Microbial predators form a new supergroup of eukaryotes.

Molecular phylogenetics of microbial eukaryotes has reshaped the tree of life by establishing broad taxonomic divisions, termed supergroups, that supersede the traditional kingdoms of animals, fungi and plants, and encompass a much greater breadth of eukaryotic diversity1. The vast majority of newly discovered species fall into a small number of known supergroups. Recently, however, a handful of species with no clear relationship to other supergroups have been described2-4, raising questions about the nature and degree of undiscovered diversity, and exposing the limitations of strictly molecular-based exploration. Here we report ten previously undescribed strains of microbial predators isolated through culture that collectively form a diverse new supergroup of eukaryotes, termed Provora. The Provora supergroup is genetically, morphologically and behaviourally distinct from other eukaryotes, and comprises two divergent clades of predators-Nebulidia and Nibbleridia-that are superficially similar to each other, but differ fundamentally in ultrastructure, behaviour and gene content. These predators are globally distributed in marine and freshwater environments, but are numerically rare and have consequently been overlooked by molecular-diversity surveys. In the age of high-throughput analyses, investigation of eukaryotic diversity through culture remains indispensable for the discovery of rare but ecologically and evolutionarily important eukaryotes.

Non-photosynthetic predators are sister to red algae.

Rhodophyta (red algae) is one of three lineages of Archaeplastida1, a supergroup that is united by the primary endosymbiotic origin of plastids in eukaryotes2,3. Red algae are a diverse and species-rich group, members of which are typically photoautotrophic, but are united by a number of highly derived characteristics: they have relatively small intron-poor genomes, reduced metabolism and lack cytoskeletal structures that are associated with motility, flagella and centrioles. This suggests that marked gene loss occurred around their origin4; however, this is difficult to reconstruct because they differ so much from the other archaeplastid lineages, and the relationships between these lineages are unclear. Here we describe the novel eukaryotic phylum Rhodelphidia and, using phylogenomics, demonstrate that it is a closely related sister to red algae. However, the characteristics of the two Rhodelphis species described here are nearly opposite to those that define red algae: they are non-photosynthetic, flagellate predators with gene-rich genomes, along with a relic genome-lacking primary plastid that probably participates in haem synthesis. Overall, these findings alter our views of the origins of Rhodophyta, and Archaeplastida evolution as a whole, as they indicate that mixotrophic feeding-that is, a combination of predation and phototrophy-persisted well into the evolution of the group.

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 n. gen. n. sp., Develocanicus komovi n. gen. n. sp., Develocanicus vyazemskyi n. sp., Cubaremonas variflagellatum n. gen. n. sp., Pirsonia chemainus nom. prov., Feodosia pseudopoda nom. prov., and Koktebelia satura nom. prov. 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.

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

BACKGROUND: The origin of animals from their unicellular ancestor was one of the most important events in evolutionary history, but the nature and the order of events leading up to the emergence of multicellular animals are still highly uncertain. The diversity and biology of unicellular relatives of animals have strongly informed our understanding of the transition from single-celled organisms to the multicellular Metazoa. Here, we analyze the cellular structures and complex life cycles of the novel unicellular holozoans Pigoraptor and Syssomonas (Opisthokonta), and their implications for the origin of animals. RESULTS: Syssomonas and Pigoraptor are characterized by complex life cycles with a variety of cell types including flagellates, amoeboflagellates, amoeboid non-flagellar cells, and spherical cysts. The life cycles also include 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. Several existing models explaining the origin of multicellular animals have been put forward, but these data are interestingly consistent with one, the "synzoospore hypothesis." CONCLUSIONS: The feeding modes of the ancestral metazoan may have been more complex than previously thought, including not only bacterial prey, but also larger eukaryotic cells and organic structures. The ability to feed on large eukaryotic prey could have been a powerful trigger in the formation and development of both aggregative (e.g., joint feeding, which also implies signaling) and clonal (e.g., hypertrophic growth followed by palintomy) multicellular stages that played important roles in the emergence of multicellular animals.

New Phylogenomic Analysis of the Enigmatic Phylum Telonemia Further Resolves the Eukaryote Tree of Life.

