True molecular phylogenetic position of the cockroach gut commensal Lophomonas blattarum (Lophomonadida, Parabasalia).

Lophomonas blattarum is a facultative commensal gut dweller of common pest cockroaches. Its cells are roughly spherical in shape with an apical tuft of ~50 flagella. Controversially, it has been implicated in human respiratory infections based on light microscopic observations of similarly shaped cells in sputum or bronchoalveolar lavage fluid. Here, we have sequenced the 18S rRNA gene of L. blattarum and its sole congener, Lophomonas striata, isolated from cockroaches. Both species branch in a fully supported clade with Trichonymphida, consistent with a previous study of L. striata, but not consistent with sequences from human samples attributed to L. blattarum.

Between a Pod and a Hard Test: The Deep Evolution of Amoebae.

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

Coprophilic amoebae and flagellates, including Guttulinopsis, Rosculus and Helkesimastix, characterise a divergent and diverse rhizarian radiation and contribute to a large diversity of faecal-associated protists.

A wide diversity of organisms utilize faecal habitats as a rich nutrient source or a mechanism to traverse through animal hosts. We sequenced the 18S rRNA genes of the coprophilic, fruiting body-forming amoeba Guttulinopsis vulgaris and its non-fruiting relatives Rosculus 'ithacus' CCAP 1571/3, R. terrestris n. sp. and R. elongata n. sp. and demonstrate that they are related to the coprophilic flagellate Helkesimastix in a strongly supported, but highly divergent 18S sister clade. PCR primers specific to both clades were used to generate 18S amplicons from a range of environmental and faecal DNA samples. Phylogenetic analysis of the cloned sequences demonstrated a high diversity of uncharacterised sequence types within this clade, likely representing previously described members of the genera Guttulinopsis, Rosculus and Helkesimastix, as well as so-far unobserved organisms. Further, an Illumina MiSeq sequenced set of 18S V4-region amplicons generated from faecal DNAs using universal eukaryote primers showed that core-cercozoan assemblages in faecal samples are as diverse as those found in more conventionally examined habitats. These results reveal many novel lineages, some of which appear to occur preferentially in faecal material, in particular cercomonads and glissomonads. More broadly, we show that faecal habitats are likely untapped reservoirs of microbial eukaryotic diversity.

Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles.

Phylogenomics demonstrates that breviate flagellates are related to opisthokonts and apusomonads.

Most eukaryotic lineages belong to one of a few major groups. However, several protistan lineages have not yet been robustly placed in any of these groups. Both the breviates and apusomonads are two such lineages that appear to be related to the Amoebozoa and Opisthokonta (i.e. the 'unikonts' or Amorphea); however, their precise phylogenetic positions remain unclear. Here, we describe a novel microaerophilic breviate, Pygsuia biforma gen. nov. sp. nov., isolated from a hypoxic estuarine sediment. Ultrastructurally, this species resembles the breviate genera Breviata and Subulatomonas but has two cell morphologies, adherent and swimming. Phylogenetic analyses of the small sub-unit rRNA gene show that Pygsuia is the sister to the other breviates. We constructed a 159-protein supermatrix, including orthologues identified in RNA-seq data from Pygsuia. Phylogenomic analyses of this dataset show that breviates, apusomonads and Opisthokonta form a strongly supported major eukaryotic grouping we name the Obazoa. Although some phylogenetic methods disagree, the balance of evidence suggests that the breviate lineage forms the deepest branch within Obazoa. We also found transcripts encoding a nearly complete integrin adhesome from Pygsuia, indicating that this protein complex involved in metazoan multicellularity may have evolved earlier in eukaryote evolution than previously thought.

Free-living Trichomonads are Unexpectedly Diverse.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates.

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

Sawyeria marylandensis (Heterolobosea) has a hydrogenosome with novel metabolic properties.

Protists that live under low-oxygen conditions often lack conventional mitochondria and instead possess mitochondrion-related organelles (MROs) with distinct biochemical functions. Studies of mostly parasitic organisms have suggested that these organelles could be classified into two general types: hydrogenosomes and mitosomes. Hydrogenosomes, found in parabasalids, anaerobic chytrid fungi, and ciliates, metabolize pyruvate anaerobically to generate ATP, acetate, CO(2), and hydrogen gas, employing enzymes not typically associated with mitochondria. Mitosomes that have been studied have no apparent role in energy metabolism. Recent investigations of free-living anaerobic protists have revealed a diversity of MROs with a wider array of metabolic properties that defy a simple functional classification. Here we describe an expressed sequence tag (EST) survey and ultrastructural investigation of the anaerobic heteroloboseid amoeba Sawyeria marylandensis aimed at understanding the properties of its MROs. This organism expresses typical anaerobic energy metabolic enzymes, such as pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenase, and associated hