Biology subjects

Keeling, P. J.

Publications and source records attributed to Keeling, P. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Phylogenetic position and expanded host range of the photosymbiont Brandtodinium nutricula (Peridiniales, Dinophyceae)

Brandtodinium nutricula is a dinoflagellate species commonly reported in symbiosis with the marine protist host lineage, Collodaria (Rhizaria). Despite the widespread abundance of this symbiosis throughout the open ocean, and clear indications that the alga should be capable of existing outside of the symbiosis, there are no reports to date of the alga in free-living form in the environment. Even though B. nutricula molecular data are publicly available, the phylogenetic position of B. nutricula remains unclear. Our taxon-rich 18S rRNA gene phylogenetic analysis revealed a close association between B. nutricula and the copepod parasite lineage, Blastodinium. We next analyzed Tara Oceans metabarcode data to assess B. nutricula abundance outside of the Collodaria symbiosis and found that B. nutricula is common in free-living form throughout the sunlit ocean. Similarly, B. nutricula does not significantly correlate with Collodaria in terms of read depth in Tara Oceans samples, despite their tight association. We also determined that B. nutricula can live symbiotically within planktonic Foraminifera using fluorescence lifetime imaging microscopy in combination with fluorescence in situ hybridization. These findings indicate that the radiolarian-B. nutricula symbiosis is less specific than previously suggested, highlighting the complexity of open ocean symbioses and the importance of weighing both host and symbiont perspectives when considering who benefits from the relationship.

ecology↗

bioRxiv2026

Fluorescence Lifetime Imaging Microscopy (FLIM) as a method to elucidate trends in marine planktonic symbioses

The Retaria are an abundant planktonic protist lineage that play a substantial role in biogeochemical cycling, primary production, and food web dynamics in open ocean ecosystems. Their impact is, in large part, due to their capacity to host photosynthetic endosymbionts. Despite the importance of these relationships in the global oceans, our knowledge about the diversity of photosymbionts associated with the planktonic Retaria is still limited due to the difficulty of collecting and analyzing these cells. Here we apply a new protocol to assess retarian photosymbiont diversity: Fluorescence Lifetime Imaging Microscopy (FLIM) is an easy-to-use method to distinguish between different sources of fluorescence during confocal microscopy. We applied FISH probes that hybridize with the most commonly reported retarian photosymbiont lineages to environmental plankton samples and used FLIM to confirm the presence or absence of our FISH probes in retarian cells. By analyzing the fluorescence lifetime of 92 photosymbionts, we found that the retarian photosymbiosis is far more complex than originally described, where Retaria commonly host multiple algal lineages per cell, and regularly host algal lineages outside of the two most common lineages. These efforts demonstrate that FLIM is an overlooked tool in the study of host-microbe associations, with significant potential to be used across diverse host lineages and environmental samples.

ecology↗

bioRxiv2026

Single cell genomics and fluorescence microscopy suggest a permanent plastid in a marine centrohelid

Photosynthetic organelles (plastids), which originated via primary endosymbiosis between an Archaeplastida ancestor and cyanobacteria, have shaped the Earths oxygen-rich atmosphere and spread across the eukaryotic tree of life through multiple secondary and higher order endosymbioses. Yet, the mechanisms driving the transition from endosymbiont to organelle remain poorly understood, highlighting the need for novel systems to elucidate the stages of endosymbiont integration and test the generality of plastid evolution models. Here, we employ single-cell genomics and catalyzed reporter deposition fluorescence in-situ hybridization (CARD-FISH) to investigate the dictyochophyte plastids of the poorly known marine centrohelid Meringosphaera across diverse geographic locations. Our analyses detected novel microdiversity of cells both containing and lacking plastids. Using phylogenomics we show that host and plastid data are perfectly congruent across a large plastid-containing clade (named MER-2), strongly indicating co-evolution. Extensive environmental screening using double CARD-FISH simultaneously targeting host and plastid further confirms that MER-2 cells almost always harbor plastids, supporting the hypothesis of permanent plastid integration and vertical transmission. Additionally, we find that both MER-2 hosts and other Meringosphaera lineages encode multiple plastid-associated genes from diverse phylogenetic origins, with many of their products predicted to be plastid-targeted. Collectively, our findings represent the first report of algae in centrohelids, a large eukaryotic group of otherwise heterotrophic predators. The discovery of new plastids is rare and underscores the importance of exploring uncultured algae to provide new insights into plastid origin and evolution.

