BACKGROUND:Traditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used / however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common.RESULTS:Coalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia.CONCLUSIONS:The number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.

The analysis of diverse data sets can yield different phylogenetic estimates that challenge systematists to explain the source of discordance. The Ambystomatidae are a classic example of this phylogenetic conflict. Previous attempts to resolve the ambystomatid species tree using allozymic, morphological, and mitochondrial sequence data have yielded different estimates, making it unclear which data source best approximates ambystomatid phylogeny. We present the first multi-locus DNA sequencebased phylogenetic study of the Ambystomatidae. Because independent loci can contain discordant gene tree histories, concatenating unlinked loci into a single data matrix can lead to strongly supported and erroneous results. Therefore, we utilized a range of analyses, including coalescent-based methods of phylogenetic estimation that account for incomplete lineage sorting and concordance-based methods that estimate the proportion of sampled loci that support a particular clade. We repeated these analyses with the removal of individual loci to determine if any locus has a disproportionate effect on our phylogenetic results. Many deep and relatively shallow clades within Ambystoma were robustly resolved. Analyses that excluded loci produced overlapping posterior distributions, suggesting no disproportionate influence of any particular locus. Our estimates differ from previous hypotheses, although there was greater similarity with previous molecular estimates, relative to morphological estimates.

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In the pre-genomic era, the relationships among species and their evolutionary histories were often determined by examining the fossil records. In the genomic era, these relationships are identified by analysing the genetic data, which also enables us to take a close-up view of the differences between the individual samples. Nevertheless, these relationships are often described by a tree-like structure or a network. In this thesis, we investigate some of the models that are used to describe these relationships. This thesis can be divided into two main parts. The first part focuses on investigating the theoretical properties of several neutral tree models that are often considered in phylogenetics and population genetics studies, such as the Yule–Harding model, the proportional to distinguishable arrangements and the Kingman coalescent models. In comparison to the first part, the other half of the thesis is more computationally oriented: we focus on developing and implementing methods of calculating gene tree probabilities of given species networks, and simulating genealogies within species networks.

Genome sequencing technologies are providing huge quantities of data for phylogenetic inference. However, most phylogenomic studies exclude gene families, because many have a complicated history of gene duplication/loss and structural change by domain shuffling, especially in deep phylogenies. Gene tree parsimony (GTP) methods, which seek the species tree that minimizes the cost of gene duplication, have been successfully applied to gene families with frequent duplication history. Their utility and performance in the context of gene families with complex histories of gene duplication and domain reshuffling remains unclear. In this study, we analyzed 4389 gene families from six angiosperm genomes encompassing a wide range of duplication rates, and a broad diversity of domain architecture. Overall species tree inference accuracy increased monotonically with the inclusion of more gene trees, and high accuracy was achieved with 50-100 gene trees. The rate of gene duplication strongly influences species tree inference accuracy, with the highest accuracy at either very low or very high rates of duplication and lowest accuracy centered around one duplication per branch in the unrooted species tree. This is the opposite of the relationship between substitution rates on tree construction accuracy, in which intermediate rates have highest accuracy. Accuracy is generally higher in gene families with high domain architecture diversity but has high variance in families with relatively low domain architecture diversity. The latter is probably due to the high variation of gene duplication number for those gene families. We close with some discussion of potential impacts of domain evolution on phylogenomic reconstruction protocols in general, including its effect on alignment.

A phylogenetic tree is used to model gene evolution and species evolution using molecular sequence data. For artifactual and biological reasons, a gene tree may differ from a species tree, a phenomenon known as gene tree-species tree incongruence. Assuming the presence of one or more evolutionary events, e.g., gene duplication, gene loss, and lateral gene transfer (LGT), the incongruence may be explained using a reconciliation of a gene tree inside a species tree. Such information has biological utilities, e.g., inference of orthologous relationship between genes. In this thesis, we present probabilistic models and methods for orthology analysis and species tree inference, while accounting for evolutionary factors such as gene duplication, gene loss, and sequence evolution. Furthermore, we use a probabilistic LGT-aware model for inferring gene trees having temporal information for duplication and LGT events. In the first project, we present a Bayesian method, called DLRSOrthology, for estimating orthology probabilities using the DLRS model: a probabilistic model integrating gene evolution, a relaxed molecular clock for substitution rates, and sequence evolution. We devise a dynamic programming algorithm for efficiently summing orthology probabilities over all reconciliations of a gene tree inside a species tree. Furthermore, we present heuristics based on receiver operating characteristics (ROC) curve to estimate suitable thresholds for deciding orthology events. Our method, as demonstrated by synthetic and biological results, outperforms existing probabilistic approaches in accuracy and is robust to incomplete taxon sampling artifacts. In the second project, we present a probabilistic method, based on a mixture model, for species tree inference. The method employs a two-phase approach, where in the first phase, a structural expectation maximization algorithm, based on a mixture model, is used to reconstruct a maximum likelihood set of candidate species trees. In the second phase, in order to select the best species tree, each of the candidate species tree is evaluated using PrIME-DLRS: a method based on the DLRS model. The method is accurate, efficient, and scalable when compared to a recent probabilistic species tree inference method called PHYLDOG. We observe that, in most cases, the analysis constituted only by the first phase may also be used for selecting the target species tree, yielding a fast and accurate method for larger datasets. Finally, we devise a probabilistic method based on the DLTRS model: an extension of the DLRS model to include LGT events, for sampling reconciliations of a gene tree inside a species tree. The method enables us to estimate gene trees having temporal information for duplication and LGT events. To the best of our knowledge, this is the first probabilistic method that takes gene sequence data directly into account for sampling reconciliations that contains information about LGT events. Based on the synthetic data analysis, we believe that the method has the potential to identify LGT highways. / <p>QC 20150529</p>

1 Phylogenetic Studies in the Polyploid Genus Curcuma L. SUMMARY Curcuma is genetically one of the most complex genera within the tropical family Zingiberaceae, with hybridization and polyploidization being the major forces in its evolution. In this thesis, I have focused mainly on the genetic background of Curcuma species variation, relationships and overall genome structure, as a key to solve long standing taxonomic problems. Results of my molecular studies on the genus Curcuma performed since 2007 represent an extension of ongoing taxonomic and nomenclatural work started by Jana Leong- Škorničková in 2000. The first part of the thesis consists of a broad, general introduction to the subject to reflect the current state of knowledge, formulate the major problems to be confronted within the genus, and summarise the major results of the studies presented in the second part of the thesis. As the main obstacles in studying Curcuma are consequences of its reticulate evolution, it is also outlines the importance of understanding the genetic background and species relationships using molecular markers. Common molecular methods used for assessing phylogenetic relationships on the intraspecific and infrageneric levels - AFLP and sequencing of selected markers from cpDNA, nrDNA and nDNA - are described, with the...

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.