In this thesis, I use 10 populations of Drosophila melanogaster, each fixed for a different visible recessive mutation to investigate two outstanding issues in sexual selection research. First, to quantifty indirect benefits of female mate choice and explore the nature of genotype x environment interactions for different fitness components, I estimated the effects of these mutations on male sexual fitness and productivity in the population's ancestral laboratory environment, as well as a novel food environment. Indirect benefits in the ancestral environment were lacking, suggesting that a good genes process is not acting. Cross-environment correlations were not conducive to indirect benefits following colonization of a novel environment. Second, to investigate the contribution of variable search effort to variance among males in mating success, I conducted mate choice trials to compare the relative mating success of mutant males in small vs. large arenas. Sexual selection against mutant males was stronger when search effort was included than when it was excluded, indicating that varying ability to find mates may increase the strength of selection against deleterious alleles.

The Cariceae (Carex & Kobresia; Cyperaceae) account for ca. 10% of Canada's native vascular plants, yet diversity within the tribe is overlooked due to its taxonomically-difficult floral morphology. DNA barcoding is a useful tool for identifying animals and the technique is currently being adapted for use in plants. This thesis develops a reference barcode database for the Cariceae of North America, north of Mexico, using the chloroplast gene matK. A species resolution rate of 39% is reported, although when broken down into political regions, this can increase to 75%. Unknown tissue samples from across North America were then identified using this reference database; 50% of these were identified to a single species matching the morphological identification, and 50% were identified to a group of taxa containing the correct identification. An ecogeographical regional database for the Arctic Archipelago was also challenged with 230 unknown samples and identified 98% to a single species.

The taxonomic status of the 25 nominal species of the Indo-Pacific flatfish genus Zebrias (Pleuronectiformes: Soleidae) was revised based on a morphological study of 531 specimens, including 36 type specimens. Two distinct, monophyletic groups were identified differing in the confluence and attachment of the caudal fin to adjacent dorsal and anal fins. Species most closely associated to the type species Pleuronectes zebra were included in the genus Zebrias while the remaining species were assigned to the genus Pseudaesopia to accommodate Zebrias species having a shallowly confluent caudal fin with adjacent fins, among other characters. Ten species are now recognized and re-described in Zebrias while six species are recognized and redescribed in Pseudaesopia. Comprehensive generic definitions and identification keys to species are provided. In addition, the closely related and monospecific genus Aesopia is briefly discussed. Finally, potentially useful characters for a future phylogenetic study are discussed.

Ultraviolet B radiation (UVBR) and chemical pollutants are environmental stressors that may affect amphibian populations. While adverse effects of UVBR and chemicals on amphibians are documented, few studies examined the effects of interactions between stressors on amphibian health. Gosner stage 25 Rana pipiers tadpoles were chronically exposed to environmentally relevant levels of UVBR (0.15-0.22 W/m2) and the estrogenic chemical 4-tert-octylphenol (OP) (0.01 nM or 10 nM) alone and in combination. Tadpoles exposed to UVBR and/or OP exhibited significantly delayed development at stage 29. Significantly fewer UVBR-treated tadpoles developed past stage 34 and metamorphosed. Effects of UVBR/OP on the thyroid system were assessed because it is the main regulator of amphibian metamorphosis. Stage 29 and 34 tadpoles were collected for gene expression analysis in tail and T3 (triiodothyronine) whole body levels (minus tail). Moreover, we examined whether pre-exposure to UVBR/OP affected the molecular and morphological responses of tadpoles to exogenous T3. OP increased the expression of several genes in tail and brain and/or their response to T3 (e.g., deiodinase 2), and the addition of UVBR can alter the effect of OP. Although UVBR had no observable effect on gene expression, developmentally delayed UVBR-treated stage 31 tadpoles exposed to T3 exhibited slowed tail resorption and accelerated hind limb development. UVBR and/or OP did not affect T3 levels of stage 29 and 34 tadpoles. However, a decrease and increase in deiodinase 2 and 3 (D2 and D3) mRNA levels, respectively, were observed in groups of tadpoles with slowed developmental rates at those stages. Since D2 activates and D3 inactivates thyroid hormones (TH), UVBR/OP mediated disruptions in development are likely caused by dysfunctions in the localized metabolism of THs. These results indicate that environmental levels of UVBR and/or OP can affect metamorphosis, potentially by disrupting the biological action of T3 and deiodinases in peripheral tissues, which could contribute to population declines.

