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Survival in the face of unpredictable environments is a challenge faced by all organisms. One solution is the evolution of mechanisms that cause stochastic switching between phenotypic states. Despite the wide range of switching strategies found in nature, their evolutionary origins and adaptive significance remain poorly understood. Recently in the Rainey laboratory, a long-term evolution experiment performed with populations of the bacterium Pseudomonas fluorescens SBW25 saw the de novo evolution of a phenotypic switching strategy. This provided an unprecedented opportunity to gain insight into the evolution and maintenance of switching strategies. The derived ‘switcher’ genotype was detected through colony level phenotypic dimorphism. Further microscopic examination revealed the cellular basis of phenotypic switching as the bistable (ON/OFF) expression of a capsule. Transposon mutagenesis demonstrated that the structural basis of the capsule was a colanic acid-like polymer encoded by the Pflu3656-wzb locus. Subsequently, whole genome re-sequencing enabled elucidation of the series of mutational events underlying the evolution of capsule bistability: nine mutations were identified in the switcher. Present in both forms of the switcher, the final mutation – a point mutation in a central metabolic pathway – was shown to be the sole mechanistic cause of capsule switching; it ‘set the stage’ for a series of molecular events directly responsible for bistability. Two models were proposed to explain capsule switching at the molecular level: the genetic amplification-reduction model, and the epigenetic feedback model. Collective results of biochemical and genetic assays proved consistent with the epigenetic model, whereby a decrease in flux through the pyrimidine biosynthetic pathway activates an already-present feedback loop. Subsequent analysis of a second switcher (evolved independently of and in parallel with the first) revealed a radically different genetic route leading to phenotypically and mechanistically similar capsule switching. In addition to providing the first empirical insight into the evolutionary bases of switching strategies, the work presented in this thesis demonstrates the power of natural selection – operating on even the simplest of organisms – to forge adaptive solutions to evolutionary challenges; in a single evolutionary step, selection took advantage of inherent intracellular stochasticity to generate an extraordinarily flexible phenotype.

Following content removed due to copyright restrictions: Lambert, D. M., King, T., Shepherd, L. D., Livingston, A., Anderson, S. & Craig, J. L. (2005). Serial population bottlenecks and genetic variation: translocated populations of the New Zealand saddleback (Philesturnus carunculatus rufusater). Conservation Genetics 6: 1–14. Perrie, L. R., Shepherd, L. D.& Brownsey, P. J. (2005). Asplenium ·lucrosum nothosp. nov.: a sterile hybrid widely and erroneously cultivated as ‘‘Asplenium bulbiferum’’ Plant systematics and evolution 250: 243–257 / Ancient DNA was used to provide a temporal perspective for examining a number of evolutionary, conservation and cultural questions involving members of the New Zealand avifauna. Ancient mitochondrial DNA (mtDNA) sequences were used to examine the past levels and patterns of genetic diversity in the five species of New Zealand kiwi (Apterygidae). Brown kiwi, particularly in the South Island, exhibited high levels of genetic structuring with nearly every population exhibiting private mitochondrial haplotypes. The extinction of a large number of brown kiwi populations has, therefore, led to the loss of a large amount of genetic variation in these species. The past ranges of great spotted kiwi and the three brown kiwi species, whose bones are morphologically indistinguishable, were determined. This information can aid conservation programmes aiming to re-introduce kiwi to regions where they are now extinct. In contrast to the high level of genetic structuring in South Island brown kiwi, the majority of little spotted kiwi samples from the South Island shared a common haplotype. The difference in phylogeography between brown kiwi and little spotted kiwi is hypothesised to relate to differences in their dispersal behaviour and/or their population histories. The addition of ancient samples of little spotted kiwi from the North Island indicated a complex relationship with great spotted kiwi. Nuclear microsatellite DNA markers were isolated from North Island brown kiwi and tested for cross amplification in the other kiwi species. Five loci were polymorphic in all kiwi species. Preliminary analyses of genotyping results indicated that the kiwi species were distinguished by assignment tests and that subdivision may occur within several of the species. An extensive reference database of modern and ancient mtDNA sequences was used to determine species and provenance of a number of unlabelled museum subfossil bones and skins. This method was also used to examine provenance of brown kiwi feathers from Maori artefacts (cloaks and baskets). Ancient DNA methodology was also used in a molecular examination of the relationships of a second endemic avian family, the New Zealand wattlebirds (Callaeatidae). Analyses of nuclear gene sequences, c-mos and RAG-1, revealed kokako, saddleback and huia comprised a strongly supported monophyletic group. A divergence time estimate for the New Zealand wattlebirds indicated that they are more likely to have arrived by transoceanic dispersal than have a Gondwanan origin. Sequences from three mtDNA genes, 12S, ND2 and cytochrome b, were also analysed but could not resolve the relationships between the three genera. Microsatellite DNA from the extinct New Zealand huia exhibited considerable genetic variation, exceeding that found in extant North Island saddleback, from which the loci were isolated. Assignment tests indicated no genetic structuring within huia, although interpretation was complicated by a lack of provenance details for many of the skins. The results presented here suggest that ancient DNA can not only provide information about the relationships of extinct taxa but also demonstrates the importance of placing the present day genetic diversity found in endangered taxa within the context of past patterns and levels of genetic variation.

