Prediction of functions of genes in a genome is a key step in all genome sequencing projects. Sequences that carry out important functions are likely to be conserved between evolutionarily distant species and can be identified using cross-species comparisons. In the absence of completed genomes and the accompanying high-quality annotations, expressed sequence tags (ESTs) from random cDNA clones are the primary tools for functional genomics. EST datasets are fragmented and redundant, necessitating clustering of ESTs into groups that are likely to have been derived from the same genes. EST clustering helps reduce the search space for sequence homology searching and improves the accuracy of function predictions using EST datasets. This dissertation is a case study that describes clustering of Bos taurus and Sus scrofa EST datasets, and utilizes the EST clusters to make computational function predictions using a comparative genomics approach. We used a novel EST clustering method, TAMUClust, to cluster bovine ESTs and compare its performance to the bovine EST clusters from TIGR Gene Indices (TGI) by using bovine ESTs aligned to the bovine genome assembly as a gold standard. This comparison study reveals that TAMUClust and TGI are similar in performance. Comparisons of TAMUClust and TGI with predicted bovine gene models reveal that both datasets are similar in transcript coverage. We describe here the design and implementation of an annotation pipeline for predicting functions of the Bos taurus (cattle) and Sus scrofa (pig) transcriptomes. EST datasets were clustered into gene families using Ensembl protein family clusters as a framework. Following clustering, the EST consensus sequences were assigned predicted function by transferring annotations of the Ensembl vertebrate protein(s) they are grouped to after sequence homology searches and phylogenetic analysis. The annotations benefit the livestock community by helping narrow down the gamut of direct experiments needed to verify function.

Nematoda is a very diverse animal phylum. Within Nematoda, species display a multitude of life styles, different reproductive strategies and parasitism has arisen independently several times. Furthermore, morphological conservation and a high rate of homoplasy have impeded the resolution of nematode systematics. To address these issues, single gene (usually the nuclear ribosomal small subunit gene) and mitochondrial gene phylogenies have been used, but the information contained within the sequence of these genes is not enough to resolve the topological relationships between clades that emerged during rapid cladogenesis. Next generation sequencing data have been shown to produce high quality genomic and transcriptomic assemblies at low cost, as a result more and more nematode species are being sequenced. Sequences were gathered or generated for 53 nematode species from ESTs, gene predictions from full genome assemblies and transcripts from RNA-Seq experiments. These sequences were screened for orthologous gene clusters, which were concatenated into a supermatrix with thousands of aminoacid sites. The analysis of the supermatrix with maximum likelihood and Bayesian inference methods sheds light into the early splitting clades of the phylogenetic tree of nematodes and the derived clades III, IV and V. Furthermore, the phylogenetic relationships within the parastitic family Onchocercidae were resolved, unveiling the evolutionary history of these important taxa. Finally, data produced in this work will be useful for subsequent evolutionary studies of the phylum Nematoda.

Resolving the phylogenetic tree of eukaryotes is an ongoing challenge for evolutionary biologists. One of the most intriguing questions is the phylogenetic status of Excavata, a group that is well supported by morphological evidence, yet usually not recovered as a clade in molecular phylogenies. The most problematic group of excavates are diplomonads (e.g., Giardia), which tend to have very highly divergent gene sequences, making any phylogenetic analyses that include these protists very susceptible to long branch attraction artifact. This thesis first explores which organisms are most closely related to diplomonads. Phylogenies of three marker genes demonstrate that enteromonads, formerly considered a possible sister group to diplomonads, are a polyphyletic group within diplomonads, suggesting complex evolution of cell morphology in this lineage. However, a large diversity of Carpediemonas-like organisms (CLOs) was discovered from marine/saline samples. Most of the major clades of CLOs had not been detected by previous environmental PCR studies. SSU rRNA gene phylogenies show that CLOs form a series of relatively short branches at the base of diplomonads. Phylogenomic analysis of eukaryotes (161 genes), incorporating EST data from 5 excavates, including 3 CLOs, shows that the non-monophyly of Excavata in phylogenomic studies is likely caused by long branch attraction artifact, since most of the methods used to suppress long branch attraction significantly weaken support for this topology. Furthermore, the shorter-branching CLOs represent valuable replacements for the long branching diplomonads; we recovered a robustly supported monophyletic Excavata, when long branches, including diplomonads (and parabasalids), were removed from the analysis. Subsequently, comparative analysis of the putative proteomes of three CLO isolates, the retortamonad Chilomastix, diplomonads and parabasalids was performed. Several putative evolutionary steps leading to the extremely reduced mitochondrial organelle of diplomonads were derived through the comparative analysis of predicted organellar proteomes. This thesis shows the importance of taxon sampling for inferring deep eukaryotic evolution. The more robust understanding of the phylogeny of Excavata, especially diplomonads and parabasalids, and the new availability of a number of deep branching relatives of diplomonads, provide a framework for comparative analyses exploring the evolution of anaerobic organelles or parasitism.

