Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 99 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

One of the important contributions genetic studies have made to conservation is the ability to resolve taxonomy and define relationships among populations. However, this can be complicated when species exhibit hybridization. Hybridization can be an important part of the evolutionary process and a critical component in a species ability to adapt to a changing environment. Most hybrid zones are observed at ecotones between two distinct habitats and this may be important in defining the role of hybrid zones in the evolutionary process. I examined hybridization among the three distinct lineages of desert tortoises in the genus Gopherus. An important aspect of this study system is the presence of areas of overlap between divergent lineages of desert tortoise which allowed me to test hypotheses about which forces influence these taxonomic boundaries. Specifically, I tested hypotheses about the contribution of physical vs. ecological segregation and the relative importance of isolation and gene flow in the formation of these disparate desert tortoise lineages. I used mtDNA sequence data and 25 microsatellite loci to perform Bayesian clustering, clinal analyses and habitat suitability modeling to infer population structure and influence of landscape features at each contact zone. In both instances, I observed ecological niche partitioning and limited hybridization at ecotones. I then used mtDNA and four nDNA loci to perform a multi-locus phylogenetic analysis to estimate the species tree among desert tortoise lineages and tested for ancestral admixture with RNA-seq data using demographic inference employed in the software package ∂a∂i. My results validate taxonomic distinction among all three lineages without evidence of ancestral introgression. These data suggest that despite the presence of contemporary hybridization and incomplete reproductive isolation, divergence among these lineages is consistent with species-level differentiation. By clarifying the evolutionary processes that influence the distribution of desert tortoise lineages, this study will directly inform efforts to preserve the evolutionary potential of these threatened species. Ultimately, understanding the evolutionary history of desert tortoises not only clarifies the forces that have driven speciation in this group, but it also contributes to our knowledge of the biogeographic history of the southwestern deserts and how diversity is maintained within them.

The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the Southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. My dissertation addresses a variety of evolutionary genetic questions using this system.Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, D. arizona, and D. navojoa) are lacking. I used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group (Appendix A). In this study I have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. I estimate the divergence time between D. mojavensis and D. arizonae to be between 0.66 and 0.99 million years ago. I performed additional population genetic analyses of these species to provide a basis for future hypothesis testing.In Appendix B, I report the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. I show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, D. arizonae is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, I show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring.The large degree of variation between isofemale lines in producing sterile hybrid sons suggests a complex genetic basis to hybrid male sterility warranting quantitative genetic analysis. Since the genes underlying hybrid male sterility in these species are not yet fixed, I am able to perform explicit genetic analysis of this reproductive isolating mechanism. In Appendix C, I present the results of mapping QTL for hybrid male sterility within species. The genetic architecture underlying hybrid male sterility when analyzed directly in the F1 is highly complex. Thus, hybrid male sterility arises as a complex trait in this system and we propose a drift-based model for the evolution of this phenotype.

Sonoran Desert Drosophila mettleri breeds in soil soaked by the necrotic cacti juices from saguaro (Carnegiea gigantea) and cardon (Pachycereus pringlei). An isolated population on Santa Catalina Island, 300 kilometers NW of the Sonoran Desert limit, was discovered breeding in several Opuntia cacti species. Host shifts are associated with the speciation process in phytophagous insects. I tested for evidence of premating isolation, postmating isolation, and ecological differences among allopatric populations of Drosophila mettleri using a variety of approaches. No sexual isolation was detected. However, Drosophila mettleri from Santa Catalina Island shows significant behavioral and physiological differences compared with Sonoran Desert populations. Furthermore, Drosophila mettleri from Santa Catalina Island was significantly genetically differentiated from all other populations in the study. Finally, I observed sufficiently significant F1 male sterility in crosses involving the Santa Catalina Island population to consider it indicative of early postzygotic isolation.

In Caenorhabditis nematodes, the androdioecious, self-fertilizing reproductive strategy has evolved relatively recently from gonochoristic, outcrossing ancestors. Transitions in mating system impact how genes are vertically transmitted between generations and horizontally between populations through gene flow. To date, species-wide studies have targeted selfing species, and we have limited understanding about how population genetic processes have shaped the genome structure and evolutionary history of outcrossing species of Caenorhabditis. To fill this gap, I investigate patterns of genetic variation and population genetic processes focusing on two outcrossing species in the genus, C. brenneri and C. remanei, using a survey of nucleotide polymorphisms in a multipopulation, multilocus context. I discover extensive genetic diversity in Caenorhabditis brenneri, termed hyperdiversity, giving this species the highest known levels of nucleotide polymorphism for any multicellular eukaryote. Genetic crosses between populations, extensive intra-locus recombination, and evidence of weak selection on codon usage all suggest that this is due to a large effective population size in the species and not an artifact of cryptic species divergence. I demonstrate that C. remanei also is characterised by high genetic variation globally, albeit not as extreme as in C. brenneri, and within each local population. Despite geographic barriers, considerable gene flow occurs between inter-continental locations. While exploring genetic diversity in C. remanei, I discovered C. sp. 23, a new, gonochoristic species reproductively isolated and highly divergent from it. Subsequently, taking advantage of this newly discovered species pair, I explore the patterns of postzygotic reproductive isolation between C. remanei and C. sp. 23. I find evidence of partial F1 hybrid inviability, strong F1 hybrid male sterility (Haldane’s rule) and strong F2 hybrid breakdown between the two. The findings from this thesis, especially the notion of hyperdiversity, can be taken advantage of to answer key questions on testing limits of natural selection, of evolution of genome complexity as well as identifying and characterising functional, non-coding regulatory elements. Discovery of a new species pair in Caenorhabditis helps establish an emerging age of speciation genetics in the worm.

