Bioavailability and nutrient effects on the trophic transfer of Cadmium (Cd) associated with microalgae to the marine amphipod, Leptocheirus plumulosus, were investigated. Cd assimilation efficiencies (AE) were measured by a pulse-chase technique using a radiotracer. Cd AE in L. plumulosus significantly varied among algal species tested, and was highest (38.8 %) for the benthic diatom Nitzschia punctata, lowest (5.9%) for the planktonic diatom Thalassiosira weissflogii, and intermediate (15.6%) for the planktonic dinoflagellate Isochrysis galbana. Instantaneous egestion rates of Cd displayed a typical biphasic pattern over 96 h of depuration. Depuration in seawater-only of L. plumulosus yielded the highest Cd AE of 35.0%, whereas AEs in which depuration occurred in natural sediment and processed sediment were only 5.3% and 4.3%, respectively. Body size, ranging from 0.5 to 2.0 mm, of L. plumulosus feeding on labeled T. weissflogii did not affect Cd AEs. Nitrate enrichment from 0-180 uM on algae significantly increased Cd AEs by L. plumulosus from 9.4-18.8% for T. weissflogii, from 10.0-27.3% for N. punctata, and from 10.0-16.2% for I. galbana. Physiological turnover (elimination) rate constants of Cd in L. plumulosus ranged from 0.016-0.025 h-1 for the three algal species, and were independent of nitrate addition. Algal fractionation revealed that nitrate enrichment strongly enhanced the fraction of Cd associated with cytoplasm, which probably contributed to the increased Cd AEs by L. plumulosus. Phosphate addition (0 - 7.5 uM) on algae showed that Cd AEs of L. plumulosus were from 26.4-35.8% for T. weissflogii, and from 15.3-18.5% for N. punctata. Phosphate enrichment did not significantly affect trophic transfer of Cd from algae to L. plumulosus. Cd fractionation in cytoplasm showed no obvious correlation with phosphate addition. Overall, there was a significant linear relationship between the Cd AE of L. plumulosus and the distribution of Cd within algal cells. My work suggests that eutrophication by nitrate enrichment, but not phosphate, has the potential to enhance the trophic transfer of metals from pelagic and benthic microalgae to grazers in coastal benthic food webs.

I evaluated hypotheses regarding the nature of habitat specialization by comparing the microhabitat distribution and demography of L. cernua and P. basiramia, two Florida rosemary scrub habitat specialist species, with their habitat generalist congeners, L. deckertii and P. robusta. Specifically, I addressed the following two hypotheses: (1) that habitat specialist species may occur in a narrower range of microhabitat conditions than habitat generalist species, and (2) that demographic parameters of habitat specialist species may be more variable than those of their habitat generalist congeners. For each pair of congeners, I compared the microhabitat distributions, variation in vital rates and population growth rates, and extinction probabilities under different climate regimes to evaluate these hypotheses. Both rosemary scrub specialist species occurred in a narrower range of bare sand microhabitat conditions than their habitat generalist congeners. Rosemary scrub specialists were significantly more likely to occur in sites with high percentage bare sand, whereas microhabitats of generalists were more variable with respect to percentage bare sand. Recruitment and survival rates of both rosemary scrub specialist species were more temporally variable than those of their habitat generalist congeners; however, plant growth rates of rosemary scrub specialist species were less variable than those of their generalist congeners. Rosemary scrub specialist species also exhibited greater temporal variation in population growth rates than their habitat generalist congeners. Both rosemary scrub specialist species had higher probabilities of quasi-extinction than their generalist congeners under every climate modeling scenario. The narrower microhabitat requirements and greater temporal variability of demographic parameters of L. cernua and P. basiramia distinguish them from their habitat generalist congeners. The restriction of P. basiramia and L. cernua to microhabitats with high percentage bare sand may limit their distribution to rosemary scrub habitat. Greater temporal variability in recruitment, survival, and population growth rates in L. cernua and P. basiramia may be associated with specialization on a narrower range of environmental conditions in these rosemary scrub specialist species. Greater temporal variability of demographic parameters in these rosemary scrub specialist species may make them more vulnerable to extinction than could be predicted solely from availability of suitable rosemary scrub habitat.

