Phylogeny of Choanozoa,Apusozoa, and Other Protozoa and Early Eukaryote Megaevolution

Abstract The primary diversification of eukaryotes involved protozoa, especially zooflagellates—flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads (Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto largely unrecognized major protozoan assemblage (core Rhizaria) in the eukaryote tree. Both 18S rRNA sequence trees and a rare deletion show that nonciliate haplosporidian and paramyxid parasites of shellfish (together comprising the Ascetosporea) are not two separate phyla, as often thought, but part of the Cercozoa, and may be related to the plant-parasitic plasmodiophorids and phagomyxids, which were originally the only parasites included in the Cercozoa. We discuss rRNA trees in relation to other evidence concerning the basal diversification and root of the eukaryotic tree and argue that bikonts and opisthokonts, at least, are holophyletic. Amoebozoa and bikonts may be sisters—jointly called anterokonts, as they ancestrally had an anterior cilium, not a posterior one like opisthokonts; this contrasting ciliary orientation may reflect a primary divergence in feeding mode of the first eukaryotes. Anterokonts also differ from opisthokonts in sterol biosynthesis (cycloartenol versus lanosterol pathway), major exoskeletal polymers (cellulose versus chitin), and mitochondrial cristae (ancestrally tubular not flat), possibly also primary divergences.     

Phylogenetic Analysis of Eukaryotes Using Heat-Shock Protein Hsp90

Most eukaryote molecular phylogenies have been based on small-subunit ribosomal RNA as its database includes the most species, but serious problems have been encountered that can make these trees misleading. More recent studies using concatenated protein sequences have increased the data per organism, reducing misleading signals from a single sequence, but taxon sampling is limited. To increase the database of protein-coding genes we sequenced the cytosolic form of heat-shock protein Hsp90 from a broad variety of previously unsampled eukaryote groups: protozoan flagellates (phyla Choanozoa, Apusozoa, Cercozoa) and all three groups of chromists (Cryptophyta, Heterokonta, Haptophyta). Gamma-corrected distance trees robustly show three groups: bacterial sequences are sister to all eukaryote sequences, which are cleanly subdivided into the cytosolic sequences and a clade comprising the chloroplast and endoplasmic reticulum (ER) Hsp90 sequences. The eukaryote cytosolic sequences comprise a robust opisthokont clade (animals/Choanozoa/fungi), a bikont clade, and an amoebozoan branch. However their topology is not robust. When the cytosolic sequences are rooted using only the ER/chloroplast clade as outgroup the amoebozoan Dictyostelium is sister to the opisthokonts forming a unikont clade in the distance tree. Congruence of this tree with that for concatenated mitochondrial proteins suggests that the root of the eukaryote tree is between unikonts and bikonts. Gamma-corrected maximum likelihood analyses of cytosolic sequences alone (519 unambiguously aligned amino acid positions) show bikonts as a clade, as do least-squares distance trees, but with other distance methods and parsimony the sole amoebozoan species branches weakly within bikonts. Choanozoa are clearly sisters to animals. Some major bikont groups (e.g. green plants, alveolates, Euglenozoa) are consistently recovered, but others (e.g. discicristates, chromalveolates) appear only in some trees; the backbone of the bikont subtree is not resolved, the position of groups represented only by single sequences being particularly unclear. Although single-gene trees will probably never resolve these uncertainties, the congruence of Hsp90 trees with other data is greater than for most other molecules and further taxon sampling of this molecule is recommended.     

Megaphylogeny,Cell Body Plans,Adaptive Zones: Causes and Timing of Eukaryote Basal Radiations1

