This thesis concerns the history and the intrafamilial delimitations of the plant family Colchicaceae. A phylogeny of 73 taxa representing all genera of Colchicaceae, except the monotypic Kuntheria, is presented. The molecular analysis based on three plastid regions—the rps16 intron, the atpB-rbcL intergenic spacer, and the trnL-F region—reveal the intrafamilial classification to be in need of revision. The two tribes Iphigenieae and Uvularieae are demon-strated to be paraphyletic. The well-known genus Colchicum is shown to be nested within Androcymbium, Onixotis constitutes a grade between Neodregea and Wurmbea, and Gloriosa is intermixed with species of Littonia. Two new tribes are described, Burchardieae and Tripladenieae, and the two tribes Colchiceae and Uvularieae are emended, leaving four tribes in the family. At generic level new combinations are made in Wurmbea and Gloriosa in order to render them monophyletic. The genus Androcymbium is paraphyletic in relation to Colchicum and the latter genus is therefore expanded. An investigation of the distribution of colchicine within the expanded Colchicaceae is conducted to evaluate the potential of colchicine as a synapomorphy of the re-circumscribed family. The results demonstrate presence of colchicine in all genera previously not examined in Colchicaceae and in the genus Burchardia, earlier reported to lack colchicine. Hence, demonstrating colchicine to be a synapomorphy for the family. An attempt to date the phylogeny of the order Liliales together with a dispersal-vicariance (DIVA) analysis indicates that the split between Colchicaceae and Alstromeriaceae-Luzuriagaceae represents a vicariance event following the disintegration of the Australian-Antarctican-South American link, ~34 million years ago. Further, the DIVA analysis indicates that Colchicaceae originated in Australia, first reached Asia and North America, and later Africa, from where they expanded to Europe and also dispersed back to Australia.

Inferring phylogeny is a difficult computational problem. Heuristics are necessary to minimize the time spent evaluating non optimal trees. In paper I, we developed an approach for heuristic searching, using a genetic algorithm. Genetic algorithms mimic the natural selections ability to solve complex problems. The algorithm can reduce the time required for weighted maximum parsimony phylogenetic inference using protein sequences, especially for data sets involving large number of taxa. Evaluating and comparing the ability of phylogenetic methods to infer the correct topology is complex. In paper II, we developed software that determines the minimum subtree prune and regraft (SPR) distance between binary trees to ease the process. The minimum SPR distance can be used to measure the incongruence between trees inferred using different methods. Given a known topology the methods could be evaluated on their ability to infer the correct phylogeny given specific data. The minimum SPR software the intermediate trees that separate two binary trees. In paper III we developed software that given a set of incongruent trees determines the median SPR consensus tree i.e. the tree that explains the trees with a minimum of SPR operations. We investigated the median SPR consensus tree and its possible interpretation as a species tree given a set of gene trees. We used a set of α-proteobacteria gene trees to test the ability of the algorithm to infer a species tree and compared it to previous studies. The results show that the algorithm can successfully reconstruct a species tree. Expressed sequence tag (EST) data is important in determining intron-exon boundaries, single nucleotide polymorphism and the coding sequence of genes. In paper IV we aligned ESTs to the genome to evaluate the quality of EST data. The results show that many ESTs are contaminated by vector sequences and low quality regions. The reliability of EST data is largely determined by the clustering of the ESTs and the association of the clusters to the correct portion of genome. We investigate the performance of EST clustering using the genome as template compared to previously existing methods using pair-wise alignments. The results show that using the genome as guidance improves the resulting EST clusters in respect to the extent ESTs originating from the same transcriptional unit are separated into disjunct clusters.

The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen. The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as rbcL, atpB and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales. To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative Osyris alba (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides. In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.

Heterodrilus is a marine group of small (3-25 mm) clitellates that occur interstitially in sandy sediments from the intertidal zone down to about 150 m depths. The taxon includes 42 valid species and has been recorded from localities in the Mediterranean Sea, the North-west Atlantic Ocean (including the Caribbean), the Galapagos Islands, and the Indo-Pacific Region. A majority of the species of Heterodrilus are characterized by having trifid anterior chaetae (i.e., chaetae with three teeth at the distal end). A few species have bifid chaetae, but these are regarded as having lost the third tooth secondarily. Within the taxon, species are distinguished by morphological characters in the internal organization of the male and female genitalia, but also by characters in the form and number of chaetae. In this thesis, two new taxa are introduced, Heterodrilus tripartitus and H. ursulae, both from the Mediterranean Sea. The ultrastructure of the cuticle in four species (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is studied and it is shown that there is interspecific variation in the morphology of the cuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The systematic position of Heterodrilus within Tubificidae is studied with molecular markers, and the results support that Heterodrilus (earlier classified as a member of Rhyacodrilinae) is positioned within Phallodrilinae. Furthermore, the phylogenetic relationships within Heterodrilus are estimated based on molecular characters from mitochondrial COI, the 16S rRNA gene, and the nuclear 18S rRNA gene, and the result indicate that the two major clades in our tree corresponds to different geographical distributions. This thesis also includes a checklist, as well as a key, to the species of Heterodrilus.