Comprehensive integration of paleontological and molecular data remains a sought-after goal of evolutionary research. This thesis presents a dataset unlike any previously studied to document changes over time in the evolutionary history of the New Zealand marine mollusc genus Alcithoe. In order to study evolutionary relationships in the Alcithoe, DNA sequence of approximately 8Kb of mitochondrial DNA was generated using universal and newly developed PCR primers. The gene composition of the resulting sequences has been thoroughly analysed, using a novel splits-based approach, to gain a clear understanding of the underlying phylogenetic signals in the data. Refinement of the phylogeny was achieved by considering subsets of both the taxa and genes. Taking these analyses into account the combined a robust phylogeny for the Alcithoe is presented for use in subsequent analyses. The Alcithoe genus includes species that are exemplars of the problem of correctly identifying species by morphological traits, in both the living and extinct taxa. Taxonomic assignments were explored in a population level analysis of the highly morphologically variable species A. wilsonae. Analyses revealed that the various recognised forms of A. wilsonae are genetically indistinguishable and that the previously recognised species A. knoxi is a synonym of A. wilsonae. This result has significant implications for the interpretation of the paleontological data, as A. knoxi specimens are known from the Tongaporutuan stage (10.92 – 6.5 Ma) of the New Zealand geological timescale. Therefore, this finding also has implications on the assignment of calibration data in molecular clock analysis. To ensure accurate estimation of divergence times and rates of molecular evolution, extensive explorations of parameter space in molecular-clock analyses were carried out. These analyses identified the most appropriate models and calibration settings for Alcithoe the dataset. The fossil data used to calibrate this analysis is amongst the most robust applied to molecular clock analyses to date. Statistical sampling uncertainty derived from the paleontological data was included in the calculation of prior distributions. Divergence dates inferred for the extant Alcithoe are largely consistent with the fossil record. However, the root of the tree was consistently inferred to be younger then expected. Rates of evolution in the species of Alcithoe included in this analysis are broadly consistent. However, some small rate differences are observed in some branches, for example, Alcithoe fusus appears to have a faster rate then the rest of the genus. This rate increase is the likely cause of topological inconsistencies observed for four closely related taxa, including A. fusus, and indicates that slight rate differences can cause phylogenetic instability when small genetic distances are involved. Direct comparison of diversification rates between the molecular and paleontological data for the Alcithoe illustrated that modern Alcithoe species have origins that are around 13 millions years younger then the oldest known Alcithoe fossils. The suggestion that A. fusus is descended from a series of fossil Leporemax species is directly contradicted by the molecular tree. In light of the molecular evidence this result highlights the problem of morphological convergence in the interpretation of fossil Alcithoe species. Comparison of the molecular and paleontological datasets was difficult for absolute speciation and extinction rates, as errors inherent to each dataset led to disparate estimates. For example, the fossil record clearly fails to record most recent speciation events observed in the molecular phylogeny, but the molecular data cannot sufficiently account for the amount of extinction evident in the fossil record. It is clear that the assumption of a constant and equal probability of speciation and extinction for all lineages is violated in the Alcithoe. However, the general long-term trends estimated for both datasets are concordant, and demonstrate an increase in both speciation and extinction rates over the Cenozoic era. The research described in this thesis represents significant progress toward the goal of more thorough integration of molecular and paleontological in the study of evolution. I have shown that reconciliation of molecular and paleontological data is not only possible, but can substantially improve the resulting interpretation of evolution. This study is the broadest analysis of the evolution in a single genus using combined molecular and paleontological data that the author is currently aware of. It illustrates the advantage of having quality paleontological data to compare to emerging molecular data, and how the molecular data can further inform the paleontological data. Furthermore, it adds support to the shift in perspective from an adversarial to a complementary approach to the consideration of molecular and paleontological data. This thesis is a comprehensive first step in the synthesis of molecular and paleontological data in the study of evolution of the New Zealand mollusc fauna, and alludes to many promising avenues for future study.