The resolution of the broad-scale tree of eukaryotes is constantly improving, but the evolutionary origin of several major groups remains unknown. Resolving the phylogenetic position of these "orphan" groups is important, especially those that originated early in evolution, because they represent missing evolutionary links between established groups. Telonemia is one such orphan taxon for which little is known. The group is composed of molecularly diverse biflagellated protists, often prevalent although not abundant in aquatic environments. Telonemia has been hypothesized to represent a deeply diverging eukaryotic phylum but no consensus exists as to where it is placed in the tree. Here, we established cultures and report the phylogenomic analyses of three new transcriptome data sets for divergent telonemid lineages. All our phylogenetic reconstructions, based on 248 genes and using site-heterogeneous mixture models, robustly resolve the evolutionary origin of Telonemia as sister to the Sar supergroup. This grouping remains well supported when as few as 60% of the genes are randomly subsampled, thus is not sensitive to the sets of genes used but requires a minimal alignment length to recover enough phylogenetic signal. Telonemia occupies a crucial position in the tree to examine the origin of Sar, one of the most lineage-rich eukaryote supergroups. We propose the moniker "TSAR" to accommodate this new mega-assemblage in the phylogeny of eukaryotes.

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 support their monophyly and position as the sister lineage to all other known alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but they are 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 and the origin of myzocytosis and plastids.

Phylogenomics supports the monophyly of the Cercozoa.

The phylum Cercozoa consists of a diverse assemblage of amoeboid and flagellated protists that forms a major component of the supergroup, Rhizaria. However, despite its size and ubiquity, the phylogeny of the Cercozoa remains unclear as morphological variability between cercozoan species and ambiguity in molecular analyses, including phylogenomic approaches, have produced ambiguous results and raised doubts about the monophyly of the group. Here we sought to resolve these ambiguities using a 161-gene phylogenetic dataset with data from newly available genomes and deeply sequenced transcriptomes, including three new transcriptomes from Aurigamonas solis, Abollifer prolabens, and a novel species, Lapot gusevi n. gen. n. sp. Our phylogenomic analysis strongly supported a monophyletic Cercozoa, and approximately-unbiased tests rejected the paraphyletic topologies observed in previous studies. The transcriptome of L. gusevi represents the first transcriptomic data from the large and recently characterized Aquavolonidae-Treumulida-'Novel Clade 12' group, and phylogenomics supported its position as sister to the cercozoan subphylum, Endomyxa. These results provide insights into the phylogeny of the Cercozoa and the Rhizaria as a whole.

Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives.

Apicomplexans are a major lineage of parasites, including causative agents of malaria and toxoplasmosis. How such highly adapted parasites evolved from free-living ancestors is poorly understood, particularly because they contain nonphotosynthetic plastids with which they have a complex metabolic dependency. Here, we examine the origin of apicomplexan parasitism by resolving the evolutionary distribution of several key characteristics in their closest free-living relatives, photosynthetic chromerids and predatory colpodellids. Using environmental sequence data, we describe the diversity of these apicomplexan-related lineages and select five species that represent this diversity for transcriptome sequencing. Phylogenomic analysis recovered a monophyletic lineage of chromerids and colpodellids as the sister group to apicomplexans, and a complex distribution of retention versus loss for photosynthesis, plastid genomes, and plastid organelles. Reconstructing the evolution of all plastid and cytosolic metabolic pathways related to apicomplexan plastid function revealed an ancient dependency on plastid isoprenoid biosynthesis, predating the divergence of apicomplexan and dinoflagellates. Similarly, plastid genome retention is strongly linked to the retention of two genes in the plastid genome, sufB and clpC, altogether suggesting a relatively simple model for plastid retention and loss. Lastly, we examine the broader distribution of a suite of molecular characteristics previously linked to the origins of apicomplexan parasitism and find that virtually all are present in their free-living relatives. The emergence of parasitism may not be driven by acquisition of novel components, but rather by loss and modification of the existing, conserved traits.

Culturing Heterotrophic Protists from the Baltic Sea: Mostly the "Usual Suspects" but a Few Novelties as Well.

