abstract: The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for all three compounds, established production routes suffer from notable inherent limitations. Here, multiple pathways to the same three products were engineered, each incorporating unique enzyme chemistries and/or stemming from different endogenous precursors. In the case of phenol, two novel pathways were constructed and comparatively evaluated, with titers reaching as high as 377 ± 14 mg/L at a glucose yield of 35.7 ± 0.8 mg/g. In the case of catechol, three novel pathways were engineered with titers reaching 100 ± 2 mg/L. Finally, in the case of MA, four novel pathways were engineered with maximal titers reaching 819 ± 44 mg/L at a glucose yield of 40.9 ± 2.2 mg/g. Furthermore, the unique flexibility with respect to engineering multiple pathways to the same product arises in part because these compounds are common intermediates in aromatic degradation pathways. Expanding on the novel pathway engineering efforts, a synthetic ‘metabolic funnel’ was subsequently constructed for phenol and MA, wherein multiple pathways were expressed in parallel to maximize carbon flux toward the final product. Using this novel ‘funneling’ strategy, maximal phenol and MA titers exceeding 0.5 and 3 g/L, respectively, were achieved, representing the highest achievable production metrics products reported to date. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2017

Coordination of gene expression within the cell requires the integrated actions of various multi-protein, gene regulatory complexes. The Mediator and BRG1 Associate Factor (BAF) complexes are large, dynamic regulatory cofactors (COF) that are made up of multiple different submodules, and play key roles in regulating gene expression. Gene-specific regulation requires that transcription factors (TFs) recruit these COF complexes to gene promoters. How separate subdomains in each complex interact with distinct sets of TFs in each cell remains an important question. In this study, to address this question, we sought to apply the nuclear extract protein-binding microarray (nextPBM) technology being developed in our lab to study interactions between TFs and subunits of the Mediator and BAF complexes. To facilitate this, we cloned, expressed and purified subdomains of proteins from the Mediator and BAF complexes. We then used the nextPBM technology to study the interactions of our subdomains with TFs in human macrophages. We identified several new interactions with TFs, and demonstrate the utility of this approach to student TF-COF interaction.

Master of Science / Department of Biology / Carolyn J. Ferguson / The genus Phlox L. presents intriguing opportunities for systematics research, and P. nana is of particular interest. Phlox nana occurs chiefly in mountains of the Chihuahuan desert to northern New Mexico, and it exhibits much morphological variation across its range. Historically, this taxon has been recognized as a single species (sometimes with infraspecific taxa), or as several species. Perhaps most interesting, variation in ploidy level (cytotypic variation) has been evidenced for P. nana. This research employed flow cytometry methods in conjunction with chromosome counts to document patterns of cytotypic variation. Intensive fieldwork in Arizona, New Mexico and Texas enabled excellent sampling, and evaluation of ploidy level for 76 populations was achieved. Diploid and tetraploid chromosome counts were made (four diploid counts; five tetraploid counts), and flow cytometry was conducted on all populations, providing evidence for diploid, tetraploid and hexaploid populations. Polyploids were found to occur in many geographical areas, and in some regions, diploids and polyploids occur in close geographical proximity (e.g., within both the Davis Mountains and the Chisos Mountains of west Texas). Genome size data are presented (with discussion of unusual populations), and geographic patterns of cytotypic variation are presented and discussed. Patterns are also briefly considered with respect to morphology and taxonomy: cytotypic variation does not readily align with historical recognition of taxonomic variation, and this work sets the stage for ongoing, detailed morphometric study. Research on particular species of Phlox benefits from an understanding of a broad phylogenetic context, and low copy nuclear DNA regions are an important resource for phylogeny development. This research further evaluated part of the NADP-dependent isocitrate dehydrogenase gene (idhB) for its usefulness in inferring relationships in Phlox. Samples were PCR amplified for idhB and cloned, and resulting sequences were added to a larger set of idhB sequence data previously developed in the lab. A total of 163 samples were included, and Bayesian Inference and Maximum Parsimony analyses were conducted for complete data sets. Phylogenetic findings are discussed in light of previous work based on chloroplast and high copy nuclear DNA regions, and challenges and utility of using idhB are discussed.