evolutionary biology↗

bioRxiv2026

Global diversity and distribution of coral-associated protists

Coral reefs are critical ecosystems and biodiversity hotspots that provide ecological stability and essential services to coastal communities. The coral holobiont, a complex symbiotic system composed of the coral animal and a diverse array of associated microbes, plays a central role in coral health and resilience. While Symbiodiniaceae and bacterial symbionts have been extensively studied, much less is known about the diversity and function of microbial eukaryotes such as protists and fungi. These organisms are increasingly recognized as important, yet remain vastly underexplored. Here, we present the first global survey of the coral-associated eukaryome using an anti-metazoan 18S rRNA primer set to bypass host DNA amplification. Our dataset includes corals and related anthozoans from the Caribbean Sea, the Red Sea, and several locations in the Pacific Ocean, spanning a broad taxonomic and geographic range, and includes both healthy and diseased specimens. They reveal a eukaryome that is not only more diverse than that of global coastal waters but also surpasses the diversity of the well-studied coral bacterial microbiome. We recover diverse microbial eukaryotic communities, including Symbiodiniaceae, other known symbionts, potential pathogens, and previously uncharacterized lineages. These results reveal consistent patterns across coral groups and geographic regions. This study provides the most comprehensive taxonomic overview of coral-associated microbial eukaryotes to date, offering new insights into their roles within the holobiont. Our findings highlight the ecological significance of microbial eukaryotes and underscore the importance of incorporating them into broader coral reef research and conservation strategies.

microbiology↗

bioRxiv2025

Regulated development of cannibalistic supergiant cells

Virtually all paradigms in developmental biology apply to differentiating cells and tissues within multicellular animals and plants. However, unicellular eukaryotes, which must simultaneously perform all roles necessary for organismal function, also form complex and specialized structures, using processes that take place exclusively within the confines of a single cell. Here, we describe a ciliate (Euplotes gigatrox sp. nov.) undergoing drastic morphological transformations within a genetically uniform population, the most spectacular being the appearance of "supergiants" that increase in size, change shape, and modify their locomotion and feeding behaviour to cannibalize clonal relatives. We explore supergiant formation from the perspective of life cycle, ecological strategy, and gene expression, demonstrating that supergiants are distinct, regulated, transcriptionally unique stages. These reversibly differentiated cells require both external and internal triggers to develop and have evolved regulatory loops to ensure coupling between environmental and physiological conditions. This system provides a blueprint for approaching both cell differentiation and functional ecology in unicellular organisms, which might open new avenues for the generalization and contextualization of known morphogenetic mechanisms, as well as the discovery of new ones.

cell biology↗

bioRxiv2025

Modern microbialites harbor an undescribed diversity of chromerid algae

Chromerid algae and the heterotrophic colpodellids together make up the chrompodellids, which are the closest known relatives to apicomplexan parasites [1]. As apicomplexans are prolific parasites of animals, including humans, their adaptation to parasitism from algal ancestors has garnered significant interest, and chromerids were instrumental in elucidating that evolution [2-5]. But the chromerids were also the first new algal group discovered in 100 years [2], and because so much attention has focused on comparisons with apicomplexans, the ecology of chromerid algae has remained surprisingly mysterious. They have predominately been considered to live only in association with corals, initially as intracellular coral symbionts. To test for a wider distribution of chromerids in nature, we have used plastid metagenomic binning combined with re-analysis of 18S rRNA metabarcoding data, which both support an expanded biogeographic range and ecological niche of chromerid algae by showing they are consistent associates of modern microbialites across the globe. Specifically, a broad and undescribed diversity of chromerid lineages is found in marine and also freshwater microbialites. This includes the recovery of a near-complete V. brassicaformis plastid genome and a second incomplete plastid genome from a new chromerid lineage from Highborne Cay thrombolites. This is the first concrete evidence that this under-sampled algal lineage lives outside some association with coral, including freshwater habitats, and sheds new light on the diversity and ecological range of the most recently discovered algal lineage.

