The Dynamic Regulatory Genome of Capsaspora and the Origin of Animal Multicellularity.

The unicellular ancestor of animals had a complex repertoire of genes linked to multicellular processes. This suggests that changes in the regulatory genome, rather than in gene innovation, were key to the origin of animals. Here, we carry out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression, and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora. Moreover, we demonstrate conservation of animal developmental transcription-factor networks and extensive network interconnection in this premetazoan organism. In contrast, however, Capsaspora lacks animal promoter types, and its regulatory sites are small, proximal, and lack signatures of animal enhancers. Overall, our results indicate that the emergence of animal multicellularity was linked to a major shift in genome cis-regulatory complexity, most notably the appearance of distal enhancer regulation.

Poly(A)-binding protein is an ataxin-2 chaperone that regulates biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.

The Origin of Metazoan Multicellularity: A Potential Microbial Black Swan Event.

The emergence of animals from their unicellular ancestors is a major evolutionary event. Thanks to the study of diverse close unicellular relatives of animals, we now have a better grasp of what the unicellular ancestor of animals was like. However, it is unclear how that unicellular ancestor of animals became the first animals. To explain this transition, two popular theories, the choanoblastaea and the synzoospore, have been proposed. We will revise and expose the flaws in these two theories while showing that, due to the limits of our current knowledge, the origin of animals is a biological black swan event. As such, the origin of animals defies retrospective explanations. Therefore, we should be extra careful not to fall for confirmation biases based on few data and, instead, embrace this uncertainty and be open to alternative scenarios. With the aim to broaden the potential explanations on how animals emerged, we here propose two novel and alternative scenarios. In any case, to find the answer to how animals evolved, additional data will be required, as will the hunt for microscopic creatures that are closely related to animals but have not yet been sampled and studied.

Divergent genomic trajectories predate the origin of animals and fungi.

Animals and fungi have radically distinct morphologies, yet both evolved within the same eukaryotic supergroup: Opisthokonta1,2. Here we reconstructed the trajectory of genetic changes that accompanied the origin of Metazoa and Fungi since the divergence of Opisthokonta with a dataset that includes four novel genomes from crucial positions in the Opisthokonta phylogeny. We show that animals arose only after the accumulation of genes functionally important for their multicellularity, a tendency that began in the pre-metazoan ancestors and later accelerated in the metazoan root. By contrast, the pre-fungal ancestors experienced net losses of most functional categories, including those gained in the path to Metazoa. On a broad-scale functional level, fungal genomes contain a higher proportion of metabolic genes and diverged less from the last common ancestor of Opisthokonta than did the gene repertoires of Metazoa. Metazoa and Fungi also show differences regarding gene gain mechanisms. Gene fusions are more prevalent in Metazoa, whereas a larger fraction of gene gains were detected as horizontal gene transfers in Fungi and protists, in agreement with the long-standing idea that transfers would be less relevant in Metazoa due to germline isolation3-5. Together, our results indicate that animals and fungi evolved under two contrasting trajectories of genetic change that predated the origin of both groups. The gradual establishment of two clearly differentiated genomic contexts thus set the stage for the emergence of Metazoa and Fungi.

Chemical factors induce aggregative multicellularity in a close unicellular relative of animals.

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals' closest unicellular living relatives-the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.

Regulation of sedimentation rate shapes the evolution of multicellularity in a close unicellular relative of animals.

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma-close unicellular relatives of animals with brief transient multicellular life stages-we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least 2 distinct physical mechanisms. First, we find extensive (>300×) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular Sphaeroforma arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in 4 additional species from the Sphaeroforma genus, suggesting that variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodeling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.

Towards understanding the origin of animal development.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development.

The origin of Metazoa: a unicellular perspective.

The first animals evolved from an unknown single-celled ancestor in the Precambrian period. Recently, the identification and characterization of the genomic and cellular traits of the protists most closely related to animals have shed light on the origin of animals. Comparisons of animals with these unicellular relatives allow us to reconstruct the first evolutionary steps towards animal multicellularity. Here, we review the results of these investigations and discuss their implications for understanding the earliest stages of animal evolution, including the origin of metazoan genes and genome function.

Cell cycle transcriptomics of Capsaspora provides insights into the evolution of cyclin-CDK machinery.

Progression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are widely conserved in eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, we investigated the transcriptional program during the cell cycle of the filasterean Capsaspora owczarzaki, a unicellular species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Reticulate evolution in eukaryotes: Origin and evolution of the nitrate assimilation pathway.

Genes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolution in eukaryotes, and in particular of horizontal gene transfer (HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryotic nitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested that the nitrate assimilation cluster of dikaryotic fungi (Opisthokonta) could have been originated and transferred from a lineage leading to Oomycota (Stramenopiles). We studied the origin and evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows that nitrate assimilation is present in more lineages than previously reported, although being restricted to autotrophs and osmotrophs. The phylogenies indicate a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. In particular, we propose a distinct and more complex HGT path between Opisthokonta and Stramenopiles than the one previously suggested, involving at least two transfers of a nitrate assimilation gene cluster. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a chimeric nitrate reductase in Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryotes with independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolution in eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.

