Abstract Mitochondria contain a maternally inherited 16,568bp genome (mtDNA) that encodes for 13 out of more than 70 subunits of complexes of the respiratory chain that produce ATP by oxidative phosphorylation (OXPHOS). As a byproduct of OXPHOS, reactive oxygen species (ROS) are formed, which may play a role in ageing. MtDNA has accumulated numerous polymorphisms during evolution, leading to haplogroups characterized by ancient polymorphisms and defined by letters. MtDNA polymorphisms are thought to be neutral, but some may be slightly deleterious or even advantageous and may influence phenotypes of complex traits. Interestingly, several complex traits such as longevity and maximal aerobic power show maternal inheritance. Associations between mtDNA polymorphisms and longevity have been reported, but no systematic study has been made of the role of mtDNA in longevity. In addition, there are no previous reports on mtDNA haplogroups in elite athletic performance. Associations are demonstrated here between mtDNA haplogroups J, K and U and longevity in Finns. Interestingly, subhaplogroup J2 and haplogroup K, which were found in increased frequency among the 225 very old subjects studied, were not found among the 52 endurance athletes but were present in 11% of the 89 sprint athletes Uncoupling of OXPHOS reduces ATP and ROS production. Thus, a mitochondrial genome with a higher level of uncoupling may promote longevity but may not be favourable in situations that require a high level of ATP production, such as elite endurance performance. A more detailed analysis also showed an association between a combination of three common mtDNA polymorphisms and longevity in both the Finns and the Japanese, providing the first epidemiological support for the assumption that the nature of a mutation is determined by interactions with other mutations in mtDNA. In addition, a systematic approach was applied to study the role of mtDNA in longevity. Association analyses of mtDNA allele combinations in longevity revealed that the mtDNA control region, the tRNA and rRNA genes and the nucleotide repeats in mtDNA may play a role in longevity, since the alleles and allele combinations that showed the strongest associations with longevity, either negative or positive, were among these genes. Differences in overall variation in mtDNA between the very old and their controls were also studied, revealing more differences at synonymous (silent) sites than at non-synonymous (amino acid altering) sites. The findings support previous data suggesting that certain mtDNA haplogroups are associated with longevity. In addition, those haplogroups that increased in frequency among the very old Finns were not found among Finnish endurance athletes. Also, a novel systematic approach was applied to study mtDNA alleles, allele combinations and overall sequence variation in longevity, suggesting that there are interactions between various mtDNA positions and that the tRNA and rRNA genes and short tandem repeats in mtDNA may play a role in longevity.

Since Darwin proposed that all species on the earth have evolved from a common ancestor, evolution has played an important role in understanding biology. While the evolutionary relationships/histories of genes are represented using trees, the genomic evolutionary history may not be adequately captured by a tree, as some evolutionary events, such as horizontal gene transfer (HGT), do not fit within the branches of a tree. In this case, phylogenetic networks are more appropriate for modeling evolutionary histories. In this dissertation, we present computational algorithms to reconstruct phylogenetic networks from different types of data. Under the assumption that species have single copies of genes, and HGT and speciation are the only events through the course of evolution, gene sequences can be sampled one copy per species for HGT detection. Given the alignments of the sequences, we propose systematic methods that estimate the significance of detected HGT events under maximum parsimony (MP) and maximum likelihood (ML). The estimated significance aims at addressing the issue of overestimation of both optimization criteria in the search for phylogenetic networks and helps the search identify networks with the ``right" number of HGT edges. We study their performance on both synthetic and biological data sets. While the studies show very promising results in identifying HGT edges, they also highlight the issues that are challenging for each criterion. We also develop algorithms that estimate the amount of HGT events and reconstruct phylogenetic networks by utilizing the pairwise Subtree-Prune-Regraft (SPR) operation from a collection of trees. The methods produce good results in general in terms of quickly estimating the minimum number of HGT events required to reconcile a set of trees. Further, we identify conditions under which the methods do not work well in order to help in the development of new methods in this area. Finally, we extend the assumption for the genetic evolutionary process and allow for duplication and loss. Under this assumption, we analyze gene family trees of proteobacterial strains using a parsimony-based approach to detect evolutionary events. Also we discuss the current issues of parsimony-based approaches in the biological data analysis and propose a way to retrieve significant estimates. The evolutionary history of species is complex with various evolutionary events. As HGT contributes largely to this complexity, accurately identifying HGT will help untangle evolutionary histories and solve important questions. As our algorithms identify significant HGT events in the data and reconstruct accurate phylogenetic networks from them, they can be used to address questions arising in large-scale biological data analyses.

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined.