It has long been recognized that climate can influence the diversity and dynamics of communities and regional assemblages. Within this thesis, I ask three questions: (1) which processes are most important in mediating climate-species richness relationships; (2) are predictions of spatial climate-richness models temporally consistent, and (3) is local community structure determined primarily by regional or local processes. Metabolic theory proposes that climate-richness relationships arise due to the temperature dependence of metabolic rate. I tested the theory's predictions for six taxa in North America. Contrary to the theory's predictions, temperature-richness relationships were curvilinear and their slopes deviated from the predicted value. This suggests that the mechanism proposed by metabolic theory does not underlie climate-richness relationships. If climate determines species richness, then climate should predict how species richness will change over time. To test this, I compared alternative methods (regression and niche modelling) of forecasting shifts in species richness given global climate change. Models were trained on butterfly richness data from the early 20th century and their predictions were compared to observed changes throughout the 20th century. Overall, regression-based approaches that incorporated spatial autocorrelation outperformed other methods. Broad-scale richness gradients could arise from climatic niche conservatism. I tested this hypothesis for treefrogs (Hylidae) by combining data on species' distributions and phylogeny. I found that while niches were conserved with respect to cold tolerance, species richness was determined by precipitation, not temperature. This suggests that the processes determining regional species composition and richness are controlled by fundamentally different climatic components. I evaluated the relative importance of regional and local processes and how there were affected by climatic gradients by examining patterns of body size dispersion at local and regional scales for hylid frogs. On average, communities were over-dispersed, but there was no increased signature of competition in the tropics. Dispersion of regional assemblages decreased in cold areas, but this was not due to an elevated tropical rate of body size evolution. Overall, regional processes explained twice as much variance in body size dispersion than did local processes. This thesis rejected several hypotheses for the link between climate and macroevolutionary patterns. In doing so, it provided new insight to the role of ecological and evolutionary processes along broad-climatic gradients.

All eukaryotes contain at least three homologous RNA polymerases (RNAPI, RNAPII, and RNAPIII), which are directed towards their target genes by interactions with RNAP-specific general transcription factors (GTFs). Although the RNAPs transcribe a subset of nuclear genes, it is unknown if they have evolved altered functionality. An in silico approach to identify putative differences in the three RNAPs was used to identify whether any functional differences likely exist by identifying amino acids that have experienced shifts in evolutionary rates and by identifying length differences between the cleft loops of the RNAPs. The difference in the levels of concerted evolution experienced by the genes transcribed by RNAPs was then exploited to test the molecular coevolution hypothesis of Dover and Flavell (1984. Cell . 38:622-623.). According to the molecular coevolution hypothesis, concertedly evolving DNA increases the evolutionary rate of any interacting proteins. We thus compared the evolutionary rates of the three RNAPs and their GTFs, since their target genes undergo different levels of concerted evolution. Finally, the origins of the 5 subunits that are specific to RNAPIII were examined, since no homologous relationships have been identified thus far for any of these subunits. Several sites that have experienced shifts in substitution rates in the ancestral RNAPs were found in all three enzymes and these sites were clustered near the active sites in all cases. Several cleft loops with different lengths (in amino acids) between the three RNAPs were also identified. The validity of the molecular coevolution hypothesis was largely confirmed via the demonstration that most subunits of RNAPI evolve faster than those of RNAPIII and most subunits of RNAPIII evolve faster than RNAPII. This is consistent with the molecular coevolution hypothesis because RNAPI experiences higher levels of concerted evolution than the target genes of RNAPIII. The evolutionary rates of the GTFs of RNAPI and RNAPIII were also higher than those of RNAPII. Finally, four of the five RNAPIII-specific subunits were identified as homologs of RNAPII GTFs, indicating that several of the GTF paralogs existed in ancestral RNAPIII and were subsequently recruited to the enzyme before the diversification of eukaryotes.

Vaccination against the Influenza A virus (IAV) is often an important and critical task for much of the population, as IAV causes yearly epidemics, and can cause even deadlier pandemics. Designing the vaccine requires an understanding of the current major circulating strains of Influenza, as well as an understanding of how those strains could change over time to become either less harmful or more deadly, or simply die out completely. An error in the prediction process can lead to a non-immunized population at risk of epidemics, or even a pandemic. Presented here is a posterior predictive approach to generate emerging influenza strains based on a realistic genomic model that incorporates natural features of viral evolution such as selection and recombination. Also introduced is a sequence sampling scheme to relieve the computational burden of the posterior predictive analysis by clustering sequences based on their pairwise similarity. Finally, the impact of “evolutionary accidents” that take the form of bursts of evolution and or of recombination on the predictive power of our procedure is tested. An analysis of the impact of these bursts is carried out in a retrospective study that focuses on the unexpected emergence of a new H3N2 strain in the 2007-08 influenza season. Measuring the R2 values of both pairwise and patristic distances, the model reaches a predictive power of ∼40%, but is not able to simulate the emergence of the target Brisbane/10/2007 sequence with a high probability. The inclusion of “evolutionary accidents” improved the algorithm’s ability to predict HA sequences, but the prediction power of the NA gene remained low.