microbiology↗

bioRxiv2025

Phylogenetic position and mitochondrial genome evolution of 'orphan' eukaryotic lineages

The phylogenetic tree of eukaryotes is divided into a handful of highly diverse supergroups; only a few so-called orphan lineages branch in uncertain positions outside of these large clades. We found that the mitochondrial genome of one such lineage, the telonemids, is considerably gene-rich, a feature observed in other orphans as well, raising the possibility that these organisms share a common history. On the contrary, our phylogenomic analyses show that orphans with gene-rich mitochondria branch into two different positions: telonemids actually belong to the established supergroup Haptista, while provorans and meteorids form a strongly supported clade with hemimastigophorans, in a novel ancient supergroup that we dub here Promethea. Comparative genomics shows that this split reflects differences between mitochondrial gene sets. Thanks to the increased number of available representatives analyzed together, our results further simplify and illuminate the evolutionary relationships between eukaryotes.

microbiology↗

bioRxiv2025

Single-cell phylogenomics identifies major groups of marine eugregarine parasites (Apicomplexa)

Gregarines are a large group of apicomplexan parasites that infect a wide range of invertebrate hosts, including diverse and speciose groups, such as annelids and arthropods. Marine eugregarines represent the majority of gregarine diversity, but remain poorly understood, especially their deepest phylogenetic relationships. To expand knowledge of marine eugregarine diversity and their evolutionary history, we surveyed marine invertebrates, with a particular focus on annelids, across multiple locations in British Columbia, Canada. From this effort, we obtained high-quality, single-cell transcriptomes from 20 different species of marine eugregarines, including nine previously described species and 11 novel ones, which more than doubles the amount of phylogenomic data for the group. These data, which comprehensively represent the known diversity of marine gregarines in annelid hosts, allowed us to construct an expanded phylogenetic tree based on small subunit ribosomal DNA sequences and a phylogenomic tree inferred from 142 proteins and 44,802 amino acid sequences. Our analyses identified five "superfamily-level" groups of marine eugregarines infecting annelid hosts: The Ancoroidea, Lecudinoidea, Loxomorphoidea n. superfam., Paralecudinoidea n. superfam., and Belladinoidea n. superfam., with the latter three newly established in this study. These findings contribute to ongoing efforts to build a robust molecular phylogenetic framework for gregarine diversity and refine gregarine classification, supporting the recognition of 11 eugregarine superfamilies. However, some of the deepest evolutionary relationships among these superfamilies remain unresolved, highlighting the need for expanded taxon sampling to better capture the true diversity of eugregarine parasites.

microbiology↗

bioRxiv2025

Fireworms (Hermodice carunculata) are a reservoir and potential vector for coral-infecting apicomplexans

Corals (Cnidaria; Anthozoa) play critical roles as habitat-forming species with a wide range, from warm shallow-water tropical coral reefs to cold-water ecosystems [1-3]. They also represent a complex ecosystem as intricate holobionts made up of microbes from all domains of the Tree of Life, that can play significant roles in host health and fitness [4]. The corallicolids are a clade of apicomplexans that infect a wide variety of anthozoans across the world, and can influence the thermal tolerance of habitat-forming corals [1, 5]. Despite their potentially important impacts on reef ecosystems, much of the basic biology and ecology of corallicolids remains unclear. Apicomplexans often have a closed life cycle, with minimal environmental exposure, and sometimes multiple hosts. Corallicolids have only been documented in anthozoan hosts, with no known secondary/reservoir hosts or vectors [6]. Here, we show that abundant corallicolid sequences are recovered from bearded fireworms (Hermodice carunculata) in tropical reef habitats off Curacao, and that they are distinct from corallicolids infecting the corals on which the fireworms were feeding at the time of their collection. The data are consistent with an active infection of fireworms, as opposed to corallicolids being a byproduct of feeding on infected corals, and we suggest that H. carunculata is potentially a vector moving corallicolids among coral hosts through its faeces. These findings not only expand our understanding of the ecological interactions within coral reef ecosystems but also highlight the potential role of host-associated parasites in shaping the resilience of reef habitats.

