Ahmed Moustafa

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Publications

• Evolution and Functional Diversification of Fructose Bisphosphate Aldolase Genes in Photosynthetic Marine Diatoms.

  Authors: Andrew E Allen, Ahmed Moustafa, Anton Montsant, Angelika Eckert, Peter G Kroth, Chris Bowler

  Molecular biology and evolution.

  Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked throughDiatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, Fructose 1,6- Bisphosphate Aldolases (FBA) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid-localized and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase; which is known to play a significant role in regulation of the diatom carbon concentrating mechanism (CCM). The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared to optimal CO(2) fixation conditions, suggestive of a distinct specialized function for each. Cytosolically-localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

• Update on Sea Slug Kleptoplasty and Plastid Maintenance in a Metazoan.

  Authors: Karen N Pelletreau, Debashish Bhattacharya, Dana C Price, Jared M Worful, Ahmed Moustafa, Mary E Rumpho

  Plant physiology.

• The making of a photosynthetic animal.

  Authors: Mary E Rumpho, Karen N Pelletreau, Ahmed Moustafa, Debashish Bhattacharya

  The Journal of experimental biology. 214(Pt 2):303-11.

  Symbiotic animals containing green photobionts challenge the common perception that only plants are capable of capturing the sun's rays and converting them into biological energy throughSymbiotic animals containing green photobionts challenge the common perception that only plants are capable of capturing the sun's rays and converting them into biological energy through photoautotrophic CO(2) fixation (photosynthesis). 'Solar-powered' sacoglossan molluscs, or sea slugs, have taken this type of symbiotic association one step further by solely harboring the photosynthetic organelle, the plastid (=chloroplast). One such sea slug, Elysia chlorotica, lives as a 'plant' when provided with only light and air as a result of acquiring plastids during feeding on its algal prey Vaucheria litorea. The captured plastids (kleptoplasts) are retained intracellularly in cells lining the digestive diverticula of the sea slug, a phenomenon sometimes referred to as kleptoplasty. Photosynthesis by the plastids provides E. chlorotica with energy and fixed carbon for its entire lifespan of ~10 months. The plastids are not transmitted vertically (i.e. are absent in eggs) and do not undergo division in the sea slug. However, de novo protein synthesis continues, including plastid- and nuclear-encoded plastid-targeted proteins, despite the apparent absence of algal nuclei. Here we discuss current data and provide hypotheses to explain how long-term photosynthetic activity is maintained by the kleptoplasts. This fascinating 'green animal' provides a unique model to study the evolution of photosynthesis in a multicellular heterotrophic organism.

• Differential gene retention in plastids of common recent origin.

  Authors: Adrian Reyes-Prieto, Hwan Su Yoon, Ahmed Moustafa, Eun Chan Yang, Robert A Andersen, Sung Min Boo, Takuro Nakayama, Ken-Ichiro Ishida, Debashish Bhattacharya

  Molecular biology and evolution.

  The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely onThe cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote-eukaryote (primary) endosymbiosis that occurred ca. 60 million years ago. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence to the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers (EGTs) to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus-Prochlorococus group and the cyanobacterial provenance of all plastid genes suggests these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/dS ratios <<1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution.

• Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence.

  Authors: Ahmed Moustafa, Andrew N Evans, David M Kulis, Jeremiah D Hackett, Deana L Erdner, Donald M Anderson, Debashish Bhattacharya

  PloS one. 5(3):e9688.