Protists are defined as eukaryotic organisms that are not animals, land plants, or fungi and make up the vast majority of eukaryotic diversity. Unfortunately, they are often overlooked and under sampled. Some rationales behind this are that they are often microscopic, most rarely the cause of human disease, and are often difficult to get into culture. Molecular phylogenetics has revolutionized our understanding of the evolutionary history of eukaryotes and without a clear picture of protist evolution our understanding would be severely hindered. Phylofisher is an easy-to-use phylogenomic software package, with an accompanying database and various utilities, intended for deep phylogenomic analyses. Here I present thorough documentation for the use of PhyloFisher. I also developed a PhyloFisher utility, nucl_matrix_constructor.py, to allow the software package to build nucleotide phylogenomic matrices. This will allow users to address more shallow evolutionary questions. I developed a metabarcoding pipeline based on Oxford Nanopore Technology’s long read sequencing platform to explore the diversity of eukaryotic organisms in environmental samples. I characterized a novel discosean amoeba. Lastly, I predicted the proteomes from publicly available, but non-annotated genomes for 19 Acanthamoeba spp. and two Balamuthia spp. and used the PhyloFisher tools documented and developed to gain a better understanding into the evolutionary history of the clade as well as provide key resources in the future research of the clade.

Societies where workers sacrifice their own reproduction and cooperatively nurture the offspring of a reproductive queen caste have originated repeatedly across the Tree of Life. The attainment of such reproductive division of labor enabled the evolution of remarkable diversity in development, behavior, and social organization in the Hymenoptera (ants, bees, and wasps). Wasps of the family Vespidae exhibit a gamut of social levels, ranging from solitary to highly social behavior. The highly social yellowjackets and hornets (Vespinae) have well developed differences in form and function between queens and workers, large colony sizes, and intricate nest architecture. Moreover, certain socially parasitic species in the Vespinae have secondarily lost the worker caste and rely entirely on the workers of a host species to ensure the survival of parasitic offspring. Understanding the evolution of behavioral traits in the Vespinae over long periods of time would be greatly enhanced by a robust hypothesis of historical relationships. In this study, I analyze targeted genes and transcriptomes to address three goals. First, infer phylogenetic relationships within yellowjackets (Vespula and Dolichovespula) and hornets (Vespa and Provespa). Second, test the hypothesis that social parasites are more closely related to their hosts than to any other species (Emery's rule). Third, test the protein evolution hypothesis, which states that accelerated evolution of protein coding genes and positive selection operated in the transition to highly eusocial behavior. The findings of this study challenge the predominant understanding of evolutionary relationships in the Vespinae. I show that yellowjacket genera are not sister lineages, instead recovering Dolichovespula as more closely related to the hornets, and placing Vespula as sister to all other vespine genera. This implies that traits such as large colony size and high paternity are mostly restricted to a particular evolutionary trajectory (Vespula) from an early split in the Vespinae. I demonstrate that obligate and facultative social parasites do not share immediate common ancestry with their hosts, indicating that socially parasitic behavior likely evolved independently of host species. Moreover, obligate social parasites share a unique evolutionary history, suggesting that their parasitic behavior might have a genetic component. Lastly, I analyze transcriptomic data to infer a phylogeny of vespid wasps and use this phylogeny to discover lineage-specific signatures of positive selection. I identify more than two hundred genes showing signatures of positive selection on the branch leading to the highly eusocial yellowjackets and hornets. These positively selected genes involve functions related mainly to carbohydrate metabolism and mitochondrial activity, in agreement with insights from studies of bees and ants. Parallels of functional categories for genes under positive selection suggests that at the molecular level the evolution of highly eusocial behavior across the Hymenoptera might have followed similar and narrow paths.