ABSTRACT. I discuss eukaryote megaphylogeny and the timing of major innovations in the light of multigene trees and the rarity of marine/freshwater evolutionary transitions. The first eukaryotes were aerobic phagotrophs, probably substratum‐associated heterotrophic amoeboflagellates. The primary eukaryote bifurcation generated unikonts (ancestrally probably unicentriolar, with a conical microtubular [MT] cytoskeleton) and bikonts (ciliary transformation from anterior cilium to ancestrally gliding posterior cilium; cytoskeleton of ventral MT bands). Unikonts diverged into Amoebozoa with anterior cilia, lost when lobosan broad pseudopods evolved for locomotion, and Choanozoa with posterior cilium and filose pseudopods that became unbranched tentacles/microvilli in holozoa and eventually the choanoflagellate/choanocyte collar. Of choanozoan ancestry, animals evolved epithelia, fibroblasts, eggs, and sperm. Fungi and Ichthyosporea evolved walls. Bikonts, ancestrally with ventral grooves, include three adaptively divergent megagroups: Rhizaria (Retaria and Cercozoa, ancestrally reticulofilose soft‐surfaced gliding amoeboflagellates), and the originally planktonic Excavata, and the corticates (Plantae and chromalveolates) that suppressed pseudopodia. Excavata evolved cilia‐generated feeding currents for grooval ingestion; corticates evolved cortical alveoli and ciliary hairs. Symbiogenetic origin and transfers of chloroplasts stimulated an explosive radiation of corticates—hard to resolve on multigene trees—and opisthokonts, and ensuing Cambrian explosions of animals and protists. Plantae lost phagotrophy and multiply evolved walls and macroalgae. Apusozoa, with dorsal pellicle and ventral pseudopods, are probably the most divergent bikonts or related to opisthokonts. Eukaryotes probably originated 800–850 My ago. Amoebozoa, Apusozoa, Loukozoa, and Metamonada may be the only extant eukaryote phyla pre‐dating Neoproterozoic snowball earth. New subphyla are established for Choanozoa and Loukozoa; Amoebozoa are divided into three revised subphyla, with Variosea transferred into Conosa.     

Hsp70 sequences indicate that choanoflagellates are closely related to animals.

Over 130 years ago, James-Clark noted a remarkable structural similarity between the feeding cells of sponges (choanocytes) and a group of free-living protists, the choanoflagellates. Both cell types possess a single flagellum surrounded by a collar of fine tentacles. The similarity led to the hypothesis that sponges, and, by implication, other animals, evolved from choanoflagellate-like ancestors. Phylogenetic analysis of ribosomal DNA neither supports nor refutes this hypothesis. Here, we report the sequence of an hsp70 gene and pseudogene from the freshwater choanoflagellate Monosiga ovata. These represent the first nuclear-encoded protein-coding sequences reported for any choanoflagellate. We find that Monosiga and most bilaterian hsp70 genes have high GC contents that may distort phylogenetic tree construction; therefore, protein sequences were used for phylogenetic reconstruction. Our analyses indicate that Monosiga is more closely related to animals than to fungi. We infer that animals and at least some choanoflagellates are part of a clade that excludes the fungi. This is consistent with the origin of animals from a choanoflagellate-like ancestor.     

Cell differentiation and morphogenesis in the colony-forming choanoflagellate Salpingoeca rosetta

It has been posited that animal development evolved from pre-existing mechanisms for regulating cell differentiation in the single celled and colonial ancestors of animals. Although the progenitors of animals cannot be studied directly, insights into their cell biology may be gleaned from comparisons between animals and their closest living relatives, the choanoflagellates. We report here on the life history, cell differentiation and intercellular interactions in the colony-forming choanoflagellate Salpingoeca rosetta. In response to diverse environmental cues, S. rosetta differentiates into at least five distinct cell types, including three solitary cell types (slow swimmers, fast swimmers, and thecate cells) and two colonial forms (rosettes and chains). Electron microscopy reveals that cells within colonies are held together by a combination of fine intercellular bridges, a shared extracellular matrix, and filopodia. In addition, we have discovered that the carbohydrate-binding protein wheat germ agglutinin specifically stains colonies and the slow swimmers from which they form, showing that molecular differentiation precedes multicellular development. Together, these results help establish S. rosetta as a model system for studying simple multicellularity in choanoflagellates and provide an experimental framework for investigating the origin of animal multicellularity and development.     