microbiology↗

bioRxiv2022

Euglenozoan kleptoplasty illuminates the early evolution of photoendosymbiosis

Kleptoplasts are distinct among photosynthetic organelles in eukaryotes (i.e, plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light- dark cycles, and 13C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 247 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis. These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early stages in the endosymbiotic origin of euglenophyte plastids also involved kleptoplasty.

evolutionary biology↗

bioRxiv2021

Monophyly of Diverse Bigyromonadea and their Impact on Phylogenomic Relationships Within Stramenopiles

Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao, Develocanicus komovi, Develocanicus vyazemskyi, Cubaremonas variflagellatum, Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 gene-matrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self- aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.

evolutionary biology↗

bioRxiv2021

Pseudofinder: detection of pseudogenes in prokaryotic genomes

Prokaryotic genomes are generally gene dense and encode relatively few pseudogenes, or nonfunctional/inactivated remnants of genes. However, in certain contexts, such as recent ecological shifts or extreme population bottlenecks (such as those experienced by symbionts and pathogens), pseudogenes can quickly accumulate and form a substantial fraction of the genome. Identification of pseudogenes is, thus, a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis. With Pseudofinders multi-pronged, reference-based approach, we demonstrate its capacity to detect a wide variety of pseudogenes, including those that are highly degraded and typically missed by gene-calling pipelines, as well newly formed pseudogenes, which can have only one or a few inactivating mutations. Additionally, Pseudofinder can detect intact genes undergoing relaxed selection, which may indicate incipient pseudogene formation. Implementation of Pseudofinder in annotation pipelines will not only clarify the functional potential of sequenced microbes, but will also generate novel insights and hypotheses regarding the evolutionary dynamics of bacterial and archaeal genomes.

bioinformatics↗

bioRxiv2021

Picozoa are archaeplastids without plastid

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we used single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids.

evolutionary biology↗

bioRxiv2020

Taxonomy of the apicomplexan symbionts of coral, including Corallicolida ord. nov., reassignment of the genus Gemmocystis, and description of new species Corallicola aquarius gen. nov. sp. nov. and Anthozoaphila gnarlus gen. nov. sp. nov.

Corals (Metazoa; Cnidaria; Anthozoa) have recently been shown to play host to a widespread and diverse group of intracellular symbionts of the phylum Apicomplexa. These symbionts, colloquially called corallicolids, are mostly known through molecular analyses, and no formal taxonomy has been proposed. Another apicomplexan, Gemmocystis cylindrus (described from the coral Dendrogyra cylindrus), may be related to corallicolids, but lacks molecular data. Here, we isolate and describe motile trophozoite (feeding) corallicolids cells using microscopic (light, SEM, and TEM) and molecular phylogenetic analysis to provide the basis for a formal description. Phylogenetic analyses using nuclear and plastid rRNA operons, and three mitochondrial protein sequences derived from single cell transcriptomes, all confirm that these organisms fall into a discrete deep-branching clade within the Apicomplexa not closely related to any known species or major subgroup. As a result, we assign this clade to a new order, Corallicolida ord. nov., and family, Corallicolidae fam. nov. We describe a type species, Corallicola aquarius gen. nov. sp. nov. from its Rhodactis sp. host, and also describe a second species, Anthozoaphila gnarlus gen. nov. sp. nov., from the coral host Madracis mirabilis. Finally, we propose reassigning the incertae sedis taxon G. cylindrus from the order Agamococcidiorida to the Corallicolida, based on similarities in morphology and host localization to that of the corallicolids.

microbiology↗

bioRxiv2020

Heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao, the Netherlands Antilles