Transfection of Capsaspora owczarzaki, a close unicellular relative of animals.

How animals emerged from their unicellular ancestor remains a major evolutionary question. New genome data from the closest unicellular relatives of animals have provided important insights into the evolution of animal multicellularity. We know that the unicellular ancestor of animals had an unexpectedly complex genetic repertoire, including many genes that are key to animal development and multicellularity. Thus, assessing the function of these genes among unicellular relatives of animals is key to understanding how they were co-opted at the onset of the Metazoa. However, such analyses have been hampered by the lack of genetic tools. Progress has been made in choanoflagellates and teretosporeans, two of the three lineages closely related to animals, whereas no tools are yet available for functional analysis in the third lineage: the filastereans. Importantly, filastereans have a striking repertoire of genes involved in transcriptional regulation and other developmental processes. Here, we describe a reliable transfection method for the filasterean Capsaspora owczarzaki We also provide a set of constructs for visualising subcellular structures in live cells. These tools convert Capsaspora into a unique experimentally tractable organism to use to investigate the origin and evolution of animal multicellularity.

The Expansion of Inosine at the Wobble Position of tRNAs, and Its Role in the Evolution of Proteomes.

The modification of adenosine to inosine at the first position of transfer RNA (tRNA) anticodons (I34) is widespread among bacteria and eukaryotes. In bacteria, the modification is found in tRNAArg and is catalyzed by tRNA adenosine deaminase A, a homodimeric enzyme. In eukaryotes, I34 is introduced in up to eight different tRNAs by the heterodimeric adenosine deaminase acting on tRNA. This substrate expansion significantly influenced the evolution of eukaryotic genomes in terms of codon usage and tRNA gene composition. However, the selective advantages driving this process remain unclear. Here, we have studied the evolution of I34, tRNA adenosine deaminase A, adenosine deaminase acting on tRNA, and their relevant codons in a large set of bacterial and eukaryotic species. We show that a functional expansion of I34 to tRNAs other than tRNAArg also occurred within bacteria, in a process likely initiated by the emergence of unmodified A34-containing tRNAs. In eukaryotes, we report on a large variability in the use of I34 in protists, in contrast to a more uniform presence in fungi, plans, and animals. Our data support that the eukaryotic expansion of I34-tRNAs was driven by the improvement brought by these tRNAs to the synthesis of proteins highly enriched in certain amino acids.

Revisiting the phylogenetic position of Caullerya mesnili (Ichthyosporea), a common Daphnia parasite, based on 22 protein-coding genes.

Caullerya mesnili is a common and virulent parasite of the water flea, Daphnia. It was classified within the Haplosporidia (Rhizaria) for over a century. However, a recent molecular phylogeny based on the 18S rRNA gene suggested it belonged to the Ichthyosporea, a class of protists closely related to animals within the Opisthokonta clade. The exact phylogenetic position of C. mesnili remained uncertain because it appeared in the 18S rRNA tree with a very long branch and separated from all other taxa, suggesting that its position could be artifactual. A better understanding of its phylogenetic position has been constrained by a lack of molecular markers and the difficulty of obtaining a suitable quantity and quality of DNA from in vitro cultures, as this intracellular parasite cannot be cultured without its host. We isolated and collected spores of C. mesnili and sequenced genomic libraries. Phylogenetic analyses of a newly generated multi-protein data set (22 proteins, 4998 amino acids) and of sequences from the 18S rRNA gene both placed C. mesnili within the Ichthyophonida sub-clade of Ichthyosporea, as sister-taxon to Abeoforma whisleri and Pirum gemmata. Our study highlights the utility of metagenomic approaches for obtaining genomic information from intracellular parasites and for more accurate phylogenetic placement in evolutionary studies.

Revision of the Capsaspora genome using read mating information adjusts the view on premetazoan genome.

The genome sequences of unicellular holozoans, the closest relatives to animals, are shedding light on the evolution of animal multicellularity, shaping the genetic contents of the putative premetazoans. However, the assembly quality of the genomes remains poor compared to the major model organisms such as human and fly. Improving the assembly is critical for precise comparative genomics studies and further molecular biological studies requiring accurate sequence information such as enhancer analysis and genome editing. In this report, we present a new strategy to improve the assembly by fully exploiting the information of Illumina mate-pair reads. By visualizing the distance and orientation of the mapped read pairs, we could highlight the regions where possible assembly errors exist in the genome sequence of Capsaspora, a lineage of unicellular holozoans. Manual modification of these errors repaired 590 assembly problems in total and reassembled 84 supercontigs into 55. Our telomere prediction analysis using the read pairs containing the pan-eukaryotic telomere-like sequence identified at least 13 chromosomes. The resulting new assembly posed us a re-annotation of 112 genes, including 15 putative receptor protein tyrosine kinases. Our strategy thus provides a useful approach for improving assemblies of draft genomes, and the new Capsaspora genome offers us an opportunity to adjust the view on the genome of the unicellular animal ancestor.

