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Speciation can be understood as a continuum occurring at different levels, from population to species. The recent molecular revolution in population genetics has opened a pathway towards understanding species evolution. At the same time, speciation patterns can be better explained by incorporating a geographic context, through the use of geographic information systems (GIS). Phaedranassa (Amaryllidaceae) is a genus restricted to one of the world’s most biodiverse hotspots, the Northern Andes. I studied seven Phaedranassa species from Ecuador. Six of these species are endemic to the country. The topographic complexity of the Andes, which creates local microhabitats ranging from moist slopes to dry valleys, might explain the patterns of Phaedranassa species differentiation. With a Bayesian individual assignment approach, I assessed the genetic structure of the genus throughout Ecuador using twelve microsatellite loci. I also used bioclimatic variables and species geographic coordinates under a Maximum Entropy algorithm to generate distribution models of the species. My results show that Phaedranassa species are genetically well-differentiated. Furthermore, with the exception of two species, all Phaedranassa showed non-overlapping distributions. Phaedranassa viridiflora and P. glauciflora were the only species in which the model predicted a broad species distribution, but genetic evidence indicates that these findings are likely an artifact of species delimitation issues. Both genetic differentiation and non-overlapping geographic distribution suggest that allopatric divergence could be the general model of genetic differentiation. Evidence of sympatric speciation was found in two geographically and genetically distinct groups of P. viridiflora. Additionally, I report the first register of natural hybridization for the genus. The findings of this research show that the genetic differentiation of species in an intricate landscape as the Andes does not necessarily show a unique trend. Although allopatric speciation is the most common form of speciation, I found evidence of sympatric speciation and hybridization. These results show that the processes of speciation in the Andes have followed several pathways. The mixture of these processes contributes to the high biodiversity of the region

It is now widely accepted that adaptation to different ecological niches can result in the evolution of new species. However, when gene flow persists speciation must overcome the antagonism between selection and recombination: Specifically, if gene flow persists, recombination will break down the genetic associations between alleles that characterise emerging species and cause reproductive isolation. Accordingly, genetic architectures that impede recombination can slow the breakdown of linkage disequilibrium and facilitate speciation. Mimicry in tropical butterflies has long been championed as an example of adaptation driving speciation. In the Neotropical genus Heliconius, distantly related pairs of unpalatable species often converge on the same bright warning-pattern to more efficiently advertise their distastefulness to predators. In contrast, closely related taxa often belong to different mimicry rings. The sister species, Heliconius melpomene and H. cydno are sympatric across much of Central and northern South America. Using artificial butterflies I reveal selection against non-mimetic hybrid colour patterns between these two species. These colour patterns are also used as mating cues and mimetic shifts may cause both pre-mating and post-mating isolation. However, shifts in colour pattern cannot drive reproductive isolation alone; rather, they must be accompanied by corresponding mate preferences. Associations between trait and preference loci may be broken down by mating and subsequent recombination. I demonstrate a genetic linkage between loci for both male and female mate preference and wing colour pattern in Heliconius cydno and H. melpomene. In addition, I present evidence for further associations between alleles affecting hybrid sterility and host-plant use and colour pattern loci. All this implies that linkage between traits that contribute to reproductive and ecological isolation is a general phenomenon in Heliconius with an underlying adaptive basis. Overall these results expose a genetic mechanism that, by impeding recombination, can facilitate speciation in the face of gene flow.