Acyl Coenzyme A: Isopenicillin N Acyl Transferase (AT) is a key enzyme in the biosynthesis of β-lactam antibiotics in penicillin producing organisms such as P. chrysogenum and A. nidulans. Its natural activity is to exchange the side chain of the low activity antibiotic IPN [18] for the phenylacetyl side chain resulting in the more active antibiotic Penicillin G [5]. The biosynthesis of β-lactams has been exploited towards producing these compounds for therapeutic use. However, increasing bacterial resistance means new analogues in this compound class are constantly sought.As well as improving current production methods of β-lactam antibiotics, AT's broad substrate specificity means it could potentially play a role in the development and production of alternative β-lactam antibiotics that are able to overcome resistance.This thesis describes the identification of an AT mutant with improved acylation activity (AAT activity) via screening of an AT library using a previously developed screening method. Approaches towards the development of a method for the identification of AT mutants with improved hydrolysis activity were also explored. The main problem to overcome in developing such a screen is the inhibitory effect of 6-APA [1], the product of hydrolysis, on AT's IAT activity. The first approach investigated the potential of increasing the sensitivity of an assay by targeting AT to the periplasm. A second approach using β-lactamases to hydrolyse 6-APA [1] thus freeing up the active site of AT was also investigated.

Marstonia lustrica is a poorly understood aquatic snail, relatively rare throughout its range and listed in the State of Massachusetts as Endangered (MNHESP 2010, Hershler et al. 1987). It is the northern-most cold temperate species of its genus, with other members of the genus occurring along the southern edge of its range and in the southeastern United States (Thompson 1977). The current range of M. lustrica appears to follow the maximum extent of the Laurentide Glacier (20–25 kya), extending from Minnesota to western Massachusetts. Research regarding the distribution, ecology, and phylogeny of M. lustrica in the State of Massachusetts and eastern New York raised the possible role of glaciers and pro-glacial lakes in the establishment and distribution of the snail, leading to the hypothesis that its distribution and evolution may be dependent upon glacial processes. A full range survey was completed in 2007 and 2008, with populations identified in 20 water bodies from Minnesota to Massachusetts, and Ohio to Ontario, Canada. Fifty-seven specimens from the 20 populations were sequenced for two mtDNA markers (COI and NDI), developing both phylogenetic trees and haplotype networks. Here I present those trees and networks, and correlate the distribution of these populations and their representative haplotypes with both glacial events and contemporary watersheds, using AMOVAs and Mantel tests to examine several phylogeographic models. In addition to the results for M. lustrica, the unexpected occurrence of several other species of Marstonia spp. found across the range of M. lustrica are presented, including M. pachyta, M. comalensis , and M. hershleri.

Mole salamanders (Ambystoma) and woodfrogs ( Lithobates sylvaticus) are abundant in New England and depend on ephemeral wetlands for breeding. Their aquatic habitats have been well studied and are protected by several local and regional regulations. State endangered species laws also protect mabled salamanders (A. opacum), Jefferson salamanders (A. jeffersonianum), and blue-spotted salamanders (A. laterale). However, these amphibians spend most of their adult lives in terrestrial habitats that remain poorly protected and elusive to researchers. In chapter 1, I developed a novel technique using passive integrated transponders for tracking small animals. I used this technique to track marbled salamanders walking up to 200 m from their breeding pond during post-breeding migrations. In Chapter 2, I examined the importance of multiple habitat variables for controlling the distributions of woodfrogs and spotted salamanders at 455 ponds in western Massachusetts. Based on a variable-comparison technique I developed, the best predictor for either species of amphibian was the amount of forest in the surrounding landscape. Both species were found more frequently in upland forests where the ponds are least protected by state and federal wetland regulations. In chapter 3, I used my data from chapter 2 and three other similar data sets to conduct an analysis of spatial scale and to parameterize a recently published resistant kernel model. The complex model parameterized by an expert panel did significantly worse than the null model. The distributions of both amphibians were best predicted by measuring the landscape at very large scales (over 1000 m). The most effective scales for conservation may be largest for organisms of intermediate dispersal capability. In chapter 4, I explored the evolution and genetics of the Jefferson/blue-spotted/unisexual salamander complex. I framed research into the fascinating unisexual reproductive system with a model that relates nuclear genome replacement, positive selection on hybrids, and biogeography of the species complex. I parameterized this model using genetic data taken from salamanders spanning Massachusetts and an individual-based breeding simulation. If paternal genomes are transmitted to offspring with the frequencies reported from laboratory experiments, then my model suggests that there must be strong selection favoring unisexuals with hybrid nuclei.