Genus Danio includes zebrafish (Danio rerio) and several other phenotypically diverse species. To understand the history of these species and how they acquired the genetic differences underlying their diverse phenotypes, I performed two phylogenomic studies using Restriction-Site Associated DNA Sequencing and DNA hybridization-based exome enrichment. The results of these studies highlight important methodological considerations applicable to future experiments across taxa. Furthermore, these studies provide detailed understanding of the relationships within Danio including extensive introgression between lineages. The extent of introgression varies across the genome with regions of high recombination at the ends of chromosomes having the most evidence for introgression. Together, this work gives vital insight into the history of a model organism and the evolutionary processes that give rise to phenotypic diversity.

As flores possuem uma grande variedade de sistemas sexuais. O mais raro deles é a androdioicia, com poucos casos conhecidos, no qual as espécies apresentam indivíduos ou com flores masculinas ou com flores bissexuais. A maioria das espécies androdioicas evoluiu de ancestrais dioicos, sendo geralmente plantas herbáceas em populações com recorrente extinção local seguida de re-colonização. No entanto, há alguns exemplos de espécies androdioicas e lenhosas cujos ancestrais são hermafroditas. Este é o caso das Annonaceae, uma família pantropical de plantas lenhosas e predominantemente hermafrodita, no qual a androdioicia é frequente. Gêneros androdioicos surgiram várias vezes em diferentes linhagens de Annonaceae, tanto na subfamília Annonoideae, quanto na subfamília Malmeoideae. A maioria dos gêneros androdioicos pertence à subfamília Malmeoideae, um grupo predominantemente asiático. Nas Américas há cinco gêneros androdioicos, todos da tribo Malmeeae, que formam um grupo monofilético, com exceção de Pseudoxandra que, embora membro de Malmeeae, não está incluído no clado dos gêneros androdioicos. Assim, a tribo Malmeeae é um excelente modelo para o estudo da androdioicia em plantas lenhosas neotropicais. Para tanto a reconstrução da filogenia de 33 táxons da tribo Malmeeae foi realizada a partir de sequências de DNA de 66 marcadores moleculares do genoma do cloroplasto, obtidas por sequenciamento de nova geração. Foram realizadas análises de máxima verossimilhança, máxima parcimônia e inferência Bayesiana. A reconstrução dos estados ancestrais de caracteres relacionados ao sistema sexual e morfologia da flor foi realizada numa abordagem Bayesiana. Análises morfo-anatômicas das flores masculinas e bissexuais de Pseudoxandra spiritus-sancti, uma espécie androdioica, foram feitas com microscopia ótica e MEV. Por último, a filogenia morfológica e a revisão taxonômica de Ephedranthus, um gênero androdioico da tribo Malmeeae, são apresentadas / Flowers have a high diversity of sexual systems. The rarest among them is androdioecy, in which species present individuals with male flowers and others with bisexual flowers. The majority of androdioecious species, usually herbaceous plants with recurrent local extinction followed by re-colonization, has evolved from dioecious ancestors. Nevertheless, some woody and androdioecious plants have hermaphrodite ancestors. This is the case of Annonaceae, a pantropical family of woody and hermaphrodite plants, in which androdioecy is frequent. Androdioecious genera have arisen several times in different Annonaceae lineages, both in Annonoideae subfamily and in Malmeoideae subfamily. The majority of androdioecious genera belong to Malmeoideae, a mostly Asian group. In the Americas there are five androdioecious genera, all from tribe Malmeeae, where they compose a monophyletic group, with the exception of Pseudoxandra, which although being a member of Malmeeae, is not included in the androdioecious genera clade. Tribe Malmeeae is, thus, an excellent model to study androdioecy in Neotropical woody plants. The phylogenetic reconstruction of 33 taxa of this group was performed using DNA sequences of 66 molecular markers of the chloroplast genome, sequenced by next generation sequencing. Maximum likelihood, Bayesian inference and maximum parsimony were the methods used for the phylogenetic analyses. The reconstruction of ancestral states were performed to characters related to sexual system and floral morphology using a Bayesian approach. Morphological and anatomical analyses of male and bisexual flowers were done using LM and SEM. A morphological phylogeny and a taxonomic revision of Ephedranthus, an androdioecious genus of tribe Malmeeae, are also presented

