Patrick John Keeling


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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Exploring the biology and evolution of dinoflagellates through rare and uncultured taxa (2022)

Dinoflagellates are a diverse group of protists with many unique traits including large genomes packaged into permanently condensed chromosomes, photosynthetic or cryptic plastids acquired vertically or horizontally in serial endosymbioses, and in some taxa, highly complex organelles like nematocysts and the eye-like ocelloid. Because these features promise to expand our understanding of eukaryotic biology, reconstructing how they evolved has become a point of interest. To infer ancestral states, robust and well-supported phylogenies generated from high-coverage transcriptomic datasets are needed. So far, these analyses have relied on transcriptome data from cultured taxa, which are mostly photosynthetic. As half of known dinoflagellate species are non-photosynthetic, current phylogenies fail to reflect the diversity that characterizes this group. Here, I generate single cell transcriptomes from over 150 rare and under-sampled dinoflagellates collected from the environment. Using these data, I explore three major heterotrophic lineages of interest: Abedinium, the Noctilucales, and the complex organelle-bearing members of the Gymnodiniales, the warnowiids. In these investigations I reveal that Abedinium is an independent, deep-branching core dinoflagellate lineage, the unique traits of the Noctilucales are derived rather than ancestral, and the heterotrophic warnowiids retain photosynthetic genes except for photosystem II and RuBisCo, suggesting that this mechanism serves an alternate function in the ocelloid. In the final chapter I generate a comprehensive dinoflagellate phylogeny that better represents the proportion of heterotrophic, athecate, and deep- branching taxa in dinoflagellates. This analysis reveals several new insights, including the early acquisition timing of two histone-like protein (HLP) types, the diversity and punctate distribution of microbial rhodopsins, and the common retention of plastid-derived electron transport genes across heterotrophic dinoflagellates.

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The evolution of bacterial symbionts and bacteriophages in microbial eukaryotes (2022)

Genome evolution of bacterial symbionts has led to different evolutionary outcomes including stable symbiosis, extinction, and rarely organelle formation (e.g., mitochondria and plastids). Symbiont genome evolution has mostly been studied in animal endosymbionts, but microbial eukaryotes (protists) contain diverse symbionts with various functional roles and evolutionary trajectories. However, symbiont genome reduction and functional relationships with protist hosts are largely unexplored. Additionally, how bacteriophage (phage) infection impacts symbiont-eukaryotic interactions and symbiont genome evolution is relatively unknown in protists. Here, I used DNA and RNA sequencing and microscopy methods to characterize endosymbionts and symbiont-infecting phages from diverse protists, including marine diplonemids, freshwater cryptomonads, and termite gut parabasalids. The diplonemid and cryptomonad endosymbionts belonged to intracellular bacterial groups Rickettsiales and Holosporaceae (Alphaproteobacteria), and they harboured an arsenal of proteins with putative eukaryotic-interacting domains (e.g., leucine-rich repeats and ankyrin repeats). The diplonemid endosymbionts had extremely small genomes with reduced metabolic potential compared to other Rickettsiales and Holosporaceae endosymbionts. Despite this, they retained a small cluster of gene transfer agent (GTA) genes also highly conserved in Rickettsiales and Holosporaceae, and evidence of GTA gene expression was found. An endosymbiont-infecting phage was also characterized in an undescribed Cryptomonas species, and the temperate phage encoded several eukaryotic-interacting proteins. Finally, phages from the hindgut of wood-feeding insects were sequenced and predicted to infect protist endosymbionts and hindgut bacteria. Overall, this work highlights the complex interactions between phages, bacteria and protists from diverse environments and provides valuable insights into the evolution of all three groups.

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The phylogeny and evolution apicomplexan parasites (2021)

