Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Invasive alien plants have had diverse ecological and social impacts on recipient ecosystems and are a major problem for land managers. Successful management demands an understanding of the ecology of invading taxa. The invasive status and impacts are documented for Prosopis populations in South Africa. However, unresolved taxonomic issues, the extent of hybridization, the applicability of morphology as a species identification approach, and the role that some traits plays in the invasion success have not been studied. This creates a gap that hinders implementation of effective management policies. In this thesis I use a phylogenetic approach to determine the taxonomic make-up of invasive Prosopis populations in South Africa (Chapter 2) and compare the results to morphological identification (Chapter 3). I also look at seedling growth rates in the context of variation in genome size and seed size (Chapter 4). Almost all regions invaded by Prosopis are characterized by taxonomic uncertainty exacerbated by the ease of inter-specific hybridization. In Chapter 2 I aim to resolve taxonomic issues of invasive Prosopis populations in South Africa using a phylogenetic approach. In addition, I aim to unravel the extent of hybridization and the species involved in South Africa. Here, I found that Prosopis populations in South Africa comprise both reported and previously unreported species, indicating a need for a reassessment of the identity of invasive taxa. Hybridization is prevalent and all confirmed species are involved. These findings call for a rethink of legislation and management approaches, e.g. the selection of classical biological control agents. Overall the extent of hybridization indicates that Prosopis species in South Africa comprise a freely inter-breeding population typical of a syngameon. Proper morphological identification of invasive species is crucial for ecological studies and management of invasions. In Chapter 3, I use the total evidence approach to assess whether morphological approaches for identification are adequate for identifying Prosopis species in South Africa. I found that Prosopis taxa in South Africa cannot be reliably distinguished using existing morphological keys. This is likely due mainly to the proliferation of hybrids with a diverse morphology. Therefore, molecular tools are crucial for confirming any morphological identities and for determining the presence of any unreported species. Genome size and seed size have been reported to be associated with invasiveness in a number of plant groups, but not often in a system with multiple hybrids like Prosopis. In Chapter 4, I first investigate the relationship between genome size and seed size in invasive populations of Prosopis spp. in South Africa and secondly I investigate how genome and seed sizes influence germination and early growth. Here I found that genome size loses its distinctness, being diluted in hybridizing populations, but can still be used to assess hybridization events themselves. Large seed size seems to be important for invasiveness as it positively influences germination and early growth. This thesis confirms the taxonomic conundrum of Prosopis species in invasive ranges. This coupled with inadequacy of morphological identification calls for a global study involving native and invasive range taxa to clarify the existing confusions. In view of the presence of unreported Prosopis species in South Africa and extensive hybridization, a rethink of the current legislation and control is needed. / AFRIKAANSE OPSOMMING: Uitheemse indringer plante het grootskaalse ekologiese en sosiale impakte op die ekosisteme wat hulle indring en stel 'n groot uitdaging vir bestuurders van natuurlike hulpbronne. Suksesvolle bestuur en bestryding van indringer plante verg deeglike kennis oor hulle ekologie. Die indringer status en impakte van Prosopis populasies in Suid Afrika is reeds voorheen beskryf. Nieteenstaande, die problematiese taksonomie, die omvang van hibridisasie, die waarde van morfologiese identifikasie, en die rol wat sekere eienskappe speel in die sukses van hierdie groep is nog nie bestudeer nie. Daar is dus 'n gaping in kennis wat die effektiewe beheer van die groep in Suid Afrika belemmer. In hierdie tesis pas ek 'n filogenetiese