Recent progress in understanding the early evolution of eukaryotes was tied to morphological identification of flagellates and heliozoans in the natural samples, isolation of their cultures and genomic and ultrastructural investigations. These protists are the smallest and least studied microbial eukaryotes but play an important role in functioning of microbial food webs. Using light and electron microscopy, we have studied the diversity of heterotrophic flagellates and centrohelid heliozoans from marine waters of Curacao (The Netherlands Antilles), and provide micrographs and morphological descriptions of observed species. Among 86 flagellates and 3 centrohelids encountered in this survey, five heterotrophic flagellates and one Centrohelid heliozoan were not identified even to the genus. Some flagellate protists have a unique morphology, and may represent undescribed lineages of eukaryotes of high taxonomic rank. The vast majority (89%) of identified flagellates are characterized by wide geographical distribution and had been reported previously from all hemispheres and various climatic regions. More than half of the species were previously observed not only from marine, but also from freshwater habitats. The parameters of the species accumulation curve indicate that our species list obtained for the Curacao study sites is far from complete, and each new sample should yield new species.

microbiology↗

bioRxiv2020

Predatory colponemids are the sister group to all other alveolates

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which unambiguously support their monophyly and position as the sister lineage to all other alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates as well as origin of myzocytosis and plastids. HighlightsO_LIPhylogenomics resolves Colponemidia as a sister group to all other alveolates C_LIO_LIThe ancestor of all alveolates was a biflagellate predator feeding by phagocytosis C_LIO_LIColponemids may illuminate the ancestral states of apicomplexans, dinoflagellates, and ciliates C_LIO_LIColponemids are geographically widespread in freshwater habitats C_LI

evolutionary biology↗

bioRxiv2019

Insights into the origin of metazoan multicellularity from predatory unicellular relatives of animals

The diversity and biology of unicellular relatives of animals has strongly informed our understanding of the transition from single-celled organisms to the multicellular Metazoa. Here we analyse the cellular structures and complex life cycles of the novel unicellular holozoans Pigoraptor and Syssomonas (Opisthokonta). Both lineages are characterized by complex life cycles with a variety of cell types, the formation of multicellular aggregations and syncytium-like structures, and an unusual diet for single-celled opisthokonts (partial cell fusion and joint sucking of a large eukaryotic prey), all of which provide new insights into the origin of multicellularity in Metazoa. The ability to feed on large eukaryotic prey could have been a powerful trigger in the formation and development both aggregative (e.g., joint feeding, which also implies signalling) and clonal (e.g., hypertrophic growth followed by palintomy) multicellular stages that played important roles in the emergence of multicellular animals.

evolutionary biology↗

bioRxiv2019

Early eukaryotic origins and metazoan elaboration of MAPR family proteins

BackgroundThe membrane-associated progesterone receptor (MAPR) family consists of heme-binding proteins containing a cytochrome b5 (cytb5) domain characterized by the presence of a MAPR-specific interhelical insert region (MIHIR) between helices 3 and 4 of the canonical cytb5-domain fold. Animals possess three MAPR families (PGRMC-like, Neuferricin and Neudesin).\n\nResultsHere we show that all animal MAPR families were already present in the common ancestor of the Opisthokonta (comprising animals and fungi as well as related protistan taxa). All three MAPR genes acquired extensions C-terminal to the cytb5 domain, either before or with the evolution of animals. The archetypical MAPR protein, progesterone receptor membrane component 1 (PGRMC1), contains phosphorylated tyrosines Y139 and Y180. The combination of Y139/Y180 appeared in the common ancestor of Cnidaria and bilaterally symmetrical animals, along with an early embryological organizer and synapsed neurons, and is strongly conserved in all bilateral animals. A predicted protein interaction motif in the PGRMC1 MIHIR is potentially regulated by Y139 phosphorylation. A multilayered model of animal MAPR function acquisition includes some pre-metazoan functions (e.g., heme binding and cytochrome P450 interactions) and some acquired animal-specific functions that involve regulation of strongly conserved protein interaction motifs acquired by early-branching animals.\n\nConclusionsThis study provides a conceptual framework for future studies, against which PGRMC1s multiple functions can perhaps be stratified and functionally dissected. In accompanying papers we show that mutational perturbation of PGRMC1 phosphorylation status of the Y180 motif is associated with dramatic changes cell pasticity assayed by protein abundances, cell morphology, mitochondrial function, genomic stability, and epigenetic status, with pathways analysis associating Y180 mutation with processes related to organizer function. These combined works reveal previously unrecognized involvement of PGRMC1 in foundational animal processes of great relevance to disease.

evolutionary biology↗