Apicomplexans are a large phylum of obligate animal parasites that contain pathogens such as Plasmodium spp. (the causative agent of malaria) and Toxoplasma gondii. While these medically relevant apicomplexans are the subject of extensive research, the bulk of the diversity of the group, particularly the lineages that infect invertebrates, remain poorly studied and largely ignored in high-throughput sequencing surveys. In this dissertation, I show that these groups are critical to gaining insights into the origins and evolution of the Apicomplexa. I begin by examining the diversity and inferred ecology of the enigmatic apicomplexan-related lineages (ARLs), and show that ARL-V is highly abundant in environmental surveys, and is tightly associated with coral tissue and mucus, suggesting that it represents a core symbiont of coral. In the following chapters, using methods of single-cell transcriptomics, I sequenced the transcriptomes of 15 invertebrate-infecting apicomplexans. Using this dataset, I constructed a robust and taxon-rich multi-gene apicomplexan phylogeny that resolves the deep phylogenetic relationships within the group, and also form a new class of apicomplexans, the Marosporida, that is sister to the Hematozoa and Coccidia. Most unexpectedly, in Chapter 2, I show that certain taxa previously classified as apicomplexans, actually represent convergently evolved animal parasites, suggesting that apicomplexan-like parasites have evolved at least four times independently. In Chapter 3, I examine the presence and function of apicoplasts (remnant plastids) across the diversity of the group using whole genome shotgun sequencing (WGS), and find that the Marosporida contain the smallest, most AT-rich, and gene poor apicoplast genomes sequenced to date. I also present the first evidence of plastids in the gregarines, and show that archigregarines retain the canonical apicomplexan plastid metabolism, whereas only one clade of marine eugregarines retains plastids that solely carry out type II fatty acid biosynthesis. Lastly in Chapter 4, I reconstruct the mitochondrial metabolism in the gregarines and squirmids, and find that eugregarines contain highly reduced respiratory chains, suggesting that they have lost their mitochondrial genomes, and possess limited energy metabolism. Altogether, the data presented here, illustrates the significance of invertebrate-infecting apicomplexans in illuminating the early evolution of the apicomplexans and myzozoans.

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The functional diversity and evolution of nuclear processes (2020)

The nucleus is a defining characteristic of eukaryotic cells which not only houses the genome but a myriad of processes that function synergistically to regulate cellular activity. Nuclear proteins are key in facilitating core eukaryotic processes such as genome compaction, nucleocytoplasmic exchange, and DNA replication, but the interconnectedness of these processes makes them challenging to dissect mechanistically. Moreover, the antiquity of the nucleus complicates evolutionary analyses, limiting our view of nuclear evolution. Despite this, a comprehensive understanding of the function and evolution of nuclear processes is essential given their central importance in disease, basic cell biology, and eukaryotic evolution. In this dissertation, I argue that insights into nuclear biology and evolution can be obtained by examining eukaryotic diversity rather than relying solely on traditional model organisms. I begin by presenting an introduction to nuclear evolution and diversity, highlighting the existence of nuclear variation across eukaryotes from a systems perspective and underscoring the potential utility of biodiversity in studying nuclear processes (Chapter 1). In the following chapters, I test my hypothesis by examining the function and evolution of different processes in a subset of divergent nuclear systems: namely, chromatin in the dinoflagellate dinokaryon, nuclear pore complexes (NPCs) in the nucleomorphs of chlorarachniophytes and cryptophytes, and DNA replication in the ciliate macronucleus. In Chapter 2, I use an experimental evolutionary approach to investigate the drivers of histone depletion in dinoflagellates, revealing the capacity for viruses to shape cellular evolution and raising questions regarding the subfunctionalization of remnant dinoflagellate histones. In Chapter 3, I reconstruct the NPCs of endosymbiotically acquired nuclei, termed nucleomorphs, in silico, and predict a highly reduced pore structure, suggesting that a complex NPC may not be required for baseline nuclear function. Lastly, in Chapter 4, I examine the diversity of motile DNA replication systems in ciliates, highlighting new models for studying DNA replication and the capacity of cytoskeletal elements to coordinate nuclear organization and processes. Ultimately this work confirms the efficacy of examining diverse nuclear systems, provides insights into the biology and evolution of nuclear processes, and encourages a re-evaluation of how we view and select model organisms.

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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

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

De novo assembly of a zoanthid transcriptome for the study of corallicolids (2023)

The discovery of widespread apicomplexan symbionts of anthozoans, known as corallicolids, has raised many questions about their evolutionary history, physiology, and ecology. However, gathering molecular data from these unicellular eukaryotes is challenging due to their small size, low abundance in host tissue, and inconsistent prevalence in host populations. This task is further complicated by the presence of contamination from host genetic material in the samples. Parazoanthus swiftii is a corallicolid host with a high infection rate and no dinoflagellate symbionts making it a potentially useful model for this association. The objective of my thesis is to develop baseline genetic resources for the study of P. swiftii and its symbionts using transcriptomics. Several P. swiftii transcriptomes were sequenced, assembled, and filtered, and BUSCO analysis detected 88% of proteins from a set of conserved orthologs. Although less than half of the transcripts were functionally annotated, 56% of predicted proteins were present across all of the samples. Two of the five samples had a diverging expression profile and lower relative expression of anthozoan transcripts. The dataset also contained 1059 potential apicomplexan genes, including three that were lineage-specific. This transcriptome will aide in the study of corallicolids and their host associations, as well as add to the number of genetic resources available for zoanthids.