Molecular evidence of fungal signatures in the marine protist Corallochytrium limacisporum and its implications in the evolution of animals and fungi

Fungi, animals, and single-celled organisms belonging to the choanozoans together constitute the supergroup Opisthokonta. The latter are considered crucial in understanding the evolutionary origin of animals and fungi. The choanozoan Corallochytrium limacisporum is an enigmatic marine protist of considerable interest in opisthokontan evolution. Several isolates of the organism were obtained from a coral reef lagoon in the Lakshadweep group of islands of the Arabian Sea. The capability of these cultures to grow on media containing inorganic nitrogen sources prompted us to examine the possible presence of fungal signatures, namely the enzyme alpha-aminoadipate reductase (alpha-AAR) involved in the alpha-aminoadipate (AAA) pathway for synthesizing lysine and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi, were detected in the organism. Phylogenetic trees based on the alpha-AAR gene suggested that Corallochytrium limacisporum is a sister clade to fungi, while those based on the C-14 reductase gene did not adequately resolve whether the organism was more closely related to fungi or animals. While many studies indicate that Corallochytrium is a sister clade to animals, we suggest that further studies are required to examine whether this protist is in fact more closely related to fungi rather than to animals.     

The predicted secretomes of Monosiga brevicollis and Capsaspora owczarzaki,close unicellular relatives of metazoans,reveal new insights into the evolution of the metazoan extracellular matrix

The extracellular matrix (ECM) is a major mediator of multi-cellularity in the metazoa. Multiple ECM proteins are conserved from sponges to human, raising questions about the evolutionary origin of ECM. Choanoflagellates are the closest unicellular relatives of the metazoa and proteins with domains characteristic of metazoan ECM proteins have been identified from the genome-predicted proteome of the choanoflagellate Monosiga brevicollis. However, a systematic analysis of M. brevicollis secretory signal peptide-containing proteins with ECM domains has been lacking. We analysed all predicted secretory signal-peptide-containing proteins of M. brevicollis for ECM domains. Nine domains that are widespread in metazoan ECM proteins are represented, with EGF, fibronectin III, laminin G, and von Willebrand Factor_A domains being the most numerous. Three proteins contain more than one category of ECM domain, however, no proteins correspond to the domain architecture of metazoan ECM proteins. The fibronectin III domains are all present within glycoside hydrolases and none contain an integrin-binding motif. Glycosaminoglycan-binding motifs identified in animal thrombospondin type 1 domains are conserved in some M. brevicollis representatives of this domain, whereas there is little evidence of conservation of glycosaminoglycan-binding motifs in the laminin G domains. The identified proteins were compared with the predicted secretory ECM domain-containing proteins of the integrin-expressing filasterean, Capsaspora owczarzaki. C. owczarzaki encodes a smaller number of secretory, ECM domain-containing proteins and only EGF, fibronectin type III and laminin G domains are represented. The M. brevicollis and C. owczarzaki proteins have distinct domain architectures and all proteins differ in their domain architecture to metazoan ECM proteins. These identifications provide a basis for future experiments to validate the extracellular location of these proteins and uncover their functions in choanoflagellates and C. owczarzaki. The data strengthen the model that ECM proteins are metazoan-specific and evolved as innovations in the last common metazoan ancestor.     

Novel cultured protists identify deep-branching environmental DNA clades of cercozoa: New Genera Tremula, Micrometopion, Minimassisteria, Nudifila, Peregrinia

We describe three new orders of filosan Cercozoa, five new deep-branching genera, eight new species of Thaumatomonas, Reckertia, Spongomonas, Rhogostoma, Agitata, Neoheteromita and Paracercomonas, sequence their 18S rDNA, and construct 18S rDNA trees for 148 Cercozoa. Our phylogeny indicates that Filosa were ancestrally gliding flagellates; non-flagellate filose amoebae evolved from them five times independently. The new genera are more closely related to environmental DNA sequences than cultured organisms. Tremula longifila, a zooflagellate glider on both flagella (unlike other Cercozoa), is the most divergent filosan (Tremulida ord. n.). Micrometopion nutans is a eukaryote-eating gliding zooflagellate like Metopion and Metromonas. Minimassisteria diva is a widespread trimorphic marine amoeboflagellate granofilosan. Peregrinia clavideferens, a non-testate, scale-bearing, filose amoeba, branches deeply in Thaumatomonadida, which are probably sisters to Spongomonadida. Nudifila producta is a filose amoeboflagellate related to Clautriavia and Marimonadida (ord. n., e.g. Pseudopirsonia, Auranticordis). We substantially revise Imbricatea, now including Spongomonadida, and Thecofilosea to include Phaeodaria. Thecofilosea and Imbricatea and Thecofilosea are sisters, both arguably ancestrally rigid gliding flagellates with ventral pseudopod-emitting grooves. Scale-free Ovulinata parva is sister to Paulinella, so imbricate silica scales can be lost. Internal hollow silica skeletons evolved twice in Thecofilosea (Ebriida, Phaeodaria) or were multiply lost. Protaspa replaces preoccupied 'Protaspis'.     