  BACKGROUND: Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA isBACKGROUND: Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by their massive genome sizes that are often several times that in humans. However, modern transcriptomic methods offer promising approaches to tackle this challenging system. Here, we used massively parallel signature sequencing (MPSS) to understand global transcriptional regulation patterns in Alexandrium tamarense cultures that were grown under four different conditions. METHODOLOGY/PRINCIPAL FINDINGS: We generated more than 40,000 unique short expression signatures gathered from the four conditions. Of these, about 11,000 signatures did not display detectable differential expression patterns. At a p-value < 1E-10, 1,124 signatures were differentially expressed in the three treatments, xenic, nitrogen-limited, and phosphorus-limited, compared to the nutrient-replete control, with the presence of bacteria explaining the largest set of these differentially expressed signatures. CONCLUSIONS/SIGNIFICANCE: Among microbial eukaryotes, dinoflagellates contain the largest number of genes in their nuclear genomes. These genes occur in complex families, many of which have evolved via recent gene duplication events. Our expression data suggest that about 73% of the Alexandrium transcriptome shows no significant change in gene expression under the experimental conditions used here and may comprise a "core" component for this species. We report a fundamental shift in expression patterns in response to the presence of bacteria, highlighting the impact of biotic interaction on gene expression in dinoflagellates.

• Genomic footprints of a cryptic plastid endosymbiosis in diatoms.

  Authors: Ahmed Moustafa, Bánk Beszteri, Uwe-G Maier, Chris Bowler, Klaus Valentin, Debashish Bhattacharya

  Science (New York, N.Y.). 324(5935):1724-6.

  Diatoms and other chromalveolates are among the dominant phytoplankters in the world's oceans. Endosymbiosis was essential to the success of chromalveolates, and it appears that the ancestral plastidDiatoms and other chromalveolates are among the dominant phytoplankters in the world's oceans. Endosymbiosis was essential to the success of chromalveolates, and it appears that the ancestral plastid in this group had a red algal origin via an ancient secondary endosymbiosis. However, recent analyses have turned up a handful of nuclear genes in chromalveolates that are of green algal derivation. Using a genome-wide approach to estimate the "green" contribution to diatoms, we identified >1700 green gene transfers, constituting 16% of the diatom nuclear coding potential. These genes were probably introduced into diatoms and other chromalveolates from a cryptic endosymbiont related to prasinophyte-like green algae. Chromalveolates appear to have recruited genes from the two major existing algal groups to forge a highly successful, species-rich protist lineage.

• Origin of saxitoxin biosynthetic genes in cyanobacteria.

  Authors: Ahmed Moustafa, Jeannette E Loram, Jeremiah D Hackett, Donald M Anderson, F Gerald Plumley, Debashish Bhattacharya

  PloS one. 4(6):e5758.

  BACKGROUND: Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopicBACKGROUND: Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS: We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX-) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena circinalis strains.

• Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica.

  Authors: Mary E Rumpho, Jared M Worful, Jungho Lee, Krishna Kannan, Mary S Tyler, Debashish Bhattacharya, Ahmed Moustafa, James R Manhart

  Proceedings of the National Academy of Sciences of the United States of America. 105(46):17867-71.

  The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesizeThe sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3' flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty ("green animals") and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

• Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic.

  Authors: Adrian Reyes-Prieto, Ahmed Moustafa, Debashish Bhattacharya

  Current biology : CB. 18(13):956-62.

  Plantae (as defined by Cavalier-Smith, 1981) [1] plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spreadPlantae (as defined by Cavalier-Smith, 1981) [1] plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid [2]. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.

• Chlamydiae has contributed at least 55 genes to Plantae with predominantly plastid functions.

  Authors: Ahmed Moustafa, Adrian Reyes-Prieto, Debashish Bhattacharya

  PLoS ONE. 3(5):e2205.

