Tracking seed dispersal using traditional, direct measurement approaches is difficult and generally underestimates dispersal distances. Variation in chloroplast haplotypes (cpDNA) offers a way to trace past seed dispersal and to make inferences about factors contributing to present patterns of dispersal. Although cpDNA generally has low levels of intraspecific variation, this can be overcome by assaying the whole chloroplast genome. Whole-genome sequencing is more expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among SNPs are lost in pooled samples and treating SNP frequencies as independent estimates of variation provides biased estimates of genetic distance. I have developed an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP frequencies from pooled samples based on network topology, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially-constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from Whetstone Prairie, in Southern Oregon, USA. In test networks, CallHap reliably recovered network topologies and haplotype frequencies. Overall, the CallHap pipeline allows for the efficient use of resources for estimation of genetic distance for studies using non-recombining, whole-genome haplotypes, such as intra-specific variation in chloroplast, mitochondrial, bacterial, or viral DNA.

<p>The great difference in the level of chloroplast variation and its geographic structure among the three main species studied here demonstrates that forest species do not form a homogeneous group. Hazel shows a genetic structure similar to many other thermophilous species and this structure, in combination with fossil evidence, indicates that the post-glacial colonisation of most of Europe originated in a refugium in western France while the Balkan and Italy were colonised from a south-eastern refugium.</p><p>In sallow and silver birch the chloroplast DNA variation and its structure does not fit with a scenario of glacial restriction to southern refugia and survival at intermediate latitudes is suggested for both species. The chloroplast DNA variation in silver birch suggests the presence of one western and one eastern European post-glacial colonisation route and limited contribution of southern populations in the colonisation of the rest of Europe. Unique haplotypes by the Ural Mountains indicates the possibility of a separate glacial origin of these populations.</p><p>The study of chloroplast DNA in species closely related to sallow and silver birch indicate that extensive hybridisation and cytoplasmic gene flow occurs within both the Salix and Betula genera in Europe. The nuclear and chloroplast phylogenies of 14 Betula species were not in complete agreement with each other or with the classical division of the Betula genus into subgenera or sections. The phylogenetic structure implies that hybridisation has played a role in the evolution of the Betula genus.</p><p>This thesis focuses on the chloroplast DNA variation in three forest tree genera: Corylus, Betula and Salix. Chloroplast PCR-RFLP is used to evaluate the post-glacial history of hazel, Corylus avellana, silver birch, Betula pendula and sallow, Salix caprea and to explore the possibility of introgression in the Salix and Betula genera. In addition, the chloroplast matK gene, its flanking regions and the nuclear ADH gene were used to study the phylogenetic relationships within the Betula genus.</p>

Wheat breeders must effectively maintain and manage existing genetic diversity in order to continue the development of superior genotypes. It is therefore fundamental that the genetic relationships and diversity within the germplasm pools be thoroughly characterized and understood. Recently, DNA-based markers have provided powerful tools for genetic diversity analysis. This study investigates the usefulness of nuclear and chloroplast simple sequence repeat (SSR) markers in characterizing Pacific Northwest wheat (Triticum aestivum) breeding germplasm, and explores the patterns of genetic relatedness revealed by these markers. The 15 chloroplast SSRs were effective in differentiating between B-type, D-type, and barley (Hordeum vulgare) cytoplasms. Genetic distance estimates were determined for each pair of lines studied and analyzed using UPGMA clustering. The markers revealed five plastomic types within the B-type cytoplasm studied. Several lines of wheat in this germplasm, including important PNW cultivars like Madsen, were found to contain D-type cytoplasm rather than the B cytoplasm of wheat. Nuclear SSR assays using 24 markers revealed three major clusters of germplasms: PNW soft white winter wheat, Western European-derived lines, and Great Plains accessions, as well as two clusters of more distantly related lines and genetic stocks. The primary defining characteristic of these clusters was regional adaptation. Subgroups of these major groups often clustered together on the basis of pedigree and market class. When nuclear and chloroplast SSR data was combined in analysis, the primary defining characteristic of the dendrogram became the type of cytoplasm rather than regional adaptation, with secondary divisions based on pedigree relationships. Cultivars released prior to 1950 were found to have a minimum of 20% of alleles in common for nuclear and chloroplast data combined, despite being unrelated via pedigree information. Heterogeneity was 2.3% for all marker/variety combinations. Overall, these sets of markers were found to be effective in characterizing the genetic relatedness of PNW wheat breeding germplasm. / Graduation date: 2003