Comprehensive integration of paleontological and molecular data remains a sought-after goal of evolutionary research. This thesis presents a dataset unlike any previously studied to document changes over time in the evolutionary history of the New Zealand marine mollusc genus Alcithoe. In order to study evolutionary relationships in the Alcithoe, DNA sequence of approximately 8Kb of mitochondrial DNA was generated using universal and newly developed PCR primers. The gene composition of the resulting sequences has been thoroughly analysed, using a novel splits-based approach, to gain a clear understanding of the underlying phylogenetic signals in the data. Refinement of the phylogeny was achieved by considering subsets of both the taxa and genes. Taking these analyses into account the combined a robust phylogeny for the Alcithoe is presented for use in subsequent analyses. The Alcithoe genus includes species that are exemplars of the problem of correctly identifying species by morphological traits, in both the living and extinct taxa. Taxonomic assignments were explored in a population level analysis of the highly morphologically variable species A. wilsonae. Analyses revealed that the various recognised forms of A. wilsonae are genetically indistinguishable and that the previously recognised species A. knoxi is a synonym of A. wilsonae. This result has significant implications for the interpretation of the paleontological data, as A. knoxi specimens are known from the Tongaporutuan stage (10.92 – 6.5 Ma) of the New Zealand geological timescale. Therefore, this finding also has implications on the assignment of calibration data in molecular clock analysis. To ensure accurate estimation of divergence times and rates of molecular evolution, extensive explorations of parameter space in molecular-clock analyses were carried out. These analyses identified the most appropriate models and calibration settings for Alcithoe the dataset. The fossil data used to calibrate this analysis is amongst the most robust applied to molecular clock analyses to date. Statistical sampling uncertainty derived from the paleontological data was included in the calculation of prior distributions. Divergence dates inferred for the extant Alcithoe are largely consistent with the fossil record. However, the root of the tree was consistently inferred to be younger then expected. Rates of evolution in the species of Alcithoe included in this analysis are broadly consistent. However, some small rate differences are observed in some branches, for example, Alcithoe fusus appears to have a faster rate then the rest of the genus. This rate increase is the likely cause of topological inconsistencies observed for four closely related taxa, including A. fusus, and indicates that slight rate differences can cause phylogenetic instability when small genetic distances are involved. Direct comparison of diversification rates between the molecular and paleontological data for the Alcithoe illustrated that modern Alcithoe species have origins that are around 13 millions years younger then the oldest known Alcithoe fossils. The suggestion that A. fusus is descended from a series of fossil Leporemax species is directly contradicted by the molecular tree. In light of the molecular evidence this result highlights the problem of morphological convergence in the interpretation of fossil Alcithoe species. Comparison of the molecular and paleontological datasets was difficult for absolute speciation and extinction rates, as errors inherent to each dataset led to disparate estimates. For example, the fossil record clearly fails to record most recent speciation events observed in the molecular phylogeny, but the molecular data cannot sufficiently account for the amount of extinction evident in the fossil record. It is clear that the assumption of a constant and equal probability of speciation and extinction for all lineages is violated in the Alcithoe. However, the general long-term trends estimated for both datasets are concordant, and demonstrate an increase in both speciation and extinction rates over the Cenozoic era. The research described in this thesis represents significant progress toward the goal of more thorough integration of molecular and paleontological in the study of evolution. I have shown that reconciliation of molecular and paleontological data is not only possible, but can substantially improve the resulting interpretation of evolution. This study is the broadest analysis of the evolution in a single genus using combined molecular and paleontological data that the author is currently aware of. It illustrates the advantage of having quality paleontological data to compare to emerging molecular data, and how the molecular data can further inform the paleontological data. Furthermore, it adds support to the shift in perspective from an adversarial to a complementary approach to the consideration of molecular and paleontological data. This thesis is a comprehensive first step in the synthesis of molecular and paleontological data in the study of evolution of the New Zealand mollusc fauna, and alludes to many promising avenues for future study.