benadering toe om die taksonomiese verwantskappe van Prosopis populasies in Suid Afrika te bepaal (Hoofstuk 2) en vergelyk my resultate met morfologiese identifikasie sleutels (Hoofstuk 3). Ek ondersoek ook saailing groei tempos in die konteks van variasie in genoom en saad groote in die groep (Hoofstuk 4). Bykans alle areas in Suid Afrika waar Prosopis voorkom word gekenmerk deur taksonomiese onsekerheid, verder bemoeilik deur die gemak waarmee spesies vrylik hibridiseer. Ek vind dat beide bekende en voorheen-onbeskryfde Prosopis spesies in Suid Afrika aangetref word en beklemtoon die behoefte om die identiteit van spesies in die land te hersien. Hibridisasie kom algemeen voor tussen alle spesies teenwoordig in Suid Afrika. Hierdie bevindinge beklemtoon dat wetgewing en beheermaatreêls hersiening benodig, byvoorbeeld in die toepassing van biologiese beheer. In samevatting kom dit voor asof hibridisasie gelei het tot 'n vrytelende Prosopis groep in Suid Afrika, tipies van 'n singameon. Ordentlike morfologiese identifikasie van indringer spesies is belangrik in enige ekologiese studie en die implementering van doeltreffende beheermaatreëls. In Hoofstuk 3 gebruik ek ʼn ‘totale bewys’ benadering om vas te stel of morfologiese eienskappe alleenlik genoegsaam is om Prosopis spesies in Suid Afrika korrek te kan identifiseer. Ek vind dat spesies nie geloofwaardig geïdentifiseer kan word nie, heel moontlik as gevolg van wydverspreide hibridisasie tussen alle spesies teenwoordig in die land. Genoom en saad groote is voorheen geassosieer met die indringer aard van verskeie plant groepe. In Hoofstuk 4 ondersoek ek die verwantskap tussen genoom en saad groote. Tweedens bepaal ek die invloed van genoom en saad groote op ontkieming en vroeë groei eienskappe van Prosopis. My bevindinge toon dat, terwyl die kenmerklikheid van genoom groote verloor word as gevolg van hibridisasie, dit steeds hibridisasie gebeurtenisse per se kan identifiseer. Groot sade het ook ʼn positiewe invloed op die ontkieming en vroeë groei eienskappe van Prosopis. Die tesis bevestig die taksonomiese onduidelikheid van indringer Prosopis taksa in Suid Afrika. Tesame met die onakkuraatheid van morfologiese sleutels beklemtoon my bevindinge die behoefte vir ʼn dringende wêreldwye studie op indringer en inheemse populasies van Prosopis om taksonomiese onsekerhede op te klaar. Die identifikasie van nuwe spesies in Suid Afrika beklemtoon ook die behoefte om huidige wetgewing en beheer van die groep in die land te hersien.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: A molecular, morphological and biological characterisation of the genus Globodera (Nematoda: Heteroderidae) in South Africa is presented. The aims of the study were to determine the spread of the genus in South Africa; to study the systematics and describe the characteristics of the group and to gain a more complete understanding of the biology of the group as agricultural pests. Surveys were conducted in the Cape Floristic Region and in all the potato-producing areas of South Africa. The surveys unearthed new species of cyst nematodes and determined the spread of Globodera rostochiensis in the country. Phylogenetic analysis of sequences from ITS-rDNA was used to infer phylogenetic relationships among cyst nematodes from South Africa. The analyses established the distinct phylogenetic positions of cyst nematode populations from South Africa relative to an array of other cyst nematode species and indicated the existence of four new species of cyst nematodes. Analysis of random amplified polymorphic DNA (RAPD) banding patterns revealed intraspecific genetic variation amongst populations of Globodera rostochiensis. In order to provide molecular protocols for the accurate identification of South African cyst nematodes, species-specific primers and restriction enzymes were tested for their ability to discriminate between local Globodera spp. A combination of the molecular, morphological and morphometric characteristics of these populations were used to describe three new species of cyst nematodes. Experiments to determine the effect of storage temperature on the viability and hatching of South African populations of G. rostochiensis, showed differences in the responses of different populations to different storage temperatures. Experiments to