M.Sc. (Zoology) / Tilapia sparrmanii (Smith) was exposed to sub-lethal doses of chromium, manganese, zinc and iron for 96 hours at an acidic pH (5), a physiological pH (7,4) and an alkaline pH (9), as well as a prolonged exposure (2 - 4 weeks) at an uncontrolled pH. In the laboratory fish were kept in aquaria which were supplied with continuously flowing borehole water. Controlled laboratory conditions existed during experimentation. Blood and liver samples of the experimental fish were sampled after exposure. The bioconcentration and the effect of the selected metals at the mentioned conditions, were thereby determined on bloodcoagulation, total and differential leucocyte counts, and liver enzYmes to investigate the possible damage to the liver. As metals have the ability to enter and concentrate in the body, the bioconcentration of each metal was determined by atomic absorption sPectrophotometry in the blood and liver. Chromium showed an increase in concentration in both the blood and liver, with an increase in pH after short term (96h) exposure. The statistical significant increases of manganese concentration in the blood over a short term were not reflected in the liver. It may be indicative of homeostatic control. A similar phenomenon was found with .zd.nc , The concentration of zinc in the liver did not reflect the progressive decrease in the blood wi th a increase in pH. Iron revealed a statistical significant increase in bioconcentration in the liver with, an increase in pH, which is indicative of the activity of the liver. The long term exposure of fish to chromium and manganese caused a significant increase in the concentration of the metals in the blood and liver. The only significant increase in concentration after exposure to iron, occured . in the Iiver. Exposure to zinc however caused a statistical significant decrease in concentration in both the blood and liver. Differential leucocyte counts reflected lymphocytosis, eosinophilia, monocytopenia and neutropenia, after short term exposure. These conditions nearly always resulted in leucopenia. Long term exposure to manganese and zinc reflected both eosinophilia and neutropenia. Chromium reflected eosinophilia, and iron, neutropenia. The only statistically significant change in the number of leucocytes after long term exposure, was caused by zinc and resulted in leucopenia. According to the photokymographic observation of blood coagulation by the thrombelastograph, exposure to the selected heavy metals lead to a prolonged clotting time and thrombocytopenia. Long term exposure (4 weeks) to manganese caused another bleeding disease, called hemophilia. It was thus evident that the exposure to heavy metals led to clotting defects, which caused bleeding.

The regulations for water fluoridation of South African municipal waters up to the optimum fluoride (F-) concentration of 0.7 mg/L, (Government Gazette, 2001) have been legislated. Fluoridation processes need accurate analytical methodology for the determination of F- , because F- has a narrow margin of safety between beneficial and toxic levels. In this work the analytical chemistry of F- was investigated comprehensively and guidelines compiled for the accurate determination of low level F- (between 0.05 to 1 mg/L) in aqueous systems. The first part of this study focused on method validation and the evaluation of the ISE and IC methods. The analytical methodologies were applied to the analysis of natural waters such as river water (Vaal and Crocodile Rivers), dam water (Hartbeespoort Dam), and drinking water (Johannesburg municipal tap water) to evaluate the performance of the chosen methods in the analysis of real samples and to assess the effect of the sample matrix on the accuracy of F- determinations. An inter-laboratory study in collaboration with the South African Bureau of Standard (SABS) was carried out to evaluate the proficiency of South African analytical laboratories and to check the proficiency of the procedures developed in this study. In the second part of this study, the development of an IC-ICP-OES and IC-ICP-MS method was investigated for the speciation of fluoro-aluminium complexes. This work was motivated by the fact that the water fluoridation could lead to remobilisation of scale from municipal pipes. Scale may contain aluminium hydroxide or oxide precipitates that can dissolve as fluoroaluminates or hydroxofluoro aluminates. The speciation of cationic fluoro-aluminates, free Al3+, AlF2+ and, AlF2 + together with the neutral AlF3 0, was based on cation exchange Ion chromatography (IC) coupled with Inductively Coupled Plasma- Optical Emission Spectroscopy (ICP-OES) and Inductively Coupled Plasma Mass spectroscopy (ICP-MS). / Prof. P.P. Coetzee

Optimization forms an intrinsic part of the design and implementation of modern systems, such as industrial systems, communication networks, and the configuration of electric or electronic components. Population-based single-solution optimization algorithms such as Particle Swarm Optimization (PSO) have been shown to perform well when a number of optimal or suboptimal solutions exist. However, some problems require algorithms that locate all or most of these optimal and suboptimal solutions. Such algorithms are known as niching or speciation algorithms. Several techniques have been proposed to extend the PSO paradigm so that multiple optima can be located and maintained within a convoluted search space. A significant number of these implementations are subswarm-based, that is, portions of the swarm are optimized separately. Niches are formed to contain these subswarms, a process that often requires user-specified parameters, as the sizes and placing of the niches are unknown. This thesis presents a new niching technique that uses the vector dot product of the social and cognitive direction vectors to determine niche boundaries. Thus, a separate niche radius is calculated for each niche, a process that requires minimal knowledge of the search space. This strategy differs from other techniques using niche radii where a niche radius is either required to be set in advance, or calculated from the distances between particles. The development of the algorithm is traced and tested extensively using synthetic benchmark functions. Empirical results are reported using a variety of settings. An analysis of the algorithm is presented as well as a scalability study. The performance of the algorithm is also compared to that of two other well-known PSO niching algorithms. To conclude, the vector-based PSO is extended to locate and track multiple optima in dynamic environments. / Thesis (PhD)--University of Pretoria, 2010. / Computer Science / unrestricted