My dissertation focuses on genome and functional evolution of photosynthetic eukaryotes and the design and implementation of computational methods and tools to enable genome-wide studies to investigate these taxa. The work described here is grouped into two major topics, 1) endosymbiosis and genome evolution, and 2) harmful algal blooms. I discuss my work related to endosymbiosis and genome evolution in chapters 2-4. Chapters 5-6 cover the work related to harmful algal blooms. In chapter 1, I introduce the state-of-art of what is known about the history of plastids and evolution of photosynthesis in eukaryotes, an overview of marine harmful algae, and the specific aims of my dissertation. In chapter 2, I describe the design and implementation of the phylogenetic sorting tool, PhyloSort and the assembly of a high-throughput phylogenomic pipeline. Together, PhyloSort and the pipeline has become a key tool for multiple subsequent studies. chapter 2 also presents a case study using these tools in which we provide an estimate of the number of cyanobacterial genes that have been transferred to the nuclear genome of Plantae through primary endosymbiotic gene transfer; I use the model unicellular green alga Chlamydomonas reinhardtii for this purpose. In chapter 3, I discuss another case of prokaryotic contribution to the nucleus of photosynthetic eukaryotes. Here, the intriguing relationship of Chlamydiae-like bacteria and plants and algae is examined in a large-scale analysis, in which we scanned all available genomes of the primary photosynthetic organisms for genes of potential Chlamydiae origin. Surprisingly, we identified more than fifty Chlamydiae-derived genes in plants and algae. Here, we propose a model for the role that a Chlamydiae-like symbiont might have played in the establishment of the primary plastid in the common ancestor of Plantae. In chapter 4, I describe a study in which we explored the complete protein models of two diatom organisms as representative for photosynthetic chromalveolates and looked for genes that might have been acquired through endosymbiotic (secondary) or horizontal transfers from red or green algae. In contradiction of the “chromalveolate hypothesis” which states that photosynthesis in chromalveolates originated via the engulfment of a red alga symbiont, our study shows an unexpected green algal contribution that is fourfold greater than that of the canonical red algal symbiont. Our data suggest that the chromalveolate history includes a previously unrecognized green algal endosymbiont that was captured and lost prior to the more recent establishment of the red alga plastid, which is widespread in extant photosynthetic chromalveolates. In chapter 5, I discuss the identification of the phylogenetic origin of the genes involved in the biosynthetic pathway of saxitoxin in cyanobacteria. Here, we used a pyrosequencing approach to sequence de novo genomes of two strains of Anabaena circinalis, one of which is saxitoxin-producing and the other is non-toxic. Using comparative and phylogenetic analyses, I show that, within the saxitoxin gene cluster, genes that encode the key and unique enzymes in the pathway are of foreign origin that originated via horizontal transfer from non-cyanobacterial sources. These genes introduced the ability to produce saxitoxin in the ancestor of the toxic cyanobacterial clade. In chapter 6, I describe a gene expression study in which we used massively parallel signature sequencing (MPSS) to investigate RNA abundance patterns in the toxic dinoflagellate Alexandrium tamarense. This work provides the first clear evidence for the utilization by dinoflagellates of transcriptional to regulation. Moreover, using MPSS, we provide an estimate of the number of the distinct genes in Alexandrium tamarense; i.e., remarkably 40,000 loci. Taken together, our data indicate that dinoflagellates possess a great metabolic flexibility that allows them to efficiently toggle between photoautotrophy and heterotrophy based on the environmental conditions.

There are ~12 supergroups of complex-celled organisms (eukaryotes), but relationships among them (including the root) remain elusive. For Paper I, I developed a dataset of 37 eukaryotic proteins of bacterial origin (euBac), representing the conservative protein core of the proto-mitochondrion. This gives a relatively short distance between ingroup (eukaryotes) and outgroup (mitochondrial progenitor), which is important for accurate rooting. The resulting phylogeny reconstructs three eukaryote megagroups and places one, Discoba (Excavata), as sister group to the other two (neozoa). This rejects the reigning “Unikont-Bikont” root and highlights the evolutionary importance of Excavata. For Paper II, I developed a 150-gene dataset to test relationships in supergroup SAR (Stramenopila, Alveolata, Rhizaria). Analyses of all 150-genes give different trees with different methods, but also reveal artifactual signal due to extremely long rhizarian branches and illegitimate sequences due to horizontal gene transfer (HGT) or contamination. Removing these artifacts leads to strong consistent support for Rhizaria+Alveolata. This breaks up the core of the chromalveolate hypothesis (Stramenopila+Alveolata), adding support to theories of multiple secondary endosymbiosis of chloroplasts. For Paper III, I studied the evolution of cox15, which encodes the essential mitochondrial protein Heme A synthase (HAS). HAS is nuclear encoded (nc-cox15) in all aerobic eukaryotes except Andalucia godoyi (Jakobida, Excavata), which encodes it in mitochondrial DNA (mtDNA) (mt-cox15). Thus the jakobid gene was postulated to represent the ancestral gene, which gave rise to nc-cox15 by endosymbiotic gene transfer. However, our phylogenetic and structure analyses demonstrate an independent origin of mt-cox15, providing the first strong evidence of bacteria to mtDNA HGT. Rickettsiales or SAR11 often appear as sister group to modern mitochondria. However these bacteria and mitochondria also have independently evolved AT-rich genomes. For Paper IV, I assembled a dataset of 55 mitochondrial proteins of clear α-proteobacterial origin (including 30 euBacs). Phylogenies from these data support mitochondria+Rickettsiales but disagree on the placement of SAR11. Reducing amino-acid compositional heterogeneity (resulting from AT-bias) stabilizes SAR11 but moves mitochondria to the base of α-proteobacteria. Signal heterogeneity supporting other alternative hypotheses is also detected using real and simulated data. This suggests a complex scenario for the origin of mitochondria.