The great difference in the level of chloroplast variation and its geographic structure among the three main species studied here demonstrates that forest species do not form a homogeneous group. Hazel shows a genetic structure similar to many other thermophilous species and this structure, in combination with fossil evidence, indicates that the post-glacial colonisation of most of Europe originated in a refugium in western France while the Balkan and Italy were colonised from a south-eastern refugium. In sallow and silver birch the chloroplast DNA variation and its structure does not fit with a scenario of glacial restriction to southern refugia and survival at intermediate latitudes is suggested for both species. The chloroplast DNA variation in silver birch suggests the presence of one western and one eastern European post-glacial colonisation route and limited contribution of southern populations in the colonisation of the rest of Europe. Unique haplotypes by the Ural Mountains indicates the possibility of a separate glacial origin of these populations. The study of chloroplast DNA in species closely related to sallow and silver birch indicate that extensive hybridisation and cytoplasmic gene flow occurs within both the Salix and Betula genera in Europe. The nuclear and chloroplast phylogenies of 14 Betula species were not in complete agreement with each other or with the classical division of the Betula genus into subgenera or sections. The phylogenetic structure implies that hybridisation has played a role in the evolution of the Betula genus. This thesis focuses on the chloroplast DNA variation in three forest tree genera: Corylus, Betula and Salix. Chloroplast PCR-RFLP is used to evaluate the post-glacial history of hazel, Corylus avellana, silver birch, Betula pendula and sallow, Salix caprea and to explore the possibility of introgression in the Salix and Betula genera. In addition, the chloroplast matK gene, its flanking regions and the nuclear ADH gene were used to study the phylogenetic relationships within the Betula genus.

Traditional Chinese medicine has a long history of using plants therapeutically including multiple species of the genus Rubus (Rosaceae). Fruits and other parts of Rubus plants have had a significant effect on human health and nutrition in both ancient and modern times. The pharmacological effects of Rubus include anti-inflammatory, antibacterial, anti-stress, anti-cancer and anti-aging properties. One of the current challenges limiting further development of Rubus resources in traditional Chinese medicine is a poor understanding of phylogenetic relationships among Rubus species in general and especially among Asian species, and also the need for additional studies of phytochemicals. Several confounding factors are frequent hybridization, polyploidy, and highly variable morphology due in part to diverse ecological conditions across species’ distributions. The goal of this study was to elucidate phylogenetic relationships among Rubus species in the predominantly Asian subgenera Idaeobatus and Malachobatus emphasizing species valued in traditional Chinese medicine. Sequences of six noncoding (plus matK) chloroplast DNA regions totaling 8,276 aligned characters were analyzed for 35 Rubus species using maximum parsimony (MP) and maximum likelihood (ML). Both analytical approaches yielded topologically identical phylogenies except for one additional grouping in the ML tree. The phylogeny has nearly complete resolution and divides the species into two primary clades; one comprises R. geoides (representing the Southern Trans-Pacific clade), R. nivalis (subg. Chamaebatus) and R. trivialis (representing subg. Rubus) and the other is composed largely of R. arcticus and R.saxatilis (subg. Cylactis), and the large subgenera Idaeobatus (raspberries) and Malachobatus. Within the latter, principally Asian clade, three unresolved lineages exist (four using MP) precluding an improved understanding of the relationships among them. However, three major subclades containing Asian species have good support. Two contain subg. Idaeobatus species only, and the third comprises members of the exclusively polyploid subgenera Malachobatus and Dalibardastrum. Examination of the presence of biochemically active terpenes reveal that triterpenes are common among subg. Idaeobatus species with diterpenes reported only in R. pungens and R. chingii (not sampled). Subg. Malachobatus species have not been as thoroughly investigated so triterpenes may also be common in these species. From these results, multiple subg. Idaeobatus and subg. Malachobatus species may be good candidates for terpene analysis as members of their respective clades possess triterpenes. Moreover, five species not currently used in traditional Chinese medicine should be evaluated as they also occur in China, and may possess medicinal value.