determine the effect of field conditions on the viability and hatching of South African populations of G. rostochiensis, indicated that a decline in viable eggs in cysts from different populations occur, but suggests that the cysts will be able to survive for much longer in these soils than was expected. Spontaneous hatch was the main contributor to the decline of viability of cysts in the soil. Recording of soil temperatures in different locations indicated that the average temperature at 20 cm depth was approximately 20°C, the optimum temperature for the reproduction of G. rostochiensis, as confirmed by in vitro reproduction tests, which also showed that multiplication and survival is influenced negatively when the temperatures rise above 25°C. Reproduction on differential potato clones confirmed the pathotype of all the South African populations of G. rostochiensis as Ro1. After assessing the reproduction of G. rostochiensis on indigenous solanaceous plants, it was concluded that none of these plants induce substantial hatch in G. rostochiensis, nor do they support multiplication of the nematode. The results of this project have an impact on inquiries at all taxonomic levels, while also having an essential practical application in nematology. Knowledge of the distribution, pathogenicity, survival potential and reproduction capacity of Globodera species in South African soils are valuable for the design of effective management strategies as well as regulatory measures. / AFRIKAANSE OPSOMMING: ‘n Molekulêre, morfologiese en biologiese karakterisering van die genus Globodera (Nematoda: Heteroderidae) in Suid-Afrika word aangebied. Die doelwitte van die studieprojek was om die verspreiding van die genus in Suid-Afrika vas te stel, om die sistematiek van die groep te bestudeer en om ‘n meer volledige begrip van die biologie van die groep te bekom. Opnames is in die Kaapse Floristiese streek en in al die aartappelproduserende gebiede in die land gedoen. Tydens die opnames is nuwe sist nematode spesies gevind en die verspreiding van Globodera rostochiensis in Suid-Afrika is vasgestel. Filogenetiese analises van die basispaar opeenvolgings van ITS-rDNS is gebruik om die filogenetiese verwantskappe tussen die sist nematodes van Suid-Afrika vas te stel. Die spesifieke filogenetiese posisies van die nematodes, relatief tot ander spesies het gedui op die teenwoordigheid van 4 nuwe spesies. ‘n Analise van “random amplified polymorphic DNA” (RAPD) bandpatrone het intraspesifieke variasie tussen populasies van G. rostochiensis uitgewys. Diagnostiese tegnieke, aan die hand van spesies-spesifieke inleiers en restriksie-ensieme, is geevalueer vir hul vermoë om Globodera spesies van mekaar te onderskei. ‘n Kombinasie van molekulére, morfologiese en morfometriese karaktertrekke is gebruik vir die beskrywing van drie nuwe Globodera spesies. Eksperimente om die effek van verskillende temperature op die lewenskragtigheid en uitbroei van Suid-Afrikaanse populasies van G. rostochiensis vas te stel, het verskille in die reaksies van die poulasies uitgewys. Eksperimente om die effek van veldtoestande op die lewenskragtigheid en uitbroei van Suid-Afrikaanse populasies van G. rostochiensis vas te stel, het gewys dat alhoewel ‘n afname in die lewenskragtigheid van eiers plaasvind, sal die siste nog steeds langer in die grond oorleef as wat verwag is. Spontane uitbroei van eiers was die grootste oorsaak van die afname in lewenskragtigheid van siste in die grond. Die monitoring van grondtemperature in verskillende lokaliteite, het gewys dat die gemiddelde temperatuur, 20 cm onder die grond, nagenoeg 20°C was. Die optimum temperatuur vir die aanteling van G. rostochiensis, soos gewys deur in vitro toetse is ook 20°C, maar ‘n skerp daling vind plaas by temperature hoer as 25°C. Aanteling op verskillende aartappel cultivars, het gewys dat die patotipe van plaaslike populasies van G. rostochiensis, Ro1 is. Toetse op inheemse Solanum plante het gewys dat die plante nie goeie gashere vir G. rostochiensis is nie. Die bevindings van hierdie studieprojek het ‘n impak op die taksonomie van die groep en kennis van die verspreiding, patogenisiteit en oorlewing van die nematodes onder Suid-Afrikaanse toestande is van waarde vir die daarstelling van effektiewe beheerstrategiee en wetstoepaslike regulasies.