In Caenorhabditis nematodes, the androdioecious, self-fertilizing reproductive strategy has evolved relatively recently from gonochoristic, outcrossing ancestors. Transitions in mating system impact how genes are vertically transmitted between generations and horizontally between populations through gene flow. To date, species-wide studies have targeted selfing species, and we have limited understanding about how population genetic processes have shaped the genome structure and evolutionary history of outcrossing species of Caenorhabditis. To fill this gap, I investigate patterns of genetic variation and population genetic processes focusing on two outcrossing species in the genus, C. brenneri and C. remanei, using a survey of nucleotide polymorphisms in a multipopulation, multilocus context. I discover extensive genetic diversity in Caenorhabditis brenneri, termed hyperdiversity, giving this species the highest known levels of nucleotide polymorphism for any multicellular eukaryote. Genetic crosses between populations, extensive intra-locus recombination, and evidence of weak selection on codon usage all suggest that this is due to a large effective population size in the species and not an artifact of cryptic species divergence. I demonstrate that C. remanei also is characterised by high genetic variation globally, albeit not as extreme as in C. brenneri, and within each local population. Despite geographic barriers, considerable gene flow occurs between inter-continental locations. While exploring genetic diversity in C. remanei, I discovered C. sp. 23, a new, gonochoristic species reproductively isolated and highly divergent from it. Subsequently, taking advantage of this newly discovered species pair, I explore the patterns of postzygotic reproductive isolation between C. remanei and C. sp. 23. I find evidence of partial F1 hybrid inviability, strong F1 hybrid male sterility (Haldane’s rule) and strong F2 hybrid breakdown between the two. The findings from this thesis, especially the notion of hyperdiversity, can be taken advantage of to answer key questions on testing limits of natural selection, of evolution of genome complexity as well as identifying and characterising functional, non-coding regulatory elements. Discovery of a new species pair in Caenorhabditis helps establish an emerging age of speciation genetics in the worm.

Selective isolation protocols of selenium (Se) species integrated to Se specific atomic absorption spectroscopy (AAS) detection were developed and optimized for Se speciation in food supplements, including selenized yeasts. By ultrafiltration, 69.18% of Se in the extract was found as a low molecular weight soluble form, the remaining 30.82% was bound to high molecular weight components. After a cation-exchange chromatography of the ultrafiltrate, 3.77% of the Se in the extract was found in the aqueous washings of the column indicating the presence of free inorganic anions of Se; the 65.41% of Se retained on the column corresponded to the free organic Se cations. The limit of detection for the HPLC-THG-AAS system was 1.85 ng of Se. Se was shown to be widely distributed over all the proteins with one sharp peak corresponding to the free forms of Se. Four major peaks were found at MW $>$ 250 000 Da (15.97% of Se recovered), between 102 330 and 117 490 Da (7.06%), between 48 977 and 53 703 Da (12.71%) and close to the dye migration band (17.25%). / Selective isolation and HPLC-AAS protocols were also developed and optimized for the determination of free organic forms e.g. selenomethionine (SeMet), selenocystine (SeCystine) and inorganic forms of selenium in aqueous solutions, and in complex matrices such as nutritional supplements and mixtures of free amino acids. The selenoamino acid in alkaline solution was first derivatized with 1-fluoro-2,4-dinitrobenzene. After removal of excess of reagent by partitioning with diethyl ether, the N-dinitrophenyl (DNP)-derivatized selenoamino acid was acidified and extracted with diethyl ether. Inorganic Se(IV) was extracted from the acidic aqueous phases by complexation with 1,2-phenylenediamine, forming a piazselenol. Se derivatives were determined selectively by HPLC-THG-AAS. A selective chromatographic mechanism based on $ pi$-electron interactions was optimized using a silica stationary phase derivatized with p-nitrophenyl moieties. Co-injections of DNP-SeMet, DNP-SeCystine and piazselenol save retention times of 3.7, 4.0 and 4.9 min, respectively, using a methanolic mobile phase containing 1.5% triethylamine and 0.013M acetic acid. Primary analytical validation parameters including stability, linearity and limits of detection were obtained using purified DNP-SeMet, DNP-SeCystine and piazselenol standards which were characterized by $ sp1$H-, $ sp{13}$C- and $ sp{77}$Se-NMR analysis and/or fast atom bombardment MS techniques. The calibration graphs for sequential dilutions of these Se standards were linear and the limits of detection from the resultant calibration graphs were 17 ng, 0.21 ng and 18.53 ng of Se, respectively. The purified DNP-SeMet and DNP-SeCystine were found to be photosensitive. The recovery of SeMet, SeCystine and inorganic Se from the stock solutions and/or nutritional supplements was virtually quantitative. In the presence of a 500-fold excess of other amino acids, the recovery of SeMet and SeCystine (96.1 $ pm$ 3.9% and 98.08 $ pm$ 4.2%, respec