The limiting factor involved in past assessments of soil bacterial diversity when using culture-independent techniques has often been the lack of sampling and replication. As a result, analyses of community structural shifts across soil environments have lacked statistical power. In this study, 23 16S rRNA gene clone libraries consisting of over 11,000 clones were constructed from soils at the Central Arizona Phoenix Long Term Ecological Research (CAP LTER) site. Subsequent ARDRA fingerprinting and partial 16S rRNA gene sequencing allowed for a more robust investigation of various components that may explain any observed variations in bacterial species composition. The designated land use type of the soils best explained the overall diversity gradient. Based on Simpsons reciprocal index, diversity was found to significantly increase when comparing urbanized and agricultural soils to open desert samples located outside the metropolitan region. Land use type appears to be a powerful indicator of overall diversity due to irrigation methods that differ greatly across land use types. Experiment-wise comparisons of complete CAP LTER clone libraries via the LIBSHUFF method yielded no statistical similarity in sequence libraries, except for two replicate libraries constructed from one urban soil. However, inter-phylum LIBSHUFF analysis of the clones also shows degrees of phylogenetic partitioning between land use categories and that open desert remnant patches located within the city limits more closely resemble those urban soils than the open deserts outside of Phoenix. Examination of constructed 16S rRNA phylogenetic trees that include CAP LTER phylotypes indicate some distinct clustering of sequences appears to be driven by land use type rather than geography, and that most of these groups may be endemic to the region. However, some ubiquitous phylotype groups were discovered and were used as templates for specific PCR primer design, allowing for the detection of ten of these groups in all soil samples analyzed. Overall, this study suggests that anthropogenic factors have altered soil bacterial communities, the biogeography of many species is controlled in some manner by land use type, and that a small subset of taxa is ecologically tolerant despite the heterogeneity of habitats within the site.

The tempo of evolution and the causes of rate variation among lineages are central foci of evolutionary biology. I evaluated two hypothesized sources of variation in molecular evolutionary rate, and I applied a variable molecular clock to estimate the timescale of diversification in three families of Neotropical birds. First, I examined the phylogenetic evidence for molecular punctuated equilibrium, the hypothesis that speciation drives accelerated molecular evolution. Recent findings that rates of DNA evolution and speciation are linked implicate molecular punctuated equilibrium as an important cause of rate variation among lineages. I used phylogenetic simulations to test this reported link, and I found that it was entirely attributable to a methodological artifact. In a review of the topic, I found no unequivocal empirical evidence for molecular punctuated equilibrium and I concluded that its predicted phylogenetic consequences are theoretically implausible. Second, I tested the metabolic rate hypothesis, which holds that mutation rate in mtDNA is correlated with mass-specific metabolic rate. This hypothesis predicts that small-bodied lineages should evolve rapidly. Previous studies verified this prediction, but none utilized adequately large samples of independent contrasts among appropriate taxa. The use of many such contrasts from bird mtDNA sequences conspicuously failed to corroborate the link between metabolic and mtDNA rates. On the contrary, high rates of nonsynonymous substitution were associated with large body mass, implicating population size as a pervasive cause of evolutionary rate variation. Third, I developed molecular phylogenies for puffbirds, jacamars, and motmots to test hypothesized area relationships in the Neotropics. I used penalized likelihood to estimate node times while accommodating significant rate variation under a set of biologically realistic assumptions. Phylogenetic patterns in each family were consistent with expansion following the formation of the Central American Landbridge and subsequent vicariance across the Andes. I applied a calibration based on the final uplift of the Isthmus of Panama, 3.1 Ma. Average estimated rates were close to the commonly cited 2% sequence divergence/Myr. Concordant area relationships were found among co-distributed species complexes; however, the timescale of divergence was variable, suggesting that common dispersal corridors rather than common vicariant events may be driving co-phylogenetic patterns.

The Alu Ya and Yb-lineages are distinct groups of Short INterspersed Elements (SINEs) found exclusively in primates. These lineages are comprised of several distinct subfamilies, which contain elements that are evolutionarily-related because they share common diagnostic mutations. Some of these Alu subfamilies are still actively mobilizing in the human genome. We have recovered 4333 Ya and Yb-lineage elements that reside in the human genome draft sequence and focused our analyses on the 4051 autosomal Alu elements. A total of 2672 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual elements for insertion presence and absence. The polymorphism rate for the insertion presence/absence on the human autosomes was 22% and 20% for the Ya and Yb-lineages, respectively. Less than 0.5% (12/2672) of the loci analyzed by PCR displayed positive results for insertions in the orthologous loci of various non-human primate genomes. DNA sequence analysis of these inserts showed that the non-human primate orthologous loci contained older, pre-existing Y, Sc, Sg, Sq or Sx Alu subfamily elements. These PCR results were the result of various evolutionary processes within the human lineage. This is the largest analysis of human Alu mobile element lineages to date. The size, evolutionary age and variable allele insertion frequencies of the Alu Ya and Yb-lineage subfamilies, makes these elements useful tools for human population genetics, human identification and primate comparative genomics.