<p>This dissertation investigates the processes of hybridation, polyploidy, and apomixis and their roles in the evolution of myriopterid ferns. First, I examine patterns of hybridization in members of the Cheilanthes yavapensis complex using a suite of techiniques, ranging from molecules to morphology--including isozymes, spore measurements, and molecular phylogenetics based on chloroplast and nuclear DNA markers--to elucidate relationships in this notorious group of ferns. Second, I utilize the rules of traditional taxonomy set by the International Code of Botanical Nomenclature to recircucmscribe and resurrect the genus Myriopteris from within cheilanthoid ferns. This revised classification is bolstered by results from my molecular phylogenetic analysis of DNA sequence data in the subsequent chapter. Then, using morphological and cytological analyses, I examine the evolution of indument, leaf and rachis shape, vernation, chromosome number, and reproductive mode across the myriopterid tree. In my concluding chapter I develop microsatellite markers for the apomictic triploid, M. lindheiemeri, and explore whether premeiotic chromosome duplication facilitates the production of genetically distinct offspring in this otherwise asexual lineage.</p> / Dissertation

<p>Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.</p> / Dissertation

abstract: Understanding the diversity, evolutionary relationships, and geographic distribution of species is foundational knowledge in biology. However, this knowledge is lacking for many diverse lineages of the tree of life. This is the case for the desert stink beetles in the tribe Amphidorini LeConte, 1862 (Coleoptera: Tenebrionidae) – a lineage of arid-adapted flightless beetles found throughout western North America. Four interconnected studies that jointly increase our knowledge of this group are presented. First, the darkling beetle fauna of the Algodones sand dunes in southern California is examined as a case study to explore the scientific practice of checklist creation. An updated list of the species known from this region is presented, with a critical focus on material now made available through digitization and global aggregation. This part concludes with recommendations for future biodiversity checklist authors. Second, the psammophilic genus Trogloderus LeConte, 1879 is revised. Six new species are described, and the first, multi-gene phylogeny for the genus is inferred. In addition, historical biogeographic reconstructions along with novel hypotheses of speciation patterns within the Intermountain Region are given. In particular, the Kaibab Plateau and Kaiparowitz Formation are found to have promoted speciation on the Colorado Plateau. The Owens Valley and prehistoric Bouse Embayment are similarly hypothesized to drive species diversification in southern California. Third, a novel phylogenomic analysis for the tribe Amphidorini is presented, based on 29 de novo partial transcriptomes. Three putative ortholog sets were discovered and analyzed to infer the relationships between species groups and genera. The existing classification of the tribe is found to be highly inadequate, though the earliest-diverging relationships within the tribe are still in question. Finally, the new phylogenetic framework is used to provide a genus-level revision for the Amphidorini, which previously contained six valid genera and 253 valid species. This updated classification includes more than 100 taxonomic changes and results in the revised tribe consisting of 16 genera, with three being described as new to science. / Dissertation/Thesis / Doctoral Dissertation Evolutionary Biology 2018