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Elastic net regression for the selection of orthogroups predictive of trophic mechanisms in diverse eukaryotes (2022)

The last eukaryotic common ancestor (LECA) was a unicellular heterotrophic organism that preyed upon bacteria through a process called phagocytosis. LECA’s descendents have evolved into many eukaryotic supergroups, and over this time endosymbiotic integration of cyanobacteria and then eukaryotic algae into diverse hosts has led to the establishment of a photosynthetic organelle, the plastid, throughout multiple distantly-related lineages. As a result, microbial eukaryotes display a range of trophic strategies - heterotrophy through feeding, photoautotrophy through photosynthesis, or a combination of both as mixotrophy. Additionally, saprotrophy, the ability to digest nutrients externally and take up products osmotrophically, has evolved convergently in multiple eukaryotic groups. With the continuous development of high-throughput and culture-independent sampling and sequencing technologies, novel eukaryotic lineages are increasingly recovered from genomic data alone. It is therefore useful to develop methods to predict biological traits from genomic data in microbial eukaryotes. Here, we train an elastic net regression model that predicts the probability of a species obtaining nutrients by heterotrophy, autotrophy, mixotrophy or saprotrophy. To do so, we collected proteomes predicted from genomes and transcriptomes of over 250 species, representing all major lineages with sequence data available. These were clustered into over 230,000 orthologous groups (orthogroups), and models were then trained on the presence or absence of these groups and tested for predictive accuracy on 304 validation species. The final model had a balanced accuracy of 91% and a Cohen’s Kappa of 88% on the validation set, and selected around 4,000 orthogroups that are either negatively or positively associated with each trophic class. The majority of these orthogroups are found in multiple eukaryotic phyla, and 34% had no homology to characterized protein databases. GO term enrichment and KEGG pathway analysis of predictive orthogroups suggests involvement in metabolism of essential compounds, with differential biosynthetic and catabolic abilities associated with each trophic class. By predicting ecological functions in diverse eukaryotes in a culture- and phylogenetic-independent manner, this model has the potential to expedite aspects of both evolutionary and ecological research, as well as assist in selecting candidate proteins that play a functional role in the trophic classes they predict.

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Transcriptome sequencing of Trichonympha from Reticulitermes hesperus and multi-protein phylogenetic analysis of selected Spirotrichonymphids, Cristamonads, and Trichonymphids (2022)

Parabasalid protists include may obligate termite gut symbionts that play important ecological roles, permitting their hosts to digest cellulose. One of the earliest-identified groups of parabasalids is the hypermastigote Trichonymphids, of which Trichonympha is the type species. The purpose of my thesis was to illustrate deep phylogenetic relationships between selected species of Trichonymphids and the other parabasalid groups Spirotrichonymphids and Cristamonads, and to compare the results of using multiple sequences against a single sequence for phylogenetic analyses in these groups. To this end, I sequenced the transcriptomes of Trichonympha cells isolated from a Pacific Coast population of Reticulitermes hesperus, and I used multi-sequence phylogenetic techniques to resolve the phylogeny of Trichonymphida and other Parabasalian groups beyond what has previously been done with the use of SSU-based phylogenetic analyses. I successfully isolated two Trichonympha cells via single-cell picking techniques and sequenced their transcriptomes. I annotated these transcriptomes for protein-coding genes and used the subsequent translated protein sequences, along with translated transcriptomes of other Trichonymphids, to construct a 14-sequence dataset for a multi-gene phylogenetic analysis. I also constructed a SSU sequence dataset for comparison. I used maximum-likelihood algorithms to construct phylogenetic trees from each dataset to illustrate the deep phylogeny of trichonymphids. The phylogeny thus generated agreed with previous phylogenies of Parabasalia, recovering Trichonymphids, Spirotrichonymphida, and Cristamonads as monophyletic. Additionally, I used SSU sequence data to confirm that the single Trichonympha species present in R. hesperus is phylogenetically distinct from Trichonympha agilis, despite previous literature claiming that T. agilis was present both in R. flavipes and R. hesperus and was the only Trichonympha in the latter. Therefore, the R. hesperus Trichonympha requires a full taxonomic description as a novel species, and the epithet T. agilis should be restricted to the species of Trichonympha in R. flavipes. I also confirmed, using COX-2 sequence data, a 2005 report from Austin et. al. synonymizing R. flavipes and R. santonensis.

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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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Current Students & Alumni

This is a small sample of students and/or alumni that have been supervised by this researcher. It is not meant as a comprehensive list.

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