  BACKGROUND: The photosynthetic organelle (plastid) originated via primary endosymbiosis in which a phagotrophic protist captured and harnessed a cyanobacterium. The plastid was inherited by theBACKGROUND: The photosynthetic organelle (plastid) originated via primary endosymbiosis in which a phagotrophic protist captured and harnessed a cyanobacterium. The plastid was inherited by the common ancestor of the red, green (including land plants), and glaucophyte algae (together, the Plantae). Despite the critical importance of primary plastid endosymbiosis, its ancient derivation has left behind very few "footprints" of early key events in organelle genesis. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into this process, we conducted an in-depth phylogenomic analysis of genomic data (nuclear proteins) from 17 Plantae species to identify genes of a surprising provenance in these taxa, Chlamydiae bacteria. Previous studies show that Chlamydiae contributed many genes (at least 21 in one study) to Plantae that primarily have plastid functions and were postulated to have played a fundamental role in organelle evolution. Using our comprehensive approach, we identify at least 55 Chlamydiae-derived genes in algae and plants, of which 67% (37/55) are putatively plastid targeted and at least 3 have mitochondrial functions. The remainder of the proteins does not contain a bioinformatically predicted organelle import signal although one has an N-terminal extension in comparison to the Chlamydiae homolog. Our data suggest that environmental Chlamydiae were significant contributors to early Plantae genomes that extend beyond plastid metabolism. The chlamydial gene distribution and protein tree topologies provide evidence for both endosymbiotic gene transfer and a horizontal gene transfer ratchet driven by recurrent endoparasitism as explanations for gene origin. CONCLUSIONS/SIGNIFICANCE: Our findings paint a more complex picture of gene origin than can easily be explained by endosymbiotic gene transfer from an organelle-like point source. These data significantly extend the genomic impact of Chlamydiae on Plantae and show that about one-half (30/55) of the transferred genes are most closely related to sequences emanating from the genome of the only environmental isolate that is currently available. This strain, Candidatus Protochlamydia amoebophila UWE25 is an endosymbiont of Acanthamoeba and likely represents the type of endoparasite that contributed the genes to Plantae.

• EST analysis of Ostreococcus lucimarinus, the most compact eukaryotic genome, shows an excess of introns in highly expressed genes.

  Authors: William Lanier, Ahmed Moustafa, Debashish Bhattacharya, Josep M Comeron

  PLoS ONE. 3(5):e2171.

  BACKGROUND: The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains manyBACKGROUND: The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied intron features and levels of expression in O. lucimarinus using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number (R = +0.0893, p = <0.0005) and intron density (number of introns/kb of CDS; R = +0.0753, p = <0.005). CONCLUSIONS/SIGNIFICANCE: In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance with other mutational and/or selective forces acting on genome size.

• PhyloSort: a user-friendly phylogenetic sorting tool and its application to estimating the cyanobacterial contribution to the nuclear genome of Chlamydomonas.

  Authors: Ahmed Moustafa, Debashish Bhattacharya

  BMC evolutionary biology. 8:6.

  BACKGROUND: Phylogenomic pipelines generate a large collection of phylogenetic trees that require manual inspection to answer questions about gene or genome evolution. A notable application ofBACKGROUND: Phylogenomic pipelines generate a large collection of phylogenetic trees that require manual inspection to answer questions about gene or genome evolution. A notable application of phylogenomics is to photosynthetic organelle (plastid) endosymbiosis. In the case of primary endosymbiosis, a heterotrophic protist engulfed a cyanobacterium, giving rise to the first photosynthetic eukaryote. Plastid establishment precipitated extensive gene transfer from the endosymbiont to the nuclear genome of the 'host'. Estimating the magnitude of this endosymbiotic gene transfer (EGT) and determining the functions of the prokaryotic genes remain controversial issues. We used phylogenomics to study EGT in the model green alga Chlamydomonas reinhardtii. To facilitate this procedure, we developed PhyloSort to rapidly search large collection of trees for monophyletic relationships. Here we present PhyloSort and its application to estimating EGT in Chlamydomonas. RESULTS: PhyloSort is an open-source tool to sort phylogenetic trees by searching for user specified subtrees that contain a monophyletic group of interest defined by operational taxonomic units in a phylogenomic context. Using PhyloSort, we identified 897 Chlamydomonas genes of putative cyanobacterial origin, of which 531 had bootstrap support values >/= 50% for the grouping of the algal and cyanobacterial homologs. CONCLUSION: PhyloSort can be applied to quantify the number of genes that support different evolutionary hypotheses such as a taxonomic classification or endosymbiotic or horizontal gene transfer events. In our application, we demonstrate that cyanobacteria account for 3.5-6% of the protein-coding genes in the nuclear genome of Chlamydomonas.

