Patrick John Keeling

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Evolution of complex organelles in dinoflagellates (2016)

Dinoflagellates are an abundant and diverse group of aquatic eukaryotes, with members that have photosynthetic, heterotrophic, or mixotrophic life strategies, as well as a number of unique cytological features. My thesis focuses on two groups of closely related dinoflagellates: polykrikoids and warnowiids. Both include heterotrophic as well as plastid-bearing members, though the number of times photosynthesis has been lost (or gained) in each group is unclear, and the presence and provenance of plastids in some species (e.g., Nematodinium sp. and Polykrikos lebouriae) have been debated. Polykrikoids and warnowiids also contain some of the most complex subcellular structures described--such as nematocysts and, in warnowiids, eye-like ocelloids. Yet these groups are rare in nature and uncultivated, and as such, the origins of their complex organelles are unclear. For my thesis, I modified existing techniques for use on single-cell environmental isolates, and applied these techniques to wild polykrikoid and warnowiid cells. By exploiting the common splice leader sequence found on dinoflagellate transcripts, I was able to amplify a single-cell transcriptome from Polykrikos lebouriae—a dinoflagellate with aberrant plastids. Coupled with single-cell genomics using multiple displacement amplification (MDA), I demonstrated that Polykrikos lebouriae has retained peridinin-type plastids, while photosynthesis has been lost in multiple other polykrikoid species independently. Using MDA and single-cell transmission electron microscopy, I also determined that the eye-like ocelloid of Nematodinium sp. is made in part from a peridinin plastid, and also from mitochondria. Specifically, single-cell focused ion beam scanning electron microscopy (FIB-SEM) allowed me to demonstrate that a retina-like portion of the ocelloid is a small part of a much larger peridinin-plastid that ramifies throughout the Nematodinium cell. Lastly, I investigated the evolution of nematocysts in Polykrikos spp. and Nematodinium sp. using a combination of transcriptomics, TEM, SEM, and FIB-SEM, and inferred that “nematocysts” in these groups evolved independently from those in cnidarians. Thus, nematocyst-like extrusive organelles appear to have evolved multiple times in eukaryotes. The data presented in this thesis show how extreme subcellular complexity has evolved in dinoflagellates through both endosymbiotic and autogenous origins.

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A genomic survey of two dinotoms (2013)

Endosymbiosis has played a major role in shaping eukaryotic cells, their success and diversity. At the base of the eukaryotic tree, an α-proteobacterium endosymbiont in a protoeukaryotic cell was converted into the mitochondrion through its reductive evolution, endosymbiotic gene transfer (EGT) and the development of a protein targeting system to direct the products of the transferred genes to this organelle. Similar events mark the plastid evolution from a cyanobacterium. However, the primary endosymbiosis of plastid, unlike the mitochondrion, was followed by the secondary and tertiary movement of this organelle between eukaryotes through analogous endosymbiotic reduction, EGT and evolution of a protein targeting system and many subsequent independent losses from different eukaryotic lineages.The obligate tertiary diatom endosymbiont in a small group of dinoflagellates called ‘dinotoms’ is exceptional in that it retains most of its ancestral characters including a large nucleus, its own mitochondria, plastids and many other eukaryotic organelles and structures in a large cytoplasm all enclosed in and separated from its dinoflagellate host by a single membrane. This level of conservation of ancestral features in the endosymbiont suggests an early stage of integration. In order to investigate the impacts of endosymbiosis on the organelle genomes and to determine the extent of EGT and the contribution of the host nuclear genome to the proteomes of the organelles, I conducted mass pyrosequencing of the A+T-rich portion of the DNA extracted from two dinotoms, Durinskia baltica and Kryptoperidinium foliaceum, and the SL cDNA library constructed for D. baltica.The plastid and mitochondrial genomes of these two dinotoms were sequenced, and the results indicated that, despite the permanent symbiosis between the host and its endosymbiont in dinotoms and in spite of small variations, the dinotom organelle genomes have changed very little from those of free-living diatoms and dinoflagellates. There was also no sign of EGT to the host in D. baltica, suggesting a strict compartmentalization in which the host mitochondria remain reliant on the host nucleus while the endosymbiont organelles, mitochondria and plastids, stay entirely dependent on the endosymbiont nucleus with no genetic exchange between the host and endosymbiont.