Retrotransposons are an active family of mobile elements within primate genomes and the Short INterspersed Element (SINE) Alu is the most abundant member. These non-autonomous elements are responsible for introducing genomic diversity on an intra- and inter- species level that is useful in studies of forensic identity, population genetics, and evolutionary biology. In a computational survey of the human sex chromosomes, 344 recently integrated Alu elements were detected and subjected to empirical testing by polymerase chain reaction to determine presence/absence polymorphism. Sixteen elements were found to be polymorphic on the X chromosome, and only one polymorphic element on the Y chromosome (previously termed YAP, Y chromosome Alu Polymorphism), across four geographically diverse populations. In line with previous research using other types of genetic markers, these results indicate a low Alu-associated diversity level on the human sex chromosomes, presumably due to reduced recombination rates and lower effective population sizes on the sex chromosomes. Alu elements often contribute to genomic instability via insertional and recombinational mutagenesis. Recently, a novel mechanism of retrotransposon-associated genomic instability was discovered, termed retrotransposition-mediated deletion. A computational search within the draft human and chimpanzee genomes found evidence of 33 retrotransposition-mediated deletion events that have eliminated approximately 9,000 nucleotides of genomic DNA. During the course of primate evolution, Alu retrotransposition may have contributed to over 3000 deletion events, eliminating approximately 900,000 bp of DNA in the process. Potential mechanisms for the creation of Alu retrotransposition-mediated deletions include L1 endonuclease-dependent retrotransposition, L1 endonuclease-independent retrotransposition, internal priming on DNA breaks, and promiscuous target primed reverse transcription (pTPRT). Approximately 0.27% of all human disease mutations are attributable to the activity of Long INterspersed Element (LINE) L1, Alu and SVA (SINE-R/VNTR/Alu) retrotransposons within our genomes. Although researchers in the field of human genetics have discovered many mutational mechanisms for retrotransposable elements, including retrotranspositional insertion, recombination, retrotransposition-mediated and gene conversion-mediated deletion, in addition to 3' transduction, their individual contribution to genetic variation within humans is still being resolved.

Long INterspersed Elements (LINEs or L1)and Short INterspersed Elements (SINEs) have played a critical role in shaping the human genomic landscape, making up about 33% of the human genomic mass. In this work, preTa, an L1 subfamily and the Ya-lineage of Alu SINEs were examined to determine their impact on the human genomic architecture and diversity. We analyzed 362 preTa L1 elements and 2318 Ya lineage Alu elements using computational methods and polymerase chain reaction (PCR) assays. PCR analysis on a geographically distinct panel of human populations indicated that 33 (14%) of preTa L1 elements and 313 (22%) of Ya-lineage Alu elements were polymorphic for insertion presence/absence. DNA sequence analysis of the preTa and the Ya-lineage subfamilies indicated an estimated average age of 2.34 and 2.27 million years respectively. This suggests that these elements began to amplify after the human lineage radiated from the non-human primates. None of the mobile elements analyzed were also located in orthologous non-human primate loci, supporting the fact that they are homoplasy free genetic characters. The continued amplification of mobile elements throughout evolutionary time and their variable insertion frequencies in diverse human populations make them robust tools for population genetics and comparative genomics.