The goal of my M.Sc. thesis research was to investigate the link between divergent adaptation and reproductive isolation in speciation, the process by which one species becomes two over time. Previous studies followed the evolution of 12 replicate experimental populations of the yeast Saccharomyces cerevisiae in either of two non-optimal environments, high-salt or low-glucose, over 500 generations. In this research, I re-sequenced the whole genomes of six diploid strains of these populations, three from high salt and three from low glucose. Among the replicates, there was remarkable degree of parallelism in the underlying determinants of adaptation including multiple mutations in PMA1, encoding the main proton exporter, in high-salt and in several genes in the ras signalling pathway in both environments. A variety of positive and negative epistatic interactions were found among mutations from within and among populations, emphasizing the importance of epistasis in adaptation and potentially in speciation.

<p>Adaptation is central to our understanding of the origin of biological diversity. Yet whether adaptive divergence promotes the formation of new lineages remains poorly understood. My dissertation addresses the role of adaptive divergence in diversification and speciation. I also investigate an alternative mechanism: dispersal, which can promote diversification and speciation through its effects on gene flow and allopatry. To address the role of divergent adaptation and dispersal in the process of diversification, I take an integrated approach, combining both comparative methods with quantitative genetics to characterize patterns of diversification and speciation in the tribe Brassiceae and genus Cakile. I start with a comparative study of the role of dispersal and adaptation in diversification, and then focus on the role of climatic and latitudinal divergence in the processes of adaptive divergence and speciation. In general, I find limited evidence for the role of divergent adaptation in the evolution of intrinsic reproductive isolation. Diversification in the tribe Brassiceae appears to be mediated by dispersal ability, while in the genus Cakile, the evolution of intrinsic reproductive isolation is largely independent of ecological divergence. Thus, while divergent adaptation to novel habitats and climate are likely occurring in Brassiceae, mediated in part by the evolution of long-distance dispersal, the evolution of intrinsic genic reproductive barriers appears to not be influenced by adaptation.</p> / Dissertation

Successful preservation of Earth’s biodiversity requires an understanding of the processes that generate new species. The generation of species without gene exchange is considered predominant; however, a growing body of evidence indicates that populations can diverge while exchanging genes, and that this may be common. Previous research hypothesized that blue-footed (Sula nebouxii) and Peruvian (S. variegata) boobies diverged from their common ancestor while exchanging genes. Here, I combine ecological and genetic perspectives to thoroughly evaluate this hypothesis. Using a panel of eight molecular markers, I estimate population differentiation for each species. I find evidence of weak population differentiation for both species, an uncommon pattern in seabirds, and argue that specialization to an unpredictable food resource has shaped contemporary population differentiation. Next, I use molecular markers and morphology to evaluate the hybrid status of five morphologically aberrant individuals. I report that all are likely F1 (first generation) hybrids, and are the product of crosses between female Peruvian boobies and male blue-footed boobies. Sex biases in pairing may occur because of an underlying preference for elaborate courtship displays. I then expand the dataset to 19 loci and use cline theory and Bayesian assignment tests to characterize the hybrid zone, to examine introgression, and to evaluate the hybrid status of the aberrant individuals. The hybrid zone is most likely maintained by strong endogenous and exogenous selection against hybrids and dispersal of parentals into the hybrid zone (a tension zone), and introgression is low for nuclear loci and absent for mitochondrial loci. Finally, I test the hypothesis that this species pair diverged from their common ancestor with gene flow using recently developed analyses and multiple loci. Divergence without gene flow is rejected and unidirectional introgression of sex-linked loci during divergence is reported. The results of this study support the hypotheses that: 1) populations can diverge while exchanging genes; 2) the Z chromosome may play a role in avian speciation; and 3) organisms specialized to variable foraging environments should exhibit low population differentiation. This study adds to our understanding of both population differentiation and speciation in seabirds, and the generation of new species more generally. / Thesis (Ph.D, Biology) -- Queen's University, 2011-04-06 13:55:32.151