Contact zone dynamics and the evolution of reproductive isolation in a North American treefrog, the spring peeper (Pseudacris crucifer)

Despite over seven decades of speciation research and 25 years of phylogeographic studies, a comprehensive understanding of mechanisms that generate biological species remains elusive. In temperate zones, the pervasiveness of range fragmentation and subsequent range expansions suggests that secondary contact between diverging lineages may be important in the evolution of species. Thus, such contact zones provide compelling opportunities to investigate evolutionary processes, particularly the roles of geographical isolation in initiating, and indirect selection against hybrids in completing (reinforcement), the evolution of reproductive isolation and speciation. The spring peeper (Pseudacris crucifer) has six well-supported mitochondrial lineages many of which are now in secondary contact. Here I investigate the evolutionary consequences of secondary contact of two such lineages (Eastern and Interior) in Southwestern Ontario using genetic, morphological, acoustical, experimental, and behavioural evidence to show accentuated divergence of the mate recognition system in sympatry. Mitochondrial and microsatellite data distinguish these two lineages but also show ongoing hybridization. Bayesian assignment tests and cline analysis imply asymmetrical introgression of Eastern lineage nuclear markers into Interior populations. Male calls are divergent between Eastern and Interior allopatric populations and show asymmetrical reproductive character displacement in sympatry. Female preference of pure lineage individuals is also exaggerated in sympatry, with hybrids showing intermediate traits and preference. I suggest that these patterns are most consistent with secondary reinforcement. I assessed levels of post-zygotic isolation between the Eastern and Interior lineages using a laboratory hybridization experiment. Hybrid tadpoles showed equal to or greater fitness than their pure lineage counterparts, but this may be countered through competition. More deformities and developmental anomalies in hybrid tadpoles further suggest post-zygotic isolation. Despite evidence for pre-mating isolation between the two lineages, isolation appears incomplete (i.e. hybridization is ongoing). I hypothesize that potentially less attractive hybrids may circumvent female choice by adopting satellite behaviour. Although mating tactics are related to body size, genetic status may play a role. I show that pure Eastern males almost always engage in calling, while hybrids adopt a satellite tactic. An absence of assortative mating, despite evidence of female preference, suggests successful satellite interception possibly facilitating introgression.

THE DESIGN AND SYNTHESIS OF SILOXANE PRECURSORS FOR CHIRAL PERIODIC MESOPOROUS ORGANOSILICA MATERIALS

The development of cost-effective and reliable methods for the synthesis and separation of asymmetric compounds is paramount in helping to meet society’s ever-growing demand for chiral small molecules. Of these methods, chiral heterogeneous supports are particularly appealing as they allow for the reuse of the chiral source. One such support, based on the synergy between chiral organic units and structurally stable inorganic silicon scaffolds are periodic mesoporous organosilicas (PMOs). In the work described herein, I examine some of the factors governing the transmission of chirality between chiral dopants and prochiral bulk phases in chiral PMO materials. In particular, the exploration of 1,1’-binaphthalene-bridged chiral dopants with a focus on the point of attachment into the materials. Moreover, the effects of ordering in the materials are examined and reveal that chirality transfer is more facile in materials with molecular-scale order then those containing amorphous walls. Secondly, the issues surrounding the synthesis and purification of aryl-triethoxysilanes as siloxane precursors are addressed. Both the introduction of a two-carbon linker and the direct attachment of allyl and mixed allyldiethoxysilane species are explored. This work demonstrates that allyldiethoxysilanes are ideal, in that they are stable enough to permit facile synthesis, while still being able to hydrolyze completely to produce well-ordered materials. Lastly, the production of new bulk phases for chiral PMO materials is examined by introducing new prochiral nitrogen-containing siloxane precursors. Biphenyldiamine and bipyridine-bridged siloxane precursors are readily synthesized on reasonable scales. Their use as the bulk siloxane precursor in the production of PMO materials however, is precluded by insufficient gelation and additional siloxane precursors are necessary for the production of ordered materials. In addition to the research detailed above that forms the body of this thesis, two short works are appended. The first details the production of polythiophene assemblies mediated through coordination nanospaces, while the second explores the production of N-heterocyclic carbene functionalized gold nanoparticles through ligand exchange.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.