The family Caulimoviridae comprises viruses with semicircular double-stranded DNA genomes encapsulated into isometric or bacilliform particles, being divided into eight genera. The genus Badnavirus is the most important due to its high number of species reported infecting cultivated plants worldwide. The present study aimed to evaluate the taxonomic/phylogenetic positioning and population genetic variability into the genus Badnavirus. Initially, a data set comprising badnavirus full-length genome sequences was obtained from the non-redundant GenBank database. Multiple nucleotide sequence alignments were obtained for the data sets: complete genome; ORF III; full (1020pb) and partial (579pb) RT/RNaseH. Pairwise sequence comparisons, phylogenetic and recombination analyses were performed for all data sets. A total of 127 isolates were obtained, representing 53 badnavirus species. Nucleotide pairwise comparisons for the data sets RT/RNaseH and ORF III showed that a number of distinct badnavirus species shared up to 82,5% of identity, higher than the 80% threshold currently used for species demarcation in the genus. Bayesian phylogenetic trees showed four well supported clusters, with clusters 1 and 3 being sister groups comprising predominantly isolates infecting sugarcane and banana. Non-tree-like evolution evidenced a complex pattern of recombination, and at least 23 independent events were detected with recombination breakpoints occurring predominantly in the ORF III and in the intergenic region. By the analysis of nucleotide diversity of the partial RT/RNaseH region in 12 badnavirus population, a high genetic variability was observed. These results showed that mutation and recombination are important evolutionary mechanisms acting on the diversification of badnaviruses, and that the partial RT/RNaseH sequence is sufficient to determine the taxonomic placement of most viral species described in this genus. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A família Caulimoviridae compreende fitovírus com genomas semicirculares de DNA de fita dupla encapsidados em partículas isométricas ou baciliformes, sendo dividida em oito gêneros. O gênero Badnavirus é o mais importante devido ao seu alto número de espécies relatadas infectando plantas cultivadas em todo o mundo. O presente estudo teve como objetivo avaliar a variabilidade genética de populações e o posicionamento taxonômico/filogenético de espécies de badnavírus. Inicialmente, sequências completas para o genoma das espécies de badnavírus foram obtidas a partir do banco de dados não-redundante GenBank. Alinhamentos múltiplos das sequências nucleotídicas foram obtidos para os seguintes conjuntos de dados: genoma completo, ORFIII, RT/RNaseH completa (1020nt) e parcial (579nt). Comparações pareadas de sequências, análises filogenéticas, de recombinação e variabilidade genética foram realizadas para os conjuntos de dados. Um total de 127 isolados foram obtidos, representando 53 espécies de badnavírus. As comparações de sequências de nucleotídeos para o conjunto de dados RT/RnaseH (completa e parcial) mostraram que algumas espécies de badnavírus relatadas como distintas apresentam entre 57,1-82,5% de identidade, superior ao limite de 80,0% atualmente utilizado para a demarcação de espécies em Badnavirus. Resultados similares foram observados para os dados da ORFIII e genoma completo, reforçando que sequências parciais do domínio RT/RNaseH são suficiente para determinar o posicionamento taxonômico da maioria das espécies descritas neste gênero. Quatro grupos (clusters 1-4) bem suportados foram observados nas árvores filogenéticas para genoma completo e ORFIII, com os cluster 1 e 3 formando grupos irmãos compreendendo espécies/isolados infectando predominantemente cana-de-açúcar (Saccharum spp.) e banana (Musa spp.). Análise de evolução em rede evidenciou alguns possíveis eventos de recombinação afetando a diversificação de espécies de badnavírus, com pelo menos 23 eventos independentes sendo detectados com pontos de recombinação ocorrendo predominantemente na ORFIII e região intergênica. Finalmente, altos índices de diversidade nucleotídica foram observados para a região RT/RnaseH parcial em populações de 12 espécies distintas de badnavírus. Estes resultados mostram que mutação e recombinação são mecanismos importantes atuando na evolução dos badnavírus.