• A phylogenomic approach for studying plastid endosymbiosis.

  Authors: Ahmed Moustafa, Cheong Xin Chan, Megan Danforth, David Zear, Hiba Ahmed, Nagnath Jadhav, Trevor Savage, Debashish Bhattacharya

  Genome informatics. International Conference on Genome Informatics. 21:165-76.

  Gene transfer is a major contributing factor to functional innovation in genomes. Endosymbiotic gene transfer (EGT) is a specific instance of lateral gene transfer (LGT) in which genetic materialsGene transfer is a major contributing factor to functional innovation in genomes. Endosymbiotic gene transfer (EGT) is a specific instance of lateral gene transfer (LGT) in which genetic materials are acquired by the host genome from an endosymbiont that has been engulfed and retained in the cytoplasm. Here we present a comprehensive approach for detecting gene transfer within a phylogenetic framework. We applied the approach to examine EGT of red algal genes into Thalassiosira pseudonana, a free-living diatom for which a complete genome sequence has recently been determined. Out of 11,390 predicted protein-coding sequences from the genome of T. pseudonana, 124 (1.1%, clustered into 80 gene families) are inferred to be of red algal origin (bootstrap support >or= 75%). Of these 80 gene families, 22 (27.5%) encode novel, unknown functions. We found 21.3% of the gene families to putatively encode non-plastid-targeted proteins. Our results suggest that EGT of red algal genes provides a relatively minor contribution to the nuclear genome of the diatom, but the transferred genes have functions that extend beyond photosynthesis. This assertion awaits experimental validation. Whereas the current study is focused within the context of secondary endosymbiosis, our approach can be applied to large-scale detection of gene transfer in any system.

• PhyloSort: a user-friendly phylogenetic sorting tool and its application to estimating the cyanobacterial contribution to the nuclear genome of Chlamydomonas

  Authors: Ahmed Moustafa, Debashish Bhattacharya

  BMC Evolutionary Biology.

  Abstract Background Phylogenomic pipelines generate a large collection of phylogenetic trees that require manual inspection to answer questions about gene or genome evolution. A notable applicationAbstract Background Phylogenomic pipelines generate a large collection of phylogenetic trees that require manual inspection to answer questions about gene or genome evolution. A notable application of phylogenomics is to photosynthetic organelle (plastid) endosymbiosis. In the case of primary endosymbiosis, a heterotrophic protist engulfed a cyanobacterium, giving rise to the first photosynthetic eukaryote. Plastid establishment precipitated extensive gene transfer from the endosymbiont to the nuclear genome of the 'host'. Estimating the magnitude of this endosymbiotic gene transfer (EGT) and determining the functions of the prokaryotic genes remain controversial issues. We used phylogenomics to study EGT in the model green alga Chlamydomonas reinhardtii . To facilitate this procedure, we developed PhyloSort to rapidly search large collection of trees for monophyletic relationships. Here we present PhyloSort and its application to estimating EGT in Chlamydomonas . Results PhyloSort is an open-source tool to sort phylogenetic trees by searching for user specified subtrees that contain a monophyletic group of interest defined by operational taxonomic units in a phylogenomic context. Using PhyloSort, we identified 897 Chlamydomonas genes of putative cyanobacterial origin, of which 531 had bootstrap support values ≥ 50% for the grouping of the algal and cyanobacterial homologs. Conclusion PhyloSort can be applied to quantify the number of genes that support different evolutionary hypotheses such as a taxonomic classification or endosymbiotic or horizontal gene transfer events. In our application, we demonstrate that cyanobacteria account for 3.5–6% of the protein-coding genes in the nuclear genome of Chlamydomonas .

• Evolutionary and functional genomics of photosynthetic eukaryotes

  Authors: Ahmed Moustafa

  Theses and Dissertations.

  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 toMy 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.