Comprehensive integration of paleontological and molecular data remains a sought-after goal of evolutionary research. This thesis presents a dataset unlike any previously studied to document changes over time in the evolutionary history of the New Zealand marine mollusc genus Alcithoe. In order to study evolutionary relationships in the Alcithoe, DNA sequence of approximately 8Kb of mitochondrial DNA was generated using universal and newly developed PCR primers. The gene composition of the resulting sequences has been thoroughly analysed, using a novel splits-based approach, to gain a clear understanding of the underlying phylogenetic signals in the data. Refinement of the phylogeny was achieved by considering subsets of both the taxa and genes. Taking these analyses into account the combined a robust phylogeny for the Alcithoe is presented for use in subsequent analyses. The Alcithoe genus includes species that are exemplars of the problem of correctly identifying species by morphological traits, in both the living and extinct taxa. Taxonomic assignments were explored in a population level analysis of the highly morphologically variable species A. wilsonae. Analyses revealed that the various recognised forms of A. wilsonae are genetically indistinguishable and that the previously recognised species A. knoxi is a synonym of A. wilsonae. This result has significant implications for the interpretation of the paleontological data, as A. knoxi specimens are known from the Tongaporutuan stage (10.92 – 6.5 Ma) of the New Zealand geological timescale. Therefore, this finding also has implications on the assignment of calibration data in molecular clock analysis. To ensure accurate estimation of divergence times and rates of molecular evolution, extensive explorations of parameter space in molecular-clock analyses were carried out. These analyses identified the most appropriate models and calibration settings for Alcithoe the dataset. The fossil data used to calibrate this analysis is amongst the most robust applied to molecular clock analyses to date. Statistical sampling uncertainty derived from the paleontological data was included in the calculation of prior distributions. Divergence dates inferred for the extant Alcithoe are largely consistent with the fossil record. However, the root of the tree was consistently inferred to be younger then expected. Rates of evolution in the species of Alcithoe included in this analysis are broadly consistent. However, some small rate differences are observed in some branches, for example, Alcithoe fusus appears to have a faster rate then the rest of the genus. This rate increase is the likely cause of topological inconsistencies observed for four closely related taxa, including A. fusus, and indicates that slight rate differences can cause phylogenetic instability when small genetic distances are involved. Direct comparison of diversification rates between the molecular and paleontological data for the Alcithoe illustrated that modern Alcithoe species have origins that are around 13 millions years younger then the oldest known Alcithoe fossils. The suggestion that A. fusus is descended from a series of fossil Leporemax species is directly contradicted by the molecular tree. In light of the molecular evidence this result highlights the problem of morphological convergence in the interpretation of fossil Alcithoe species. Comparison of the molecular and paleontological datasets was difficult for absolute speciation and extinction rates, as errors inherent to each dataset led to disparate estimates. For example, the fossil record clearly fails to record most recent speciation events observed in the molecular phylogeny, but the molecular data cannot sufficiently account for the amount of extinction evident in the fossil record. It is clear that the assumption of a constant and equal probability of speciation and extinction for all lineages is violated in the Alcithoe. However, the general long-term trends estimated for both datasets are concordant, and demonstrate an increase in both speciation and extinction rates over the Cenozoic era. The research described in this thesis represents significant progress toward the goal of more thorough integration of molecular and paleontological in the study of evolution. I have shown that reconciliation of molecular and paleontological data is not only possible, but can substantially improve the resulting interpretation of evolution. This study is the broadest analysis of the evolution in a single genus using combined molecular and paleontological data that the author is currently aware of. It illustrates the advantage of having quality paleontological data to compare to emerging molecular data, and how the molecular data can further inform the paleontological data. Furthermore, it adds support to the shift in perspective from an adversarial to a complementary approach to the consideration of molecular and paleontological data. This thesis is a comprehensive first step in the synthesis of molecular and paleontological data in the study of evolution of the New Zealand mollusc fauna, and alludes to many promising avenues for future study.

Experimental microbial populations provide powerful models for testing the most challenging problems in evolutionary biology. In the midst of the genome sequencing revolution microbial evolutionary genetics has flourished; promising high-resolution explanations for the underlying causes of evolutionary phenomena. This thesis describes four investigations into the adaptation of Pseudomonas fluorescesns SBW25 to a spatially structured environment. The first builds upon a large body of experimental work characterising the genetic and phenotypic causes of the ability of divergent Wrinkley Spreader (WS) types to colonise the air-liquid interface in spatially structured microcosms. The mws and aws genetic loci are described, which together with the previously described wsp locus, account for the location of the causal mutation for all known WS genotypes. It was found that if these loci were deleted from the P. fluorescens genome, it could still evolve the WS phenotype via a previously undiscovered locus (sws). This study provides the first explicit evidence that genetic biases can influence the outcome of evolution. The second study used a novel method to sample WS genotypes without the biasing effects of natural selection; the distribution of the fitness effects of these genotypes was measured and analysed from a unique perspective. The distribution of fitness effects of new mutations is found to best fit the normal distribution, facilitating the extension of the mutational landscape model of adaptation to include all possible adaptive walks. The third study investigates the underlying causes of genetic biases on evolution; many WS genotypes are obtained at different time points during colonisation of the air-liquid interface (including WS obtained without selection) and the causal mutations of many of these mutants determined. Together these results allowed the elucidation of the relative effects of natural selection, genetic architecture and mutation rate on evolutionary outcomes. The final study considers the WS mat as the product of cooperative interactions, and uses a group selection experiment to investigate the potential of WS mats to evolve group level adaptations. A novel strategy is developed to overcome cheating types, considered the main barrier to the evolution of group level complexity. Furthermore, WS groups evolved specialised cell types, the first example of a de novo evolution of a division of labour, a hallmark of complexity.