As flores possuem uma grande variedade de sistemas sexuais. O mais raro deles é a androdioicia, com poucos casos conhecidos, no qual as espécies apresentam indivíduos ou com flores masculinas ou com flores bissexuais. A maioria das espécies androdioicas evoluiu de ancestrais dioicos, sendo geralmente plantas herbáceas em populações com recorrente extinção local seguida de re-colonização. No entanto, há alguns exemplos de espécies androdioicas e lenhosas cujos ancestrais são hermafroditas. Este é o caso das Annonaceae, uma família pantropical de plantas lenhosas e predominantemente hermafrodita, no qual a androdioicia é frequente. Gêneros androdioicos surgiram várias vezes em diferentes linhagens de Annonaceae, tanto na subfamília Annonoideae, quanto na subfamília Malmeoideae. A maioria dos gêneros androdioicos pertence à subfamília Malmeoideae, um grupo predominantemente asiático. Nas Américas há cinco gêneros androdioicos, todos da tribo Malmeeae, que formam um grupo monofilético, com exceção de Pseudoxandra que, embora membro de Malmeeae, não está incluído no clado dos gêneros androdioicos. Assim, a tribo Malmeeae é um excelente modelo para o estudo da androdioicia em plantas lenhosas neotropicais. Para tanto a reconstrução da filogenia de 33 táxons da tribo Malmeeae foi realizada a partir de sequências de DNA de 66 marcadores moleculares do genoma do cloroplasto, obtidas por sequenciamento de nova geração. Foram realizadas análises de máxima verossimilhança, máxima parcimônia e inferência Bayesiana. A reconstrução dos estados ancestrais de caracteres relacionados ao sistema sexual e morfologia da flor foi realizada numa abordagem Bayesiana. Análises morfo-anatômicas das flores masculinas e bissexuais de Pseudoxandra spiritus-sancti, uma espécie androdioica, foram feitas com microscopia ótica e MEV. Por último, a filogenia morfológica e a revisão taxonômica de Ephedranthus, um gênero androdioico da tribo Malmeeae, são apresentadas / Flowers have a high diversity of sexual systems. The rarest among them is androdioecy, in which species present individuals with male flowers and others with bisexual flowers. The majority of androdioecious species, usually herbaceous plants with recurrent local extinction followed by re-colonization, has evolved from dioecious ancestors. Nevertheless, some woody and androdioecious plants have hermaphrodite ancestors. This is the case of Annonaceae, a pantropical family of woody and hermaphrodite plants, in which androdioecy is frequent. Androdioecious genera have arisen several times in different Annonaceae lineages, both in Annonoideae subfamily and in Malmeoideae subfamily. The majority of androdioecious genera belong to Malmeoideae, a mostly Asian group. In the Americas there are five androdioecious genera, all from tribe Malmeeae, where they compose a monophyletic group, with the exception of Pseudoxandra, which although being a member of Malmeeae, is not included in the androdioecious genera clade. Tribe Malmeeae is, thus, an excellent model to study androdioecy in Neotropical woody plants. The phylogenetic reconstruction of 33 taxa of this group was performed using DNA sequences of 66 molecular markers of the chloroplast genome, sequenced by next generation sequencing. Maximum likelihood, Bayesian inference and maximum parsimony were the methods used for the phylogenetic analyses. The reconstruction of ancestral states were performed to characters related to sexual system and floral morphology using a Bayesian approach. Morphological and anatomical analyses of male and bisexual flowers were done using LM and SEM. A morphological phylogeny and a taxonomic revision of Ephedranthus, an androdioecious genus of tribe Malmeeae, are also presented