The study of cultured strains has a long tradition in protistological research and has greatly contributed to establishing the morphology, taxonomy, and ecology of many protist species. However, cultivation-independent techniques, based on 18S rRNA gene sequences, have demonstrated that natural protistan assemblages mainly consist of hitherto uncultured protist lineages. This mismatch impedes the linkage of environmental diversity data with the biological features of cultured strains. Thus, novel taxa need to be obtained in culture to close this knowledge gap. In this study, traditional cultivation techniques were applied to samples from coastal surface waters and from deep oxygen-depleted waters of the Baltic Sea. Based on 18S rRNA gene sequencing, 126 monoclonal cultures of heterotrophic protists were identified. The majority of the isolated strains were affiliated with already cultured and described taxa, mainly chrysophytes and bodonids. This was likely due to "culturing bias" but also to the eutrophic nature of the Baltic Sea. Nonetheless, ~ 12% of the isolates in our culture collection showed highly divergent 18S rRNA gene sequences compared to those of known organisms and thus may represent novel taxa, either at the species level or at the genus level. Moreover, we also obtained evidence that some of the isolated taxa are ecologically relevant, under certain conditions, in the Baltic Sea.

Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista.

Assembling the global eukaryotic tree of life has long been a major effort of Biology. In recent years, pushed by the new availability of genome-scale data for microbial eukaryotes, it has become possible to revisit many evolutionary enigmas. However, some of the most ancient nodes, which are essential for inferring a stable tree, have remained highly controversial. Among other reasons, the lack of adequate genomic datasets for key taxa has prevented the robust reconstruction of early diversification events. In this context, the centrohelid heliozoans are particularly relevant for reconstructing the tree of eukaryotes because they represent one of the last substantial groups that was missing large and diverse genomic data. Here, we filled this gap by sequencing high-quality transcriptomes for four centrohelid lineages, each corresponding to a different family. Combining these new data with a broad eukaryotic sampling, we produced a gene-rich taxon-rich phylogenomic dataset that enabled us to refine the structure of the tree. Specifically, we show that (i) centrohelids relate to haptophytes, confirming Haptista; (ii) Haptista relates to SAR; (iii) Cryptista share strong affinity with Archaeplastida; and (iv) Haptista + SAR is sister to Cryptista + Archaeplastida. The implications of this topology are discussed in the broader context of plastid evolution.

The Morphology, Ultrastructure and SSU rRNA Gene Sequence of a New Freshwater Flagellate, Neobodo borokensis n. sp. (Kinetoplastea, Excavata).

A small free-living freshwater bacteriotrophic flagellate Neobodo borokensis n. sp. was investigated by electron microscopy and analysis of its SSU ribosomal RNA gene. This protist has paraxonemal rods of typical bodonid structure in the flagella, mastigonemes on the proximal part of the posterior flagellum, two nearly parallel basal bodies, a compact kinetoplast, and discoid mitochondrial cristae. The flagellar pocket is supported by three microtubular roots (R1, R2 and R3) originating from the kinetosome. The cytopharynx is supported by the root R2, a microtubular prism, cytopharynx associated additional microtubules (CMT) and cytostome associated microtubules (FAS) bands. Symbiotic bacteria and small glycosomes were found in the cytoplasm. Cysts have not been found. The flagellate prefers freshwater habitats, but tolerates salinity up to 3-4‰. The overall morphological and ultrastructural features confirm that N. borokensis represents a new species of the genus Neobodo. Phylogenetic analysis of SSU rRNA genes is congruent with the ultrastructure and strongly supports the close relationship of N. borokensis to Neobodo saliens, N. designis, Actuariola, and a misidentified sequence of "Bodo curvifilus" within the class Kinetoplastea.

Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae.