<p>The fern family Cystopteridaceae has been a thorn in the side of fern phylogeneticists, on many levels. Until this thesis, its basic existence (as a deeply isolated clade) and composition were unrecognized, hypotheses as to the relationships of its constituents within the broader fern tree-of-life were wildly inconsistent, the relationships of its genera to each other were contested, the species limits within those genera weakly understood, and the relationships among those species unknown. This thesis first establishes the broad evolutionary context for the family, which is that it is the first-diverging branch in Eupolypods II (it is sister to the rest of the eupolypod II clade). Eupolypods II is a large clade, containing nearly a third of extant fern species, making the Cystopteridaceae's position pivotal to a full understanding of fern evolution. </p><p>The evolution of the Eupolypods II is marked by an "ancient, rapid radiation" at the base of the clade, which helps to explain the difficulty that this broad group has historically posed to evolutionary biologists. Molecular data from five plastid loci show that Eupolypods II is comprised of 10 deeply divergent lineages, each worthy of recognition at the rank of family: Cystopteridaceae, Rhachidosoraceae, Diplaziopsidaceae, Hemidictyaceae, Aspleniaceae, Thelypteridaceae, Woodsiaceae, Onocleaceae, Blechnaceae, and Athyriaceae. The ancestors of Cystopteridaceae diverged from those of the rest of the clade approximately 100 million years ago, and the family is now comprised of five extant genera: Acystopteris, Cystoathyrium (the only genus for which we lack molecular data--it may be extinct), Cystopteris, Gymnocarpium, and ×Cystocarpium.</p><p>Within the family, the relationships of Cystoathyrium are unknown. Acystopteris is sister to Cystopteris, and those two genera, together, are sister to Gymnocarpium. Gymnocarpium is the maternal parent of ×Cystocarpium, so that genus falls within Gymnocarpium in phylogenetic trees based on maternally transmitted loci (i.e., plastid or mitochondrial loci). Plastid data resolve a basal trichotomy in Gymnocarpium, among the G. disjunctum clade, the G. robertianum clade, and core Gymnocarpium. The earliest diverging branch of core Gymnocarpium is the morphologically anomalous G. oyamense, followed by a split that separates G. appalachianum and G. jessoense parvulum (on one side) from G. remotepinnatum and G. jessoense jessoense, on the other. In Acystopteris, the first division surprisingly separates A. taiwaniana (which is frequently treated as a variety of A. japonica) from A. japonica + A. tenuisecta (which are morphologically very distinct from each other).</p><p>The evolution of Cystopteris is, as expected, more complex. The first lineage to diverge from the rest of the genus is the one that gave rise to C. montana. The next division, however, is unclear; molecular data infer a trichotomy among the sudetica clade (containing C. sudetica, C. moupinensis, and C. pellucida), the bulbifera clade (containing C. bulbifera and its related allopolyploids C. tennesseensis and C. utahensis), and the C. fragilis complex. Within the C. fragilis complex relationships (and species limits) get particular messy. The diploid species of eastern North America--C. protrusa--is sister to the rest of the complex, but after that point the major named species (including C. fragilis and C. tenuis) cease to be monophyletic, being found on both sides of a major split, alongside such taxa as the Australian/New Zealand C. tasmanica, the Hawaiian C. douglasii, and the Mexican C. membranifolia and C. millefolia.</p><p>In the context of the deep divergence of Gymnocarpium from Cystopteris, and the complicated species-level patterns of relationship within each genus, it is particularly surprising that molecular data confirm that ×Cystocarpium is a hybrid between Gymnocarpium dryopteris and a European tetraploid member of the Cystopteris fragilis complex. The ancestors of Cystopteris diverged from those of Gymnocarpium approximately 58 million years ago, meaning that the ×Cystocarpium hybridization event (which happened very recently) united genomes that contain, between them, over 100 million years of independent evolution. This breadth of divergence makes ×Cystocarpium the most extreme example of wide hybridization currently documented, with important implications for the pace of evolution of reproductive isolation, and thus for species formation.</p><p>This thesis ends with a tentative synopsis of the Cystopteridaceae (Appendix E). The family, as construed here, contains five genera and approximately 36 species (three in Acystopteris, one in Cystoathyrium, ~25 in Cystopteris, seven in Gymnocarpium, and one in ×Cystocarpium), plus two named subspecies (one each in Cystopteris and Gymnocarpium), and eight named sterile hybrids (three in Cystopteris and five in Gymnocarpium). Each of these tallies is highly subjective--much further research, with an emphasis on cytological and low-copy nuclear data, is necessary before we can hope to have any confidence in the species limits and finer-scale evolutionary patterns in this family.</p> / Dissertation

<p>The truffle genus Tuber (Ascomycota, Pezizales, Tuberaceae) produces underground mushrooms widely sought as edible fungi. Tuber species are distributed throughout Northern hemisphere forests and form obligate ectomycorrhizal symbiosis with trees within the Pinaceae, Fagaceae, Betulaceae, and Juglandaceae. </p><p>The transition to a truffle form (from an epigeous form) has occurred independently, multiple times in both the Ascomycetes and Basidiomycetes. One instance has given rise to the Tuberaceae, which is composed entirely of obligate ectomycorrhizal species. Attempts to cultivate European truffle species T. melanosporum, T. aestivum, and T. borchii are underway in North America and other parts of the world and have been met with mixed success.</p><p>The overarching goal of my dissertation is to address the systematics, ecology, and biogeography of Tuber within a phylogenetic framework. Multiple loci were sequenced from Tuber ascoma collected worldwide including ectomycorrhizae, though an emphasis was placed on sampling taxon within North American. Maximum likelihood, maximum parsimony, and Bayesian inference were used for phylogenetic reconstructions. </p><p>A taxonomic and phylogenetic overview of the family Tuberaceae is presented in Chapter 1. Tuber is resolved as monophyletic. In Chapter 2, through greater taxon sampling including epigeous and hypogeous Helvellaceae outgroups and related South American taxa, a resolved multi-gene phylogeny of the Tuberaceae and putative epigeous ancestor of Tuber is presented. A previously unknown South American lineage that contains both epigeous and hypogeous taxa is resolved as sister to the Tuberaceae. Chapter 3 is focused on issues of cryptic speciation and taxonomy within the Tuber gibbosum clade. The four species resolved in the Gibbosum clade appear to be endemic to the Pacific Northwest and associated primarily with Gymnosperms. Chapter 4 is a meta-analysis of all known Tuber ITS rDNA sequences (e.g. from Genbank and generated from herbarium collections) available at the time. These were placed within the Tuber phylogeny to assess species diversity, long-distance dispersal, and host associations. In total, 120 phylotypes were detected (based on a 96% similarity criterion). Tuber shows high levels of continental endemism. I hypothesize that species shared between continents and having low ITS variability (<1%) are the result of recent human-mediated introduction events. Chapters 5 and 6 are focused on the ectomycorrhizal ecology of the economic truffle T. lyonii, which is native to Eastern and Southern North America. There is a phenomenon of Tuber lyonii fruiting in pecan orchards. Pecans (Carya illinoinensis) are in the Juglandaceae, an understudied ectomycorrhizal plant family. I sampled the ectomycorrhizal communities of pecan orchards (associated with the production of the North American truffle species Tuber lyonii). In Chapter 5 I discuss four Tuber taxa discovered in these pecan orchards, their abundance and haplotype diversity. Chapter 6 examines the ectomycorrhizal communities across the five pecan orchards sampled. I show that multiple Tuber species, including Tuber lyonii, are dominant in the ectomycorrhizal community. Chapters 7 and 8 focus on black truffles in the Melanosporum clade. In Chapter 7 I document that Tuber indicum has been introduced into North America multiple times, and through ectomycorrhizal synthesis I demonstrate that this Asian species can associate readily with angiosperm and gymnosperm hosts endemic to North American. In Chapter 8 I describe a quick and reliable method for the determination of Tuber melanosporum. The method is based on direct PCR and species-specific primers and is very useful for rapid diagnostics. I have adapted this approach for other truffle and mushroom species. </p><p>Three major findings emerge from my dissertation research: 1) Tuber is more diverse than previously realized; 2) Tuber exhibits high levels of regional and continental endemism; 3) Taxonomic issues remain in many species complexes worldwide (including the Tuber candidum complex in North America, the Tuber excavatum complex in Europe, the Tuber indicum complex in Asia). Taxonomic challenges also remain regarding species known only from ectomycorrhizal or anamorphic states. The discovery of additional Tuber species is expected as the truffle flora of undersampled regions become better studied and incorporated into the Tuberaceae phylogeny.</p> / Dissertation