Molecular phylogeny of centrohelid heliozoa,a novel lineage of bikont eukaryotes that arose by ciliary loss

Abstract Recent molecular and cellular evidence indicates that eukaryotes comprise three major lineages: the probably ancestrally uniciliate protozoan phylum Amoebozoa; the ancestrally posteriorly uniciliate opisthokont clade (animals, Choanozoa, and fungi); and a very diverse ancestrally biciliate clade, the bikonts—plants, chromalveolates, and excavate and rhizarian Protozoa. As Heliozoa are the only eukaryote phylum not yet placed on molecular sequence trees, we have sequenced the 18S rRNA genes of three centrohelid heliozoa, Raphidiophrys ambigua, Heterophrys marina, and Chlamydaster sterni, to investigate their phylogenetic position. Phylogenetic analysis by distance and maximum likelihood methods allowing for intersite rate variation and invariable sites confirms that centrohelid heliozoa are a robust clade that does not fall within any other phyla. In particular, they are decisively very distant from the heterokont pedinellid chromists, at one time thought to be related to heliozoa, and lack the unique heterokont signature sequence. They also appear not to be specifically related to either Amoebozoa or Radiolaria, with which they have sometimes been classified, so it is desirable to retain Heliozoa as a separate protozoan phylum. Even though centrohelids have no cilia or centrioles, the centrohelid clade branches among the bikont eukaryotes, but there is no strong bootstrap support for any particular position. Distance trees usually place centrohelids as sisters to haptophytes, whereas parsimony puts them as sisters to red algae, but there is no reason to think that either position is correct; both have very low bootstrap support. Quartet puzzling places them with fairly low support as sisters to the apusozoan zooflagellate Ancyromonas. As Ancyromonas is the only other eukaryote that shares the character combination of flat plate-like mitochondrial cristae and kinetocyst-type extrusomes with centrohelids, this position is biologically plausible, but because of weak support and conflict between trees it might not be correct. Irrespective of their precise position, our trees (together with previous evidence that Chlamydaster sterni has the derived dihydrofolate reductase/thymidylate synthetase gene fusion unique to bikonts) indicate that centrohelid heliozoa are ancestrally derived from a bikont flagellate by the loss of cilia. The centroplast that nucleates their axonemal microtubules is therefore almost certainly homologous with the centrosome of ciliated eukaryotes and should simply be called a centrosome.     

Evolution of the MAGUK protein gene family in premetazoan lineages

Background  

Cell-to-cell communication is a key process in multicellular organisms. In multicellular animals, scaffolding proteins belonging to the family of membrane-associated guanylate kinases (MAGUK) are involved in the regulation and formation of cell junctions. These MAGUK proteins were believed to be exclusive to Metazoa. However, a MAGUK gene was recently identified in an EST survey of Capsaspora owczarzaki, an unicellular organism that branches off near the metazoan clade. To further investigate the evolutionary history of MAGUK, we have undertook a broader search for this gene family using available genomic sequences of different opisthokont taxa.     

Insights into the evolutionary origin and genome architecture of the unicellular opisthokonts Capsaspora owczarzaki and Sphaeroforma arctica

Molecular phylogenetic analyses have recently shown that the unicellular amoeboid protist Capsaspora owczarzaki is unlikely to be a nucleariid or an ichthyosporean as previously described, but is more closely related to Metazoa, Choanoflagellata, and Ichthyosporea. However, the specific phylogenetic relationship of Capsaspora to other protist opisthokont lineages was poorly resolved. To test these earlier results we have expanded both the taxonomic sampling and the number of genes from opisthokont unicellular lineages. We have sequenced the protein-coding genes elongation factor 1-alpha (EF1-alpha) and heat shock protein 70 (Hsp70) from C. owczarzaki and the ichthyosporean Sphaeroforma arctica. Our maximum likelihood (ML) and Bayesian analyses of a concatenated alignment of EF1-alpha, Hsp70, and actin protein sequences with a better sampling of opisthokont-related protist lineages indicate that C. owczarzaki is not clearly allied with any of the unicellular opisthokonts, but represents an independent unicellular lineage closely related to animals and choanoflagellates. Moreover, we have found that the ichthyosporean S. arctica possesses an EF-like (EFL) gene copy instead of the canonical EF1-alpha, the first so far described in an ichthyosporean. A maximum likelihood phylogenetic analysis shows that the EF-like gene of S. arctica strongly groups with the EF-like genes from choanoflagellates. Finally, to begin characterizing the Capsaspora genome, we have performed pulsed-field gel electrophoresis (PFGE) analyses, which indicate that its genome has at least 12 chromosomes with a total genome size in the range of 22-25 Mb.     