MicroRNAs are important biomolecules for regulating biological processes. Moreover, the secondary structure of microRNA is important for its activity and has been used previously as a mean for finding unknown microRNAs. A phylogenetic study of the microRNA secondary structure reveals more information than its primary sequence, because the primary sequence can undergo mutations that give rise to different phylogenetic relationships, whereas the secondary structure is more robust against mutations and therefore sometimes  more informative. Here we constructed a phylogenetic tree entirely based on microRNA secondary structures using tools PHYLIP (Felsenstein, 1995) and RNAforester (Matthias Höchsmann, 2003, Hochsmann et al., 2004), and compared the overall topology and clusters with the phylogenetic tree constructed using microRNA sequence. The purpose behind this comparison was to investigate the sequence and structure similarity in phylogenetic context and also to investigate if functionally similar microRNA genes are closer in their structure-derived phylogenetic tree. Our phylogenetic comparison shows that the sequence similarity has hardly any effect on the structure similarity in the phylogenetic tree. MicroRNAs that have similar function are closer in the phylogenetic tree based on secondary structure than its respective sequence phylogeny. Hence, this approach can be very useful in predicting the functions of the new microRNAs whose function is yet to be known, since the function of the miRNAs heavily relies on its secondary structure.

The thesis is divided into two parts, the phylogeny of the Geothelphusa spp. from Taiwan and the adaptive strategy of Geothelphusa ancylophallu from the badland in the southwestern Taiwan. Through SEM studies, the male first gonopod (G1) microstructures of 22 species of Geothelphusa are compared. From the Numerical Analysis of 11 G1 measurements, it shows that Geothelphusa spp can be divided into the eastern and the western geographic groups in Taiwan. In addition, the main differences in G1 micro-morphology among Geothelphusa, including the rings of scales, the numbers of simple seta on the terminal segment, and the ratios of total length of G1 divided by the terminal segment length of G1. Twenty-seven species of Geothelphusa from type locality in Taiwan were compared via the sequences of mtDNA COI (658 bp). The results indicate that the ancestors of Geothelphusa landed onto the northwestern Taiwan about 3.74 mya. After that, Geothelphusa dispersed from northern Taiwan into the eastern and the western geographic groups, this result was consisted with the previous conclusion obtained by G1 SEM studies. The eastern geographic group was further dispersed to the northeast and evolved to the present-day Ilan Geothelphusa spp. Another subgroup distributed down south to the eastern mountains where are now Lishan area. The last subgroup reached more southern to the Hengtsuen peninsula, Lanyu, and Greenisland. The western geographic group was further dispersed down south and evolved to the present Geothelphusa olea and G. nanhsi which then evolved to G. caesia¡BG. wutai and G. tsayae. Another subgroup evolved to G. ancylophallus which then evolved to G. lili¡BG. shernshan¡BG. neipu and G. pingtung. The genetic structures of Geothelphusa in Kaoping River area were studied further via the sequences of mtDNA COI (658bp). The results indicate that G. tsayae have three main subgroups according to the river they live on (Nantsishan River, Laolong River, Eyeliou River). Among them, the Eyeliou-River subgroup has the sequences divergence of 1.2% to the G. wutai, with supporting rate of 73%-82%, indicating that they may belong to the same species. In addition, Laolong-River subgroup has the sequences divergence of 0.9% to the G. caesia group, with supporting rate of 60%, indicating that they may be the same species as well but more evidences are needed to clarify the situation. The adaptive strategy of the crabs Geothelphusa ancylophallu, distribute near the badland in the southwestern Taiwan, was studied. The strategies are as follows: 1) the breeding activity correlated to precipitation, mainly occurring in spring and summer, and the maximum in June. 2) The female crabs breed 32.5 offspring in average and the mean carapace width of the first stage crabs (C1) was 5.3mm. The C1 stayed with their mother for about 4 days. 3) G. ancylophallu finished building holes before the dry season and the holes could be used until the next wet season. The holes are used for drought-resistance and defending predators during the dry season and mating and breeding during the wet season. 4) G. ancylophallu could defense their holes. The residents spent 152.3 ¡Ó 110.1 seconds driving the invaders out of their holes and attacked them actively when near their holes. The residents¡¦ alert to sound from the ground. 5) Through the experiment of hole covering, 67.0% of the valid holes were used more than 4 months, with the longest of 13 months. The moving distance of G. ancylophallu at night was 146.8 ¡Ó 73.8 cm (n = 38). The G. ancylophallu are fidelity to their holes, especially in dry season.