The ecological communities we observe today are a product of the bidirectional interactions between ecological and evolutionary processes. Although the effects of ecological processes on population divergence and speciation have been studied extensively, far less is known about the effects of divergence and speciation on ecological dynamics. This is especially true for effects of ecological speciation processes on higher trophic levels. In this thesis I focus on how divergence in the European whitefish (Coregonus lavaretus) affects a guild of six piscivorous bird species. Previous studies have indicated that population densities of these species are higher on lakes with polymorphic whitefish than on lakes with monomorphic whitefish. Here I test the hypothesis that the high densities of piscivores is a response to the rich food resource provided by dwarf whitefish ecotypes, which are of suitable size and occur in very high abundance.To test this hypothesis I analyzed fecal samples from piscivorous birds in lakes with polymorphic whitefish, using samples from lakes with monomorphic whitefish as controls. With the method of ddPCR (digital droplet Polymerase Chain Reaction) the amount of DNA from different prey fish species in the droppings of six fish-eating birds was quantified and converted to proportional abundances. The results shows that the proportion of whitefish in the diet of the entire fish-eating guild was significantly higher in lakes with polymorphic whitefish (44%) than in lakes with monomorphic whitefish (18%). Species-level analyses showed that this result also holds for both black-throated loon (Gavia artica) and red-throated loon (Gavia stellata). Common merganser (Mergus merganser), red-throated merganser (Mergus serrator) and terns (Sterna paradisaea and Sterna hirundo) did not show any difference between the two lake categories. Thus, my study supports the idea that the evolution of small-sized whitefish ecotypes provides a profitable food source for piscivorous birds. However, the finding that only some species of piscivorous bird populations seem to rely heavily on dwarfed whitefish as food suggests that also some other aspect of the speciation process may favor these species. Thus, more studies are needed to further assess what effects polymorphic whitefish have on piscivorous bird populations.

This thesis develops high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-HG-ICP-MS) method used for the analysis of glutathione arsenic complexes in biological samples. The aim of the thesis was to verify the suitability of this methods and to perform pilot studies on analysis of the enzymatic methylation assay containing glutathione and urine. Inclusion of post-column hydride generation step resolves the problem of changing sensitivity of ICP-MS with gradient elution. Using the standards of glutathione complexes, it was verified that the HPLC-HG-ICP-MS method can provide both qualitative and quantitative analysis of these complexes. The limit of detection was found at 5 pg/ml. Analysis of the methylation assay of arsenic with glutathione showed that only DMAsGS complex occurs in the assay during methylation. It was verified that the presence of the enzyme is required for the complex formation. In the samples of urine from unexposed people analyzed by HPLC- HG-ICP-MS and hydride generation-cryotrapping-inductively coupled plasma mass spectrometry (HG-CT-ICP-MS), only the presence of free pentavalent arsenic species was found, whereas neither glutathione complexes nor trivalent species could be observed.

Speciationisusuallyaslowprocessoccurringoverthousandstomillionsofyears.Thismakes speciation research difficult because no direct observation or manipulation is possible. At best, we can gain some insight by inferring the population history and structure in very fine detail by investigating genetic markers in multiple individuals of the nascent species. Today, speciationresearchisinanunprecedentedpositionthankstotheadventofhigh-throughput sequencingmethods,whichmakeiteasier and cheaper than ever before to evaluate multiple markers in many individuals. Speciation is not a straightforward process that happens in the same way every time, but rather a phenomenon occurring when genetic and ecological circumstancesactinginsymphonyultimatelyleadtoreproductiveisolationoftwosubpopula- tions. This is why it is important to study multiple model systems to understand the general principles behind speciation. We worked with two species of nightingales (Luscinia luscinia andL.megarhynchos)thatdivergedapproximately1.8Mya,likelyduetoglacialfluctuations in Europe. Our main goal was to use these new high-throughput sequencing methods to (1) detect interspecific hybrids between the species, (2) estimate levels of interspecific gene flow,(3)findareasofthenightingalegenomethatunderliereproductiveisolationand,finally, (4)...