Evolutionary origin of synapses and neurons – Bridging the gap

The evolutionary origin of synapses and neurons is an enigmatic subject that inspires much debate. Non‐bilaterian metazoans, both with and without neurons and their closest relatives already contain many components of the molecular toolkits for synapse functions. The origin of these components and their assembly into ancient synaptic signaling machineries are particularly important in light of recent findings on the phylogeny of non‐bilaterian metazoans. The evolution of synapses and neurons are often discussed only from a metazoan perspective leaving a considerable gap in our understanding. By taking an integrative approach we highlight the need to consider different, but extremely relevant phyla and to include the closest unicellular relatives of metazoans, the ichthyosporeans, filastereans and choanoflagellates, to fully understand the evolutionary origin of synapses and neurons. This approach allows for a detailed understanding of when and how the first pre‐ and postsynaptic signaling machineries evolved.     

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54

SEA (s ea urchin sperm protein, e nterokinase, a grin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor‐type protein phosphatase IA‐2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA‐2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan‐like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single‐cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε‐sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin‐like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin‐like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans.     

Early evolution of eukaryote feeding modes,cell structural diversity,and classification of the protozoan phyla Loukozoa,Sulcozoa, and Choanozoa

I discuss how different feeding modes and related cellular structures map onto the eukaryote evolutionary tree. Centrally important for understanding eukaryotic cell diversity are Loukozoa: ancestrally biciliate phagotrophic protozoa possessing a posterior cilium and ventral feeding groove into which ciliary currents direct prey. I revise their classification by including all anaerobic Metamonada as a subphylum and adding Tsukubamonas. Loukozoa, often with ciliary vanes, are probably ancestral to all protozoan phyla except Euglenozoa and Percolozoa and indirectly to kingdoms Animalia, Fungi, Plantae, and Chromista. I make a new protozoan phylum Sulcozoa comprising subphyla Apusozoa (Apusomonadida, Breviatea) and Varisulca (Diphyllatea; Planomonadida, Discocelida, Mantamonadida; Rigifilida). Understanding sulcozoan evolution clarifies the origins from them of opisthokonts (animals, fungi, Choanozoa) and Amoebozoa, and their evolutionary novelties; Sulcozoa and their descendants (collectively called podiates) arguably arose from Loukozoa by evolving posterior ciliary gliding and pseudopodia in their ventral groove. I explain subsequent independent cytoskeletal modifications, accompanying further shifts in feeding mode, that generated Amoebozoa, Choanozoa, and fungi. I revise classifications of Choanozoa, Conosa (Amoebozoa), and basal fungal phylum Archemycota. I use Choanozoa, Sulcozoa, Loukozoa, and Archemycota to emphasize the need for simply classifying ancestral (paraphyletic) groups and illustrate advantages of this for understanding step-wise phylogenetic advances.     

The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell–cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.     

The origins of multicellularity: a multi-taxon genome initiative

The emergence of multicellular organisms from single-celled ancestors -- which occurred several times, independently in different branches of the eukaryotic tree -- is one of the most profound evolutionary transitions in the history of life. These events not only radically changed the course of life on Earth but also created new challenges, including the need for cooperation and communication between cells, and the division of labor among different cell types. However, the genetic changes that accompanied the several origins of multicellularity remain elusive. Recently, the National Human Genome Research Institute (NHGRI) endorsed a multi-taxon genome-sequencing initiative that aims to gain insights into how multicellularity first evolved. This initiative (which we have termed UNICORN) will generate extensive genomic data from some of the closest extant unicellular relatives of both animals and fungi. Here, we introduce this initiative and the biological questions that underpin it, summarize the rationale guiding the choice of organisms and discuss the anticipated benefits to the broader scientific community.