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Evolution of free-living relatives of apicomplexan parasites (2013)

As obligate parasites of animals and humans, apicomplexan parasites contain many unique characteristics that are critical to their lifestyle, but bear little resemblance to other eukaryotes. Several free-living relatives of apicomplexans represent a great potential in understanding early apicomplexan evolution. The photosynthetic Chromera velia provides a particular promise in addressing the long-contentious origin of the apicomplexan plastid. The data presented here provides evidence that the photosynthetic plastids in Chromera velia and another novel alga, CCMP3155 (later named Vitrella brassicaformis), are closely and specifically related to the apicomplexan plastid, and that they together are related to plastids in dinoflagellates. The ancestral plastid went through an unusual reduction in gene content and acquired unique features such as Rubisco II and transcript oligouridylylation. The plastid genome in C. velia is interesting on its own. Two proteins of Photosystem I and ATP synthase have been split to two fragments, which are independently expressed. The genome also appears to exist prevailingly as a linear monomer. These, and additional unprecedented features, redefine our understanding of plastid genome architecture and point to intra-chromosomal recombination as a putative driving force. Assessing environmental distribution of the newly-discovered Chromera and Vitrella leads to a discovery of six additional apicomplexan-related linages (ARLs) comprising 1,316 sequences primarily from coral reefs environments. The most abundant lineage, ARL-V, is novel and exclusively associated with coral tissue and surface samples. ARL-V is present in at least 20 species of symbiotic corals across time and space, which suggests that its relationship with corals is of potential significance to the reef ecosystem. Successful culturing of five Colponema isolates provides the first molecular data for another apicomplexan relative. The genus represents two independent lineages, one of which is the closest sister to apicomplexans and dinoflagellates. Mitochondrial genome data from both lineages reveals a gene-rich content and suggests that a linear monomeric structure with telomeres was ancestral to all alveolates. Altogether, this data illustrates the significance of Chromera, Vitrella, Colponema and several uncultured lineages in illuminating early evolution in apicomplexans and alveolates.

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Molecular evolution of the eukaryotic translation elongation factor, EFL (2009)

The eukaryotic translation elongation factor EFL (for EF-Like) is a paralogue of the better-known elongation factor 1-alpha (EF-1α), which brings aminoacyl-tRNAs to the ribosome during translation. This essential protein was thought to be ubiquitous in eukaryotes until the recent discovery of EFL in a small number of diverse, mainly unicellular, eukaryotic organisms that were found to lack EF-1α. Because of the great evolutionary distances between EFL-encoding lineages and the near mutual exclusivity of the two proteins, the observed complex distribution of EFL was initially attributed entirely to multiple lateral gene transfers. In the enclosed chapters, the distribution of EFL was characterized in more detail in four distantly related eukaryotic lineages at both fine and broad taxonomic scales in order to better understand the effects that endosymbiotic gene transfer, differential loss, and lateral gene transfer have had on the molecular evolution of EFL. Endosymbiotic transfer of EFL was detected in the chlorarachniophytes, a group of algae whose secondary plastids retain a vestigial nucleus, known as a nucleomorph, in their reduced eukaryotic cytoplasm, known as the periplastid compartment (PPC). The endosymbiotically transferred EFL carries a bipartite targeting sequence similar to those of plastid-targeted proteins in this group and to plastid- and PPC-targeting sequences in cryptomonads to direct it to the PPC, suggesting similarities in the way these two lineages have solved their shared challenge of targeting to complex plastids with nucleomorphs. No clear phylogenetic evidence for lateral transfer of EFL has yet emerged; rather, differential loss of EFL and EF-1α from an ancestral state of co-occurrence was characterized in euglenozoans and detected in publicly available data from heterokonts and opisthokonts, unexpectedly revealing a significant role for this process in shaping the complex distribution of EFL and EF-1α. This finding serves as a cautionary reminder that adequate taxon sampling and a robust organismal phylogenetic hypothesis are crucial in order to correctly infer lateral gene transfer.

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Master's Student Supervision (2010 - 2018)
Labyrinthulomycetes diversity meta-analysis (2016)

Labyrinthulomycetes are a group of ubiquitous stramenopiles that inhabit a wide range of habitats and play important ecological roles as nutrient recyclers and sometimes disease causing agents. Even though they have had a long history of being studied, their diversity has not yet been fully explored. The lack of a comprehensive reference database with up-to-date phylogeny also hinders any pursuits in understanding the ecological distribution of this group. This study was designed with the purpose of constructing a curated reference database and a phylogenetic tree based on existing 18S rDNA data, and then using this database to uncover any hidden diversity and novelty among Labyrinthulomycetes and provide a reference guidance for future identification. Using the newly-created reference database, I also analyzed high-throughput environmental sequencing data from two databases. My results reveal extensive diversity within the Labyrinthulomycetes, and recover many previously unknown environmental sequences, greatly expanding our knowledge of the ecological distribution of this group. The high-throughput environmental sequencing data analysis also shows some of the newly identified environmental clades to be particularly abundant in the ocean. The phylogenetic framework I have provided in this study, together with the metadata I have compiled, will serve as a useful tool for future ecological and evolutionary studies of this widespread lineage.

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