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

This thesis examined aspects of the reproductive and molecular biology of Eucalyptus marginata (jarrah). The aims were to develop protocols for controlled pollination, that could be used in clonal orchard trees to breed jarrah seedlings that have a known genetic resistance to Phytophthora cinnamomi (dieback), for use in rehabilitation after mining and logging. An intimate knowledge of the breeding biology of jarrah was necessary to achieve this aim. The project also aimed to increase knowledge of the genetic diversity and structure of jarrah, in order to make informed decisions regarding the collection of material to be used for clonal propagation. Previous research has had little success in producing viable seed from any controlled pollinations, but clonal material resistant to P. cinnamomi has been produced using tissue culture. The question posed in this thesis was ‘Can we improve breeding and propagation techniques of jarrah?’ Techniques were developed for testing of in vitro pollen viability and pollen storage, pollination and fertilisation success after controlled pollinations, including determination of stigma receptivity and development of bud isolation techniques using alfoil. The variation in female fertility between genotypes was examined. The use of paclobutrazol was explored as a method of increasing the level of viable seed production in clonal orchard trees. The use of fertiliser as well as the growth retardant was also explored to see if it increased the level of seed production even more. Genetic diversity, genetic differentiation and phylogeny within Eucalyptus marginata were examined using nuclear and chloroplast DNA analysis with Restricted Fragment Length Polymorphisms. While it was first thought that the fertilisation rate was quite low, it was confirmed that the fertilisation rate is similar to other eucalypt species. The zygote abortion rate was quite high in one clone, but one wild tree had a similar seed production rate to other eucalypt species. The zygote and endosperm appeared to be different in the clone and the wild tree observed. The level of seed production was examined in clones and wild trees and it was found that the level was often quite low, particularly in the clones (0 – 13% in clones, 0 – 18% in wild trees) in comparison with other Eucalyptus species, and varied between genotypes. The use of a growth retardant such as paclobutrazol may increase the production of viable seed, if it is applied during autumn. The results were inconclusive for the fertiliser/paclobutrazol experiment, since the paclobutrazol was applied during spring which was the worst time of year for increasing seed production. There were differences between genotypes in reaction to both the paclobutrazol and the fertiliser/paclobutrazol. Genetic diversity was moderate in comparison with other Eucalyptus species, and there was a low level of genetic differentiation between populations in the nuclear genome. No differentiation was observed between the morphologically recognised subspecies in the nuclear genome, but differentiation between the populations on the Swan Coastal Plain and populations on the Darling Plateau was seen in the chloroplast genome, indicating that there was historical separation of these two areas. The conclusions arising from this work are that while controlled pollinations are possible in Eucalyptus marginata the clones that were used in these experiments have often behaved differently to the wild trees in the time of anthesis and levels of viable seed production, and in one clone (5J119) the zygote and endosperm nuclei appeared to be very different to the zygote and endosperm nuclei of a wild tree. Further investigation is necessary to see if these differences are related to the low level of seed production observed in the clonal populations. Paclobutrazol may be worth exploring further as a means of increasing seed production. Material to be used for rehabilitation and seed orchards can be collected from a wide area in the main distribution of the species, although trees on the Swan Coastal Plain are distinct from the trees in the main forest area in the chloroplast genome.

Thesis (M.S.)--Wake Forest University. Dept. of Biology, 2003. / Vita. Includes bibliographical references (leaves 20-22).

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv & Cons Biol) / Salicornia tegetaria is an endemic salt marsh macrophyte that is widely distributed in estuaries along the South African coast. The aims of the study were to understand the phylogeography of the species, compare the biomass allocation in two regions and to determine phenological patterns of S. tegetaria between the warm and cool temperate biogeographical regions. The phylogeography of S. tegetaria was studied using the noncoding chloroplast DNA region rpS16 and nuclear rDNA ITS region. Five samples each were collected from eighteen estuaries stretching from Orange River in the Northern Cape to Mngazana Estuary in the Eastern Cape. Above- and belowground biomass was collected and physico-chemical conditions measured at Olifants, Berg and Langebaan Estuaries in the cool temperate, and Heuningnes, Nahoon and Kwelera Estuaries in the warm temperate biogeographical regions. The growth and flowering phenology of S. tegetaria in relation to environmental conditions was investigated in the cool temperate Langebaan Estuarine Embayment and compared to findings in the warm temperate, permanently open Kowie Estuary. The physico-chemical gradient found between the cool and warm temperate biogeographical regions may be useful to study climate change effects on plant species. The comparison of similar habitats in each region may provide insight into how different climate regimes may affect biomass allocation and phenology.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv & Cons Biol) / Salicornia tegetaria is an endemic salt marsh macrophyte that is widely distributed in estuaries along the South African coast. The aims of the study were to understand the phylogeography of the species, compare the biomass allocation in two regions and to determine phenological patterns of S. tegetaria between the warm and cool temperate biogeographical regions. The phylogeography of S. tegetaria was studied using the noncoding chloroplast DNA region rpS16 and nuclear rDNA ITS region. Five samples each were collected from eighteen estuaries stretching from Orange River in the Northern Cape to Mngazana Estuary in the Eastern Cape. Above- and belowground biomass was collected and physico-chemical conditions measured at Olifants, Berg and Langebaan Estuaries in the cool temperate, and Heuningnes, Nahoon and Kwelera Estuaries in the warm temperate biogeographical regions. The growth and flowering phenology of S. tegetaria in relation to environmental conditions was investigated in the cool temperate Langebaan Estuarine Embayment and compared to findings in the warm temperate, permanently open Kowie Estuary. The physico-chemical gradient found between the cool and warm temperate biogeographical regions may be useful to study climate change effects on plant species. The comparison of similar habitats in each region may provide insight into how different climate regimes may affect biomass allocation and phenology.