Thesis (PhD (Botany and Zoology))—-Stellenbosch University, 2008. / Streptocarpus Lindl. (Gesneriaceae DC.) is a genus of herbaceous plants containing approximately 160 species, of which the majority occur in Africa and Madagascar. They are largely restricted to shaded and moist habitats such as primary forest and rock outcrops. The genus contains considerable morphological variation, with subgenus Streptocarpella containing caulescent species and subgenus Streptocarpus mostly consisting of acaulescent growth forms, mainly the unifoliates, plurifoliates and rosulates. Preliminary molecular analyses conducted using nuclear ITS sequence data suggested that subgenus Streptocarpus evolved in tropical central Africa, before radiating in several independent waves into southern Africa. Streptocarpus has therefore only recently spread into South Africa. Amongst the South African species, 11 morphologically similar species were identified as being closely related, together forming the Cape primrose clade, based on the analysis of nuclear ITS sequence data. However, these analyses only contained a few South African species, and the ITS data did not provide enough resolution of relationships within this clade. In this study nuclear and chloroplast sequence data as well as nuclear microsatellite data were therefore employed to unravel the complex relationships amongst the South African Streptocarpus species. The analyses indicate that 16 rosulate, palynologically similar species (S. primulifolius, S. rexii, S. johannis, S. baudertii, S. modestus, S. formosus, S. gardenii, S. lilliputana, the S. cyaneus complex [S. cyaneus, S. parviflorus, S. fenestra-dei, S. kunhardtii and S. roseo-albus], S. floribundus, S. aylae and S. kentaniensis), the core Cape primrose species, are closely related, while five unifoliate/plurifoliate, palynologically more variable species (S. denticulatus, S. dunnii, S. pusillus, S. rimicola and S. bolusii) consistently emerged as more distantly related to the core Cape primrose species. However, the positions of a further ten species (S. meyeri, S. montigena, S. fanniniae, S. caeruleus, S. longiflorus, S. polyanthus, S. saundersii, S. porphyrostachys, S. grandis and S. vandeleurii) were more complex in the analyses, indicating that hybridization has played a role in their evolution. Five of these species (S. meyeri, S. montigena, S. fanniniae, S. caeruleus and S. longiflorus) are, however, palynologically homogenous and rosulates, and therefore are probably more closely related to the core Cape primrose species, while the other five (S. polyanthus, S. saundersii, S. porphyrostachys, S. grandis and S. vandeleurii) are unifoliates/plurifoliates that are palynologically more heterogenous, and are probably more distantly related to the core Cape primrose species. Amongst the core Cape primrose species, S. primulifolius emerged as being ancestral or having hybridized with many of the other species, while the S. cyaneus complex forms a geographically and genetically more isolated group. However, evolutionary relationships amongst these species were to a certain extent obscured by incomplete lineage sorting caused by limited interpopulation gene flow, frequent hybridization and rapid speciation. The analyses confirmed that the Pondoland Centre forests constitute important Pleistocene refugia, and revealed some of the historical migration routes along which the species had radiated.

Psiguria Arn. is a small genus of Neotropical vines in the Cucurbitaceae that grows in both wet and dry tropical forests from southern Mexico to Paraguay, and on Caribbean islands. The genus is estimated to be very young with natural history characteristics that have contributed to confusing species circumscriptions. The unique relationship of plants in the group with their butterfly pollinators makes Psiguria an interesting and important genus in tropical ecosystems. Both molecular and morphological approaches were used to investigate the monophyly of Psiguria, to elucidate the number of species in the genus, to discover sister relationships, and to identify characteristics for delineating species. Toward that end, an intensive screening of 141 primer combinations in search of phylogenetically informative low-copy nuclear markers was conducted along with a molecular phylogenetic analysis and a complete taxonomic revision of Psiguria. From the screening study, three potentially phylogenetically informative low-copy nuclear markers were discovered for Psiguria, 11 were found to be potentially useful in rosids, and 32 in other angiosperms. DNA sequences for eight chloroplast intergenic spacers (ndhF-rpL32, ndhC-trnV, rps16-trnQ, trnS-trnG, psbZ-trnM, psbM-trnD, rpoB-trnC, and psbE-petL), ITS, and the nuclear serine/threonine phosphatase intron were obtained for 70 samples of Psiguria plus 14 outgroups. Phylogenetic analyses support the monophyly of Psiguria and a sister relationship between P. umbrosa and P. warscewiczii. In the final chapter, two reviews on the genus are presented – one encapsulating the nomenclatural history, and one summarizing 35 years of ecological and natural history studies. In addition, morphological characters were databased, descriptions were written, and maps of geographic distribution were produced for all species. Considering both molecular and morphological data, six species of Psiguria are defined. To distinguish those species missing identifiable morphological characters, a set of DNA barcodes was developed. At least four chloroplast regions are required to differentiate species (ndhC-trnV, rps16- trnQ, rpoB-trnC, and ndhF-rpL32). Because of the absence of many morphological characters, two taxonomic keys are presented – one using male flowers, and the other using the set of DNA barcodes along with consistent leaf characteristics and geographic distribution. / text