Two phylogenies of Phalaenopsis (Orchidaceae) are presented, one from combined chloroplast DNA data and one from a nuclear actin gene. We used these phylogenies to assess and modify the classification of Phalaenopsis and to examine several morphological characters and geographical distribution patterns. Our results support Christenson’s (2001) treatment of Phalaenopsis as a broadly defined genus that includes the species previously placed in the genera Doritis and Kingidium. Some of Christenson’s subgeneric groups needed to be recircumscribed to reflect a natural classification. We recognized four subgenera and six sections, subgenera Aphyllae, Parishianae (with sections Conspicuum, Delisiosae, Esmeralda, and Parishianae), Phalaenopsis, and Polychilos (with sections Fuscatae and Polychilos). In order to find a set of universally amplifiable, phylogenetically informative, single-copy nuclear regions, we conducted a whole genome comparison of the rice (Oryza sativa) and Arabidopsis thaliana genomes. We constructed a database of both genomes and searched for pairs of sequences using criteria we felt would ensure primers that would reliably amplify using standard PCR protocols. We tested the most promising 142 primer pairs in the lab on eighteen taxa and found four potentially informative markers in Phalaenopsis and one in Helianthus. Our results indicated that it will be difficult to find universal nuclear markers, however our database provides an important tool for finding informative nuclear markers within specific groups. The full set of primer combinations is available online at, “The Conserved Primer Pair Project,” http://aug.csres.utexas.edu:8080/cpp/index.html. We used fourteen Phalaenopsis species and seven horticultural hybrids to create a real dataset with which to test phylogenetic network reconstruction methods. We tested the performance of Neighbor-Net, implemented in SplitsTree, under four different categories of complexity: one hybrid, two independent hybrids (hybrids with no parents in common), three independent hybrids, and two non-independent hybrids (one parent was shared between hybrids). Neighbor-Net was able to predict accurately the parents of hybrids in only about half of the datasets we tested, and there were so many false positives that it was impossible to distinguish the hybrids from the species. We plan to use this dataset to test methods, such as RIATA and RGNet, when they become available. / text

Culture-independent DNA sequencing is being used to recover genetic material directly from environmental samples. This has spurred large-scale community efforts to catalogue the diversity of life and its geographic distribution using molecular data. These initiatives stand to revolutionize our understanding of the processes that shape biodiversity and may ultimately provide critical information for setting public health, environmental, and economic policies. To achieve these aims new tools are required to effectively explore these large biogeographic datasets. This thesis introduces a novel technique for visualizing hierarchically organized data in a geographic context that illustrates the influence of a geographic or environmental gradient on the phylogenetic relationships between organisms or the similarity of biological communities. This technique is incorporated into GenGIS, open-source software that supports the integration of digital map data with genetic sequences and environmental information from multiple sample sites. GenGIS addresses the need for an interactive geospatial analysis environment capable of handling large biogeographic datasets where a wealth of sequence data is available for each sample site. This is accomplished through a rich set of analysis options that produce georeferenced visualizations for data exploration and hypothesis generation. Studies conducted by myself and other research groups have used GenGIS to investigate the diversity of viruses, bacteria, plants, animals, and even language families. I then explore measures of beta diversity that aim to assess the influence of geographic or environmental gradients on the similarity of biological communities. This thesis examines phylogenetic beta-diversity measures that determine community variation by considering the relationships between organisms in a phylogenetic tree. A large comparative study is performed in order to assess specific properties and performance characteristics of these measures. Many measures of phylogenetic beta diversity were found to be robust to sequence clustering, the addition of an outlying basal lineage, root placement, and the presence of rare organisms. Additionally, performance was found to differ substantially under different models of community variation. This thesis then describes how an important class of phylogenetic beta-diversity measures can be calculated over phylogenetic networks in order to account for uncertainty and conflict in inferred ancestral relationships.