This study is an examination of geographic variation and evolutionary history of Stelgidopteryx swallows. These swallows comprise two recognized species: Northern Rough-winged Swallow (S. serripennis) and Southern Rough-winged Swallow (S. ruficollis). A third species, Yucatan Rough-winged Swallow (S. ridgwayi)is also commonly recognized. The species are largely allopatric, except for a contact zone in Costa Rica. Using plumage and molecular variation, I examined the likelihood of two (or three) different species of rough-winged swallows, the genetic interrelationships among taxa, and their biogeographic history. Geographic plumage variation reveals a latitudinal cline across the genus from North to South America. Specimens from throughout the range of Stelgidopteryx show that most published subspecies should be synonymized as clinal variants. Molecular data (mitochondrial cytochrome-b sequences, microsatellite allele frequencies) show a pattern consistent with a species-level division between S. serripennis and S. ruficollis . The data also suggest that the Yucatan swallow is distinct. Phylogenetic trees from the sequence data divide Stelgidopteryx into northern and southern clades consistent with S. serripennis and S. ruficollis. The Yucatan clade, S. ridgwayi, is sister to the northern group. Microsatellite data indicate allelic frequency differences between the groups, but none fixed. Population genetic analyses among individuals within S. serripennis and S. ruficollis reveal genetic structure possibly worthy of taxonomic recognition. Measures of the population parameter theta indicate high allelic diversity in each group and suggest S. ruficollis is the younger population. The Northern Rough-winged Swallow exhibits broad clinal variation, but little subspecific subdivision. The Yucatan Rough-winged Swallow, is phenotypically and genetically dissimilar to the other groups, further supporting species-status. The Southern Rough-winged Swallow contains at least two subspecies, including decolor on the Pacific slope of Central America, and ruficollis throughout the rest of its range. The widespread distribution and contact zone in Costa Rica are consistent with secondary contact between the northern and southern swallows. The emergence of the Panamanian landbridge could have contributed to the formation of the contact zone. Pleistocene climatic shifts could also have played a role in isolating birds on opposite slopes of Central America allowing divergence between S. r. uropygialis and S. r. decolor.

The rapidly increasing amount of sequence data has brought about a new appreciation for the tremendous influence mobile elements have had in shaping eukaryotic genomes. Despite their ubiquity, however, the factors governing the proliferation of mobile elementsor, in some cases, the lack of proliferationacross diverse taxa remain poorly understood. Analysis of Alu activity in humans and chimpanzees since their divergence indicates a two-fold increase in human Alu activity compared to that of the chimpanzee. This human retrotransposition increase is accompanied by a roughly two-fold higher level of chimpanzee Alu diversity. We prepose a model, wherein smaller effective population sizes in humans brought about a shift in host-element dynamic, ultimately leading to increased Alu activity in humans. We also survey Alu-associated diversity on the human sex chromosomes in order to examine whether Alu elements behave similarly to genetic marker systems. Our results suggest that, comparable to other genetic systems, Alu elements exhibit reduced diversity on the sex chromosomes. Our data provide no evidence for retrotransposon targeted biology influencing Alu insertion frequencies. We go on to synthesize several recent advances in the mobile element field and propose a novel hypothesis concerning how retrotransposon lineages manage to largely lie below the radar of population-level negative selection.

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. This study presents a biochemical characterization of a novel MarR homolog, HucR (hypothetical uricase regulator), from the DNA damage-resistant eubacterium, Deinococcus radiodurans. Circular dichroism spectroscopy suggests that HucR has ~47% alpha-helix and 10% beta-strand conformation at 25 deg C, and undergoes a transition to a disordered state with Tm = 51.1 ± 0.0 deg C. HucR binds as a homodimer with high sequence-specificity to a single site in its promoter region (hucO) with an apparent Kd = 0.29 ± 0.02 nM. DNaseI and hydroxyl-radical footprinting indicate HucR binding site sizes of ~24 bp and 21 bp, respectively. The binding site contains a pseudopalindromic sequence comprised of 8 bp inverted repeats separated by 2 bp that overlaps predicted promoter elements for hucR and a putative uricase (dr1160). Specific phenolic weak acids, notably uric acid, antagonize HucR-hucO complex formation. In vivo, uric acid increases transcript levels of hucR and dr1160, ~1.6-fold, and stimulates uricase activity 1.5-fold. HucR-hucO complex formation involves protein conformational changes and a decrease in the helical twist of the DNA duplex. Intrinsic fluorescence measurements show that uric acid induces HucR conformational changes, and its apparent Kd = 11.6 ± 3.7 micromolar and Hill coefficient of 0.7 ± 0.1 suggest negative cooperativity. An amino acid substitution in the predicted HucR wing (HucR-R118A) reduces DNA-binding affinity ~5-fold (Kd = 1.60 ± 0.14 nM), whereas a substitution in the predicted recognition helix (HucR-S104A) does not significantly alter DNA-binding affinity (Kd = 0.23 ± 0.03 nM). Each mutation decreases complex stability on the gel, but does not affect sequence-specificity. Intrinsic fluorescence spectra suggest altered conformations of the HucR-variants and altered mechanisms of DNA association. The mutations at HucR positions 118 and 104 also alter a predicted weak ligand-binding site, as indicated by minor changes in uric acid affinities for HucR-R118A and HucR-S104A (Kd = 9.7 ± 3.2 micromolar and 7.4 ± 0.5 micromolar, respectively) and modest attenuations of protein-hucO complex formation in response to uric acid.