A Profile Hidden Markov Model (HMM) is a statistical model for representing a multiple sequence alignment (MSA). Profile HMMs are important tools for sequence homology detection and have been used in wide a range of bioinformatics applications including protein structure prediction, remote homology detection, and sequence alignment. Profile HMM methods result in accurate alignments on datasets with evolutionarily similar sequences; however, I will show that on datasets with evolutionarily divergent sequences, the accuracy of HMM-based methods degrade. My dissertation presents a new statistical model for representing an MSA by using a set of HMMs. The family of HMM (fHMM) approach uses multiple HMMs instead of a single HMM to represent an MSA. I present a new algorithm for sequence alignment using the fHMM technique. I show that using the fHMM technique for sequence alignment results in more accurate alignments than the single HMM approach. As sequence alignment is a fundamental step in many bioinformatics pipelines, improvements to sequence alignment result in improvements across many different fields. I show the applicability of fHMM to three specific problems: phylogenetic placement, taxonomic profiling and identification, and MSA estimation. In phylogenetic placement, the problem addressed is how to insert a query sequence into an existing tree. In taxonomic identification and profiling, the problems addressed are how to taxonomically classify a query sequence, and how to estimate a taxonomic profile on a set of sequences. Finally, both profile HMM and fHMM require a backbone MSA as input in order to align the query sequences. In MSA estimation, the problem addressed is how to estimate a ``de novo'' MSA without the use of an existing backbone alignment. For each problem, I present a software pipeline that implements the fHMM specifically for that domain: SEPP for phylogenetic placement, TIPP for taxonomic profiling and identification, and UPP for MSA estimation. I show that SEPP has improved accuracy compared to the single HMM approach. I also show that SEPP results in more accurate phylogenetic placements compared to existing placement methods, and SEPP is more computationally efficient, both in peak memory usage and running time. I show that TIPP more accurately classifies novel sequences compared to the single HMM approach, and TIPP estimates more accurate taxonomic profiles than leading methods on simulated metagenomic datasets. I show how UPP can estimate ``de novo'' alignments using fHMM. I present results that show UPP is more accurate and efficient than existing alignment methods, and estimates accurate alignments and trees on datasets containing both full-length and fragmentary sequences. Finally, I show that UPP can estimate a very accurate alignment on a dataset with 1,000,000 sequences in less than 2 days without the need of a supercomputer. / Computer Sciences / text

No description available.

Hepatitis C virus (HCV) causes severe illness in millions of people worldwide, but the epidemic strains responsible for most infections arose within the past hundred years and represent only a small part of total HCV diversity. In this thesis I combine laboratory and computational methods to study HCV in Africa. I aim to characterize its current genetic diversity and its historical transmission prior to the global HCV epidemic. In Chapter 2 I begin by screening samples from the Democratic Republic of the Congo (DRC) for HCV and the related human pegivirus. I find high HCV sequence diversity, including a putative new subtype, and find significantly higher HCV prevalence in those born before 1950. Chapter 3 continues this screening, and combines the sequences obtained with those from online databases. Using molcular clock methods I estimate that genotype 4 originated in central Africa around 1733, and that multiple lineages, including subtype 4a which dominates the HCV epidemic in Egypt, have moved to north Africa since ~1850. In Chapter 4 I analyse sequences sampled from an elderly population in Kinshasa to estimate HCV’s transmission history there during the 20th century. The results indicate a rapid increase in HCV transmission between 1950 and 1970 in multiple independent lineages. Possible causes of this increase are discussed. This study population also exhibits high HCV genetic diversity, including the second genotype 7 sample discovered to date. Finally, Chapter 5 uses a range of sequencing techniques, including RNAseq, to characterise two putative HCV recombinants from Cameroon. I confirm that both sequences are recombinants, and generate a full genome sequence for one. I also develop new tools to distinguish between dual infection and recombination in next-generation sequencing data, and discuss how recombination might affect HCV diversity and treatment.