BACKGROUND: Protist communities inhabiting oxygen depleted waters have so far been characterized through both microscopical observations and sequence based techniques. However, the lack of cultures for abundant taxa severely hampers our knowledge on the morphology, ecology and energy metabolism of hypoxic protists. Cultivation of such protists has been unsuccessful in most cases, and has never yet succeeded for choanoflagellates, even though these small bacterivorous flagellates are known to be ecologically relevant components of aquatic protist communities. RESULTS: Quantitative data for choanoflagellates and the vertical distribution of Codosiga spp. at Gotland and Landsort Deep (Baltic Sea) indicate its preference for oxygen-depleted zones. Strains isolated and cultivated from these habitats revealed ultrastructural peculiarities such as mitochondria showing tubular cristae never seen before for choanoflagellates, and the first observation of intracellular prokaryotes in choanoflagellates. Analysis of their partial 28S rRNA gene sequence complements the description of two new species, Codosiga minima n. sp. and C. balthica n. sp. These are closely related with but well separated from C. gracilis (C. balthica and C. minima p-distance to C. gracilis 4.8% and 11.6%, respectively). In phylogenetic analyses the 18S rRNA gene sequences branch off together with environmental sequences from hypoxic habitats resulting in a wide cluster of hypoxic Codosiga relatives so far only known from environmental sequencing approaches. CONCLUSIONS: Here, we establish the morphological and ultrastructural identity of an environmental choanoflagellate lineage. Data from microscopical observations, supplemented by findings from previous culture-independent methods, indicate that C. balthica is likely an ecologically relevant player of Baltic Sea hypoxic waters. The possession of derived mitochondria could be an adaptation to life in hypoxic environments periodically influenced by small-scale mixing events and changing oxygen content allowing the reduction of oxygen consuming components. In view of the intricacy of isolating and cultivating choanoflagellates, the two new cultured species represent an important advance to the understanding of the ecology of this group, and mechanisms of adaptations to hypoxia in protists in general.

The Morphology, Ultrastructure and Molecular Phylogeny of a New Soil-Dwelling Kinetoplastid Avlakibodo gracilis gen. et sp. nov. (Neobodonida; Kinetoplastea).

Kinetoplastids represent a stockpile of undiscovered protist diversity. Free-living members of this group have been studied less intensively compared to their important parasitic relatives. We have isolated a new soil-dwelling bacteriotrophic kinetoplastid, which is described here as a new genus and new species, Avlakibodo gracilis gen. et sp. nov. Phylogenetic analysis of 18S rRNA genes showed highly supported sister relationship of this protist with the clade uniting Neobodo borokensis, Neobodo curvifilus, Neobodo saliens, Actuariola framvarensis, some Neobodo designis isolates and several environmental sequences, with high statistical support. We have reconstructed the organization of the microtubular cytoskeleton of A. gracilis and determined the origins of the main bands of microtubules. Characteristic ultrastructural features include cytostome associated microtubules (FAS), cytopharynx associated additional microtubules (CMT), microtubular prism (nemadesm) and three microtubular roots (R1, R2 and R3).

On the origin of TSAR: morphology, diversity and phylogeny of Telonemia.

Telonemia is a poorly known major phylum of flagellated eukaryotes with a unique combination of morphological traits. Phylogenomics recently revealed the phylogenetic position of telonemids as sister to SAR, one of the largest groups of eukaryotes, comprising Stramenopiles, Alveolata and Rhizaria. Due to this key evolutionary position, investigations of telonemids are of critical importance for elucidating the origin and diversification of an astounding diversity of eukaryotic forms and life strategies. To date, however, only two species have been morphologically characterized from Telonemia, which do not represent this genetically very diverse group. In this study, we established cultures for six new telonemid strains, including the description of five new species and a new genus. We used these cultures to update the phylogeny of Telonemia and provide a detailed morphological and ultrastructural investigation. Our data elucidate the origin of TSAR from flagellates with complex morphology and reconstruction of the ancestral structure of stramenopiles, alveolates and rhizarians, and their main synapomorphic characters. Since telonemids are a common component of aquatic environments, the features of their feeding, behaviour and ecological preferences observed in clonal cultures and the results of global metabarcoding analysis contribute to a deeper understanding of organization of microbial food webs.

The origin of Metazoa: a transition from temporal to spatial cell differentiation.