Abstract Mitochondrial DNA (mtDNA) is a maternally inherited 16.6 kbp circular genome that codes for 13 subunits of the mitochondrial respiratory chain, 2 rRNAs and 22 tRNAs. The mutation rate in mtDNA is high and therefore, mutations have accumulated sequentially to lineages that have diverged tens of thousands of years ago. The neutral theory predicts that a proportion of these variations may be slightly deleterious, associated with diseases and selected against, but the issue is still controversial. This study reports an analysis of selection against mutations in mtDNA. First, the population prevalence of one of the most pathogenic mtDNA mutations, the common MELAS mutation (3243A>G), was determined in a population-based screening setting in Northern Ostrobothnia, and the reproductive capacity, or genetic fitness, of women with the mutation was estimated in order to measure for the first time the degree of host-level selection against this highly pathogenic mutation. The frequency of 3243A>G was high, as the minimum estimate for the prevalence was 10.2/100,000, and this together with the geographical distribution of maternal ancestors of the mutation carriers suggested that nuclear genes may be involved in the population history of the mutation. Surprisingly, the genetic fitness of mutation carriers was not reduced, suggesting that the average host-level selection against carriers is not strong. Second, all available complete human mtDNA sequences worldwide (N=847) were collected into a database and analysed for evidence to support the hypothesis concerning slightly deleterious mutations and selective constraints imposed by lineage-specific interactions. 465 distinct missense and 6 nonsense mutations were identified. 48% of the amino acid replacements changed the polarity, 44% hydropathy, 32% aliphaticity, 26% size, 13% aromaticity, and 8% charge. Nonconservative amino acid replacements were found to be more common among the evolutionarily recent mutations than among the older ones, and mutations that have arisen more than once during human evolution showed different properties from the remaining ones. The major continent-specific mtDNA lineages were analysed in terms of nucleotide diversity indices, neutrality tests and nonsynonymous/synonymous rate ratios, and patterns suggesting selective constraints possibly due to lineage-specific interactions were identified. Moreover, a general correlation between nucleotide position and nucleotide polymorphism was identified in the mtDNA. The results are compatible with the assumption that selection has a marked role in human mtDNA evolution and that selective constraints may vary between populations, so that the pathogenic potential of a given mutation may depend markedly on the presence of other, interacting mutations.

<p>This PhD thesis, consisting of an introduction, four papers, and some supplementary results, studies the problem of finding an <i>optimal realization</i> of a given finite metric space: a weighted graph which preserves the metric's distances and has minimal total edge weight. This problem is known to be NP-hard, and solutions are not necessarily unique.</p><p>It has been conjectured that <i>extremally weighted</i> optimal realizations may be found as subgraphs of the <i>hereditarily optimal realization</i> Γ_d, a graph which in general has a higher total edge weight than the optimal realization but has the advantages of being unique, and possible to construct explicitly via the <i>tight span</i> of the metric.</p><p>In Paper I, we prove that the graph Γ_d is equivalent to the 1-skeleton of the tight span precisely when the metric considered is <i>totally split-decomposable</i>. For the subset of totally split-decomposable metrics known as <i>consistent</i> metrics this implies that Γ_d is isomorphic to the easily constructed <i>Buneman graph</i>.</p><p>In Paper II, we show that for any metric on at most five points, any optimal realization can be found as a subgraph of Γ_d.</p><p>In Paper III we provide a series of counterexamples; metrics for which there exist extremally weighted optimal realizations which are not subgraphs of Γ_d. However, for these examples there also exists at least one optimal realization which is a subgraph.</p><p>Finally, Paper IV examines a weakened conjecture suggested by the above counterexamples: can we always find some optimal realization as a subgraph in Γ_d? Defining <i>extremal</i> optimal realizations as those having the maximum possible number of shortest paths, we prove that any embedding of the vertices of an extremal optimal realization into Γ_d is injective. Moreover, we prove that this weakened conjecture holds for the subset of consistent metrics which have a 2-dimensional tight span</p>

This PhD thesis, consisting of an introduction, four papers, and some supplementary results, studies the problem of finding an optimal realization of a given finite metric space: a weighted graph which preserves the metric's distances and has minimal total edge weight. This problem is known to be NP-hard, and solutions are not necessarily unique. It has been conjectured that extremally weighted optimal realizations may be found as subgraphs of the hereditarily optimal realization Γd, a graph which in general has a higher total edge weight than the optimal realization but has the advantages of being unique, and possible to construct explicitly via the tight span of the metric. In Paper I, we prove that the graph Γd is equivalent to the 1-skeleton of the tight span precisely when the metric considered is totally split-decomposable. For the subset of totally split-decomposable metrics known as consistent metrics this implies that Γd is isomorphic to the easily constructed Buneman graph. In Paper II, we show that for any metric on at most five points, any optimal realization can be found as a subgraph of Γd. In Paper III we provide a series of counterexamples; metrics for which there exist extremally weighted optimal realizations which are not subgraphs of Γd. However, for these examples there also exists at least one optimal realization which is a subgraph. Finally, Paper IV examines a weakened conjecture suggested by the above counterexamples: can we always find some optimal realization as a subgraph in Γd? Defining extremal optimal realizations as those having the maximum possible number of shortest paths, we prove that any embedding of the vertices of an extremal optimal realization into Γd is injective. Moreover, we prove that this weakened conjecture holds for the subset of consistent metrics which have a 2-dimensional tight span

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