The genus Schefflera is the largest in Araliaceae, with approximately 900 species. Recent studies have shown that Schefflera is polyphyletic and represents no fewer than five distinct clades, each corresponding to a specific geographic region including Asia, continental Africa and Madagascar, Melanesia, the Neotropics, and a small clade distributed throughout several islands in the insular Pacific Ocean. The Afro-Malagasy clade contains nearly 50 species distributed throughout tropical, sub-Saharan Africa, Madagascar, the Comoros, and the Seychelles islands. Previous studies have suggested that this group is monophyletic, identifying two smaller subclades within Afro-Malagasy Schefflera corresponding roughly to informal groups identified as “Meiopanax” and “Sciodaphyllum” on the basis of morphology. Using sequence data from nuclear rDNA spacers and plastid markers derived from 32 of the 48 currently circumscribed species of Afro-Malagasy Schefflera, this study tested the monophyly of Afro-Malagasy Schefflera and of each of its two proposed subclades. Trees based on this molecular data were used to examine patterns of morphological evolution and biogeography among species in the clade. Results support the monophyly of Afro-Malagasy Schefflera and both subclades, which correspond closely to “Meiopanax” and “Sciodaphyllum” which are herein referred to as Neocussonia and Astropanax, respectively. Additional interspecific relationships were examined, which provides evidence for hybridization among several species. Schefflera myriantha, the most widely distributed species of Afro-Malagasy Schefflera, is paraphyletic with respect to two other species, S. humblotiana and S. monophylla. Many morphological features historically used to distinguish species of Afro-Malagasy Schefflera appear to be evolutionarily labile, with a history of gains and losses (e.g., reduction in leaflet number, which occurs independently in both subclades). Biogeographic analyses suggest an African ancestry for the entire Afro-Malagasy Schefflera clade, and for both subclades, with two independent divergence events to Madagascar.

Microbial communities are recognized as major drivers of global biogeochemical processes. However, the genetic diversity and composition, as well as processes leading to the origin and diversification of these communities in space and time, are poorly understood. Character- ization of microbial communities using high-throughput sequencing of 16S tags shows that Operational Taxonomic Unit (OTU) abundances can be approximated by a gamma distribu- tion, which suggests structuring around small numbers of highly abundant OTUs and a large proportion of rare OTUs. The current methods used to characterize how communities are structured rely on multivariate statistics, which operate on pair-wise distance matrices. My analyses demonstrate that use of these methods, by reducing a highly-dimensional data set (tens of samples, thousands of OTUs), results in a significant loss of information. I demon- strate that, in some cases, up to 80% of the least abundant OTUs may be removed while still recovering the same community relationships; this indicates these metrics are biased toward the highly abundant OTUs. I also demonstrate that the observed patterns of OTU abundance detected from microbial communities can be robustly modeled using techniques similar to those used to model the presence and absence of genes in genome evolution. Using simulation studies, I show that general Markov models in a Bayesian inference framework out- perform traditional, multivariate ecological methods in recovering true community structure. Applying this new methodology to Atlantic Ocean communities uncovered a distance-decay effect which was not revealed by the traditional methods; applying to communities discov- ered on Hog Island point toward mechanisms of thicket establishment. Although the ocean data set operated on a much larger, continental scale, characterization of the sequence data generated from the nutrient-poor soil on Hog Island, a barrier island off the Virginia coast, allows for a better characterization of the processes affecting these communities on a much smaller scale. Finally, using 16S data from the Human Vaginal Microbiome Project, gener- ated here at VCU under the umbrella of the overall NIH HMP initiative, I give examples of the quality control, analysis and visualization pipeline that I developed to support the efforts of this project. In conclusion, my analyses of the metagenomic sequence data from bacterial communities sampled from different environments demonstrate that the proper identification of the biological processes influencing these communities requires the development and im- plementation of new statistical and computational methodologies that take advantage of the extensive amount of information generated in next-generation, high-throughput sequencing projects.

In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.