For over a century, Haeckel's Gastraea theory remained a dominant theory to explain the origin of multicellular animals. According to this theory, the animal ancestor was a blastula-like colony of uniform cells that gradually evolved cell differentiation. Today, however, genes that typically control metazoan development, cell differentiation, cell-to-cell adhesion, and cell-to-matrix adhesion are found in various unicellular relatives of the Metazoa, which suggests the origin of the genetic programs of cell differentiation and adhesion in the root of the Opisthokonta. Multicellular stages occurring in the complex life cycles of opisthokont protists (mesomycetozoeans and choanoflagellates) never resemble a blastula. Here, we discuss a more realistic scenario of transition to multicellularity through integration of pre-existing transient cell types into the body of an early metazoon, which possessed a complex life cycle with a differentiated sedentary filter-feeding trophic stage and a non-feeding blastula-like larva, the synzoospore. Choanoflagellates are considered as forms with secondarily simplified life cycles.

First finding of free-living representatives of Prokinetoplastina and their nuclear and mitochondrial genomes.

Kinetoplastids are heterotrophic flagellated protists, including important parasites of humans and animals (trypanosomatids), and ecologically important free-living bacterial consumers (bodonids). Phylogenies have shown that the earliest-branching kinetoplastids are all parasites or obligate endosymbionts, whose highly-derived state makes reconstructing the ancestral state of the group challenging. We have isolated new strains of unusual free-living flagellates that molecular phylogeny shows to be most closely related to endosymbiotic and parasitic Perkinsela and Ichthyobodo species that, together with unidentified environmental sequences, form the clade at the base of kinetoplastids. These strains are therefore the first described free-living prokinetoplastids, and potentially very informative in understanding the evolution and ancestral states of morphological and molecular characteristics described in other kinetoplastids. Overall, we find that these organisms morphologically and ultrastructurally resemble some free-living bodonids and diplonemids, and possess nuclear genomes with few introns, polycistronic mRNA expression, high coding density, and derived traits shared with other kinetoplastids. Their genetic repertoires are more diverse than the best-studied free-living kinetoplastids, which is likely a reflection of their higher metabolic potential. Mitochondrial RNAs of these new species undergo the most extensive U insertion/deletion editing reported so far, and limited deaminative C-to-U and A-to-I editing, but we find no evidence for mitochondrial trans-splicing.

New Lineage of Microbial Predators Adds Complexity to Reconstructing the Evolutionary Origin of Animals.

The origin of animals is one of the most intensely studied evolutionary events, and our understanding of this transition was greatly advanced by analyses of unicellular relatives of animals, which have shown many "animal-specific" genes actually arose in protistan ancestors long before the emergence of animals [1-3]. These genes have complex distributions, and the protists have diverse lifestyles, so understanding their evolutionary significance requires both a robust phylogeny of animal relatives and a detailed understanding of their biology [4, 5]. But discoveries of new animal-related lineages are rare and historically biased to bacteriovores and parasites. Here, we characterize the morphology and transcriptome content of a new animal-related lineage, predatory flagellate Tunicaraptor unikontum. Tunicaraptor is an extremely small (3-5 µm) and morphologically simple cell superficially resembling some fungal zoospores, but it survives by preying on other eukaryotes, possibly using a dedicated but transient "mouth," which is unique for unicellular opisthokonts. The Tunicaraptor transcriptome encodes a full complement of flagellar genes and the flagella-associated calcium channel, which is only common to predatory animal relatives and missing in microbial parasites and grazers. Tunicaraptor also encodes several major classes of animal cell adhesion molecules, as well as transcription factors and homologs of proteins involved in neurodevelopment that have not been found in other animal-related lineages. Phylogenomics, including Tunicaraptor, challenges the existing framework used to reconstruct the evolution of animal-specific genes and emphasizes that the diversity of animal-related lineages may be better understood only once the smaller, more inconspicuous animal-related lineages are better studied. VIDEO ABSTRACT.

Microscopical Studies on Ministeria vibrans Tong, 1997 (Filasterea) Highlight the Cytoskeletal Structure of the Common Ancestor of Filasterea, Metazoa and Choanoflagellata.