A global metabarcoding analysis expands molecular diversity of Platyhelminthes and reveals novel early-branching clades.

Understanding biological diversity is crucial for ecological and evolutionary studies. Even though a great part of animal diversity has already been documented, both morphological surveys and metabarcoding analyses have previously shown that some animal groups, such as Platyhelminthes, may harbour hidden diversity. To better understand the molecular diversity of Platyhelminthes, one of the most diverse and biomedically important animal phyla, we here combined data from six marine and two freshwater metabarcoding expeditions that cover a broad variety of aquatic habitats and analysed the data by phylogenetic placement. Our results show that a great part of the hidden diversity is located in early-branching clades such as Catenulida and Macrostomorpha, as well as in late-diverging clades such as Proseriata and Rhabdocoela. We also report the first freshwater record of Gnosonesimida, a group previously thought to be exclusively marine. Finally, we identified two putative novel freshwater Platyhelminthes clades that branch between well-defined orders of the phylum. Thus, our analyses of several environmental datasets confirm that a large part of the diversity of Platyhelminthes remains undiscovered, point to groups with more potential novel species and identify freshwater environments as potential reservoirs for novel species of flatworms.

Sterol and genomic analyses validate the sponge biomarker hypothesis.

Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650-540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago.

Characterization of a group I Nme protein of Capsaspora owczarzaki-a close unicellular relative of animals.

Nucleoside diphosphate kinases are enzymes present in all domains of life. In animals, they are called Nme or Nm23 proteins, and are divided into group I and II. Human Nme1 was the first protein identified as a metastasis suppressor. Because of its medical importance, it has been extensively studied. In spite of the large research effort, the exact mechanism of metastasis suppression remains unclear. It is unknown which of the biochemical properties or biological functions are responsible for the antimetastatic role of the mammalian Nme1. Furthermore, it is not clear at which point in the evolution of life group I Nme proteins acquired the potential to suppress metastasis, a process that is usually associated with complex animals. In this study we performed a series of tests and assays on a group I Nme protein from filasterean Capsaspora owczarzaki, a close unicellular relative of animals. The aim was to compare the protein to the well-known human Nme1 and Nme2 homologs, as well as with the homolog from a simple animal-sponge (Porifera), in order to see how the proteins changed with the transition to multicellularity, and subsequently in the evolution of complex animals. We found that premetazoan-type protein is highly similar to the homologs from sponge and human, in terms of biochemical characteristics and potential biological functions. Like the human Nme1 and Nme2, it is able to diminish the migratory potential of human cancer cells in culture.

Parvularia atlantis gen. et sp. nov., a Nucleariid Filose Amoeba (Holomycota, Opisthokonta).

The opisthokonts constitute a eukaryotic supergroup divided into two main clades: the holozoans, which include animals and their unicellular relatives, and the holomycotans, which include fungi, opisthosporidians, and nucleariids. Nucleariids are phagotrophic filose amoebae that phenotypically resemble more their distant holozoan cousins than their holomycotan phylogenetic relatives. Despite their evolutionary interest, the diversity and internal phylogenetic relationships within the nucleariids remain poorly studied. Here, we formally describe and characterize by molecular phylogeny and microscopy observations Parvularia atlantis gen. et sp. nov. (formerly Nuclearia sp. ATCC 50694), and compare its features with those of other nucleariid genera. Parvularia is an amoebal genus characterized by radiating knobbed and branching filopodia. It exhibits prominent vacuoles observable under light microscopy, a cyst-like stage, and completely lacks cilia. P. atlantis possesses one or two nuclei with a central nucleolus, and mitochondria with flat or discoid cristae. These morphological features, although typical of nucleariids, represent a combination of characters different to those of any other described Nuclearia species. Likewise, 18S rRNA-based phylogenetic analyses show that P. atlantis represents a distinct lineage within the nucleariids.

Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla.

The modification of adenosine to inosine at position 34 of tRNA anticodons has a profound impact upon codon-anticodon recognition. In bacteria, I34 is thought to exist only in tRNAArg, while in eukaryotes the modification is present in eight different tRNAs. In eukaryotes, the widespread use of I34 strongly influenced the evolution of genomes in terms of tRNA gene abundance and codon usage. In humans, codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Here we extend the analysis of distribution of codons that are recognized by I34 containing tRNAs to all phyla known to use this modification. We find that the preference for codons recognized by such tRNAs in genes with highly biased codon compositions is universal among eukaryotes, and we report that, unexpectedly, some bacterial phyla show a similar preference. We demonstrate that the genomes of these bacterial species contain previously undescribed tRNA genes that are potential substrates for deamination at position 34.