<p>Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus <italic>Cladonia grayi</italic> and its green algal partner <italic>Asterochloris</italic> sp. This work uses the model lichen system <italic>Cladonia grayi</italic> and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.</p><p>Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.</p><p>In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from <italic>Cladonia grayi</italic> were shown to be functional by expression of the lichen genes in a strain of <italic>Saccharomyces cerevisiae</italic> in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. </p><p>In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of <italic>Cladonia grayi</italic> including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.</p> / Dissertation

<p>Morphological characters from the gametophyte and sporophyte generations have been used in land plants to infer relationships and construct classifications, but sporophytes provide the vast majority of data for the systematics of vascular plants. In bryophytes both generations are well developed and characters from both are commonly used to classify these organisms. However, because morphological traits of gametophytes and sporophytes can have different genetic bases and experience different selective pressures, taxonomic emphasis on one generation or the other may yield incongruent classifications. The moss order Hookeriales has a controversial taxonomic history because previous classifications have focused almost exclusively on either gametophytes or sporophytes. The Hookeriales provide a model for comparing morphological evolution in gametophytes and sporophytes, and its impact on alternative classification systems. Sometimes, placement of certain groups within Hookeriales remains challenging even at the molecular level. That is the case of the genus <italic>Calyptrochaeta</italic>. We study diversification dynamics in this genus to elucidate possible mechanisms obscuring its placement and we address biogeographic questions using the Tropical Conservatism scenario as our null hypothesis. Furthermore, to better understand biogeographic patterns in the Southern Hemisphere, infraspecific molecular patterns are compared in two species of the genus <italic>Calyptrochaeta</italic> (i.e., <italic>C. apiculata</italic> and <italic>C. asplenioides</italic>) and vicariance and recent long distance dispersal are tested to explain the disjunct distributions observed in these species. </p><p>In this study we reconstruct relationships among pleurocarpous mosses in or associated to the Hookeriales, in <italic>Calyptrochaeta</italic>, and within <italic>Calyptrochaeta</italic>. Six molecular markers are explored in total from all three genome compartments to reconstruct the evolution of morphological characters and habitat preferences in our phylogenies. Divergence times are estimated in a Bayesian framework using a relaxed molecular clock, and diversification rates are calculated on the chronograms resulting from these estimations. </p><p>As a result, we found that the Hookeriales, as currently circumscribed, are monophyletic and that both sporophyte and gametophyte characters are labile. We documented parallel changes and reversals in traits from both generations. We show that diversification rates in <italic>Calyptrochaeta</italic> have changed through its history. Also, though we lack support to clearly reject the tropical conservatism hypothesis, our data point to a more complex scenario where both temperate and tropical species can be ancient and give rise to one another, since shifts between tropical and temperate regions seem to be possible in any direction. Finally, we have show that recent long distance dispersal best explains the distribution of both <italic>C. apiculata</italic> and <italic>C. asplenioides</italic> in the Southern Hemisphere.</p> / Dissertation