Ministeria vibrans (Filasterea) is a tiny amoeboid species described by Tong in 1997. It has been sporadically found in different habitats, and cultured strains were established. M. vibrans is well characterised by molecular phylogeny but until now was not ultrastructurally investigated in detail. Here, we provide the ultrastructure for this species based on a new strain isolated from oxygen-depleted water of the Baltic Sea. A thin vibrating flagellum could be observed but no vibrating movement of the cell body and no stalk. Our first ultrastructural study of a filasterean taxon revealed radial microvilli supported by bundles of microfilaments. Two centrioles located in the nuclear pit can migrate to the cell periphery and transform into the kinetid: the centriole orthogonal to the kinetosome with a fibrillar root and a basal foot that initiates microtubules. Microvilli in Ministeria suggest their presence in the common ancestor of Filasterea and Choanoflagellata. The kinetid structure of Ministeria is similar to that of the choanocytes of the most deep-branching sponges, differing essentially from the kinetid of choanoflagellates. Thus, kinetid and microvilli of Ministeria illustrate features of the common ancestor of three holozoan groups: Filasterea, Metazoa and Choanoflagellata.

Evolutionary Relationship of the Scale-Bearing Kraken (incertae sedis, Monadofilosa, Cercozoa, Rhizaria): Combining Ultrastructure Data and a Two-Gene Phylogeny.

The genus Kraken represents a distinct lineage of filose amoebae within the Cercozoa. Currently a single species, Kraken carinae, has been described. SSU rDNA phylogeny showed an affiliation to the Cercomonadida, branching with weak support at its base, close to Paracercomonas, Metabolomonas, and Brevimastigomonas. Light microscopical analyses showed several unique features of the genus Kraken, but ultrastructure data were lacking. In this study, K. carinae has been studied by electron microscopy, these data conjoined with a two-gene phylogeny were used to give more insight into the evolutionary relationship of the genus Kraken within Cercozoa. The data confirmed the absence of flagella, but also showed novel characteristics, such as the presence of extrusomes, osmiophilic bodies, and mitochondria with flat cristae. Surprising was the presence of single-tier scales which are carried by cell outgrowths, much of what is expected of the last common ancestor of the class Imbricatea. The phylogenetic analyses however confirmed previous results, indicating Kraken as a sister group to Paracercomonas in Sarcomonadea with an increased but still low support of 0.98 PP/63 BP. Based on the unique features of Kraken we establish the Krakenidae fam. nov. that we, due to contradictory results in morphology and phylogeny, assign incertae sedis, Monadofilosa.

Novel Predators Reshape Holozoan Phylogeny and Reveal the Presence of a Two-Component Signaling System in the Ancestor of Animals.

Our understanding of the origin of animals has been transformed by characterizing their most closely related, unicellular sisters: the choanoflagellates, filastereans, and ichthyosporeans. Together with animals, these lineages make up the Holozoa [1, 2]. Many traits previously considered "animal specific" were subsequently found in other holozoans [3, 4], showing that they evolved before animals, although exactly when is currently uncertain because several key relationships remain unresolved [2, 5]. Here we report the morphology and transcriptome sequencing from three novel unicellular holozoans: Pigoraptor vietnamica and Pigoraptor chileana, which are related to filastereans, and Syssomonas multiformis, which forms a new lineage with Corallochytrium in phylogenomic analyses. All three species are predatory flagellates that feed on large eukaryotic prey, and all three also appear to exhibit complex life histories with several distinct stages, including multicellular clusters. Examination of genes associated with multicellularity in animals showed that the new filastereans contain a cell-adhesion gene repertoire similar to those of other species in this group. Syssomonas multiformis possessed a smaller complement overall but does encode genes absent from the earlier-branching ichthyosporeans. Analysis of the T-box transcription factor domain showed expansion of T-box transcription factors based on combination with a non-T-box domain (a receiver domain), which has not been described outside of vertebrates. This domain and other domains we identified in all unicellular holozoans are part of the two-component signaling system that has been lost in animals, suggesting the continued use of this system in the closest relatives of animals and emphasizing the importance of studying loss of function as well as gain in major evolutionary transitions.