Background: Long terminal repeats (LTRs, consisting of U3-R-U5 portions) are important elements of retroviruses and related retrotransposons. They are difficult to analyse due to their variability. The aim was to obtain a more comprehensive view of structure, diversity and phylogeny of LTRs than hitherto possible. Results: Hidden Markov models (HMM) were created for 11 clades of LTRs belonging to Retroviridae (class III retroviruses), animal Metaviridae (Gypsy/Ty3) elements and plant Pseudoviridae (Copia/Ty1) elements, complementing our work with Orthoretrovirus HMMs. The great variation in LTR length of plant Metaviridae and the few divergent animal Pseudoviridae prevented building HMMs from both of these groups. Animal Metaviridae LTRs had the same conserved motifs as retroviral LTRs, confirming that the two groups are closely related. The conserved motifs were the short inverted repeats (SIRs), integrase recognition signals (5' TGTTRNR ... YNYAACA 3'); the polyadenylation signal or AATAAA motif; a GT-rich stretch downstream of the polyadenylation signal; and a less conserved AT-rich stretch corresponding to the core promoter element, the TATA box. Plant Pseudoviridae LTRs differed slightly in having a conserved TATA-box, TATATA, but no conserved polyadenylation signal, plus a much shorter R region. The sensitivity of the HMMs for detection in genomic sequences was around 50% for most models, at a relatively high specificity, suitable for genome screening. The HMMs yielded consensus sequences, which were aligned by creating an HMM model (a 'Superviterbi' alignment). This yielded a phylogenetic tree that was compared with a Pol-based tree. Both LTR and Pol trees supported monophyly of retroviruses. In both, Pseudoviridae was ancestral to all other LTR retrotransposons. However, the LTR trees showed the chromovirus portion of Metaviridae clustering together with Pseudoviridae, dividing Metaviridae into two portions with distinct phylogeny. Conclusion: The HMMs clearly demonstrated a unitary conserved structure of LTRs, supporting that they arose once during evolution. We attempted to follow the evolution of LTRs by tracing their functional foundations, that is, acquisition of RNAse H, a combined promoter/polyadenylation site, integrase, hairpin priming and the primer binding site (PBS). Available information did not support a simple evolutionary chain of events.

The superfamily of G protein-coupled receptors (GPCRs) is one of the largest protein families of mammalian genomes and can be divided into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin. GPCRs participate in most major physiological functions, contributing to the fact that they are important targets in drug discovery. In paper I we mined the human and mouse genomes for new Adhesion GPCR genes. We found two new human genes (GPR133 and GPR144) and 17 mouse Adhesion genes, bringing the number up to 33 human and 31 mouse genes. In paper II we describe 53 new splice variants for human Adhesion receptors supported by expressed sequence tags (EST) data. 29 of these variants seem to code for functional proteins, several of which lack one or more functional domains in the N-termini. Lack of certain domains is likely to affect ligand binding or interaction with other proteins. Paper III describes the Glutamate GPCR in human, mouse, Fugu, and zebrafish. We gathered a total of 22 human, 79 mouse, 30 Fugu, and 32 zebrafish sequences and grouped these into eight clans using phylogenetic methods. The report provides an overview of the expansion or deletions among the different branches of the Glutamate receptor family. Paper IV focuses on the trace amine (TA) clan of Rhodopsin GPCRs. We identified 18 new rodent genes, 57 zebrafish genes, and eight Fugu genes belonging to the clan. Chromosomal mapping together with phylogenetic relationships suggests that the family arose through several mechanisms involving tetraploidisation, block duplications, and local duplication events. Paper V provides a comprehensive dataset of the GPCR superfamily of human and mouse containing 495 mouse and 400 human non-olfactory GPCRs. Phylogenetic analyses showed that 329 of the receptors are found in one-to-one orthologous pairs, whereas other receptors may have originated from species-specific expansions.

Collaboration and communication between conventionally diverse fields can allow for deeper understanding and clearer analysis of the concepts within each. Two fields traditionally seen as dichotomous are those of art and science. Historically they approach problems in opposite ways. However, I would argue that they in fact investigate very similar questions, hoping to discover the ways that the world works. It makes sense, then, that historically these fields have sometimes been able to interact. Artists have engaged with science by creating work through scientific processes including crossbreeding flowers, genetically modifying organisms, and sequencing nucleotides. Others have referenced scientific ideas, like those of order or sustainability, through more traditional methods. My thesis project, Adenine Uracil Guanine, is a sculptural installation portraying the phylogenetic tree of all life in a three-dimensional form. Borrowing from the aesthetic of mobiles, the sculpture takes a recognizably itinerant form, referencing the fluidity and malleability of evolution. The structure’s white base, alluding to the sterility and cleanliness of a phylogenetic tree’s aim to diagrammatize change, is overlayed by a system of colored bands. These bands reference the nucleotide sequences upon which phylogenetic trees are based. By using an artistic lens to view the scientific process of evolution and its elucidation and representation, I hope to continue to encourage a dialogue between the two fields.

The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested. / October 2008