues to the mutational mechanisms present in human sperm, and may lead to the improved risk assessment for genetic counseling. These data represent the first reports of mutational frequencies in the TP53 and KRAS genes in normal human semen specimens, and demonstrate that single base mutational frequencies for different genes can be obtained directly from semen samples.

The study of pedestrian locomotion in crustaceans has largely focused on forward walking macrurans, or sideway walking brachyurans. The spider crab, Libinia emarginata is a brachyuran that, unlike its close relatives, preferentially walks forward. The phylogenetic position, behavioral preference, and amenability to experimental techniques make spider crabs an attractive model for comparative studies of crustacean locomotion. This dissertation looks at the neuroethology of forward walking in L. emarginata. I described the skeletal, muscular, and neural anatomy of the walking machinery of L. emarginata and found adaptations at each level that reflect its walking preference. The ranges of motion of leg joints aiding in forward locomotion were larger for spider crabs than for sideway walking crabs. The leg segments housing the musculature moving these joints were also larger. The proximal leg musculature consists of multiple muscle heads that can be activated independently during locomotion. The motor neurons innervating this musculature exhibited features of distantly related species that walk forward. Unlike many brachyurans, spider crabs use all ten legs during walking. Kinematic characterization of forward walking in L. emarginata showed that anterior and posterior limbs perform different functions during walking. Cross-correlation analysis among the leg joints of spider crabs revealed that distant joints have stronger coupling than adjacent ones. Neuroethology studies of pedestrian locomotion use multiple approaches. In order to understand how adaptive behavior is produced, it is necessary to study how the neural, muscular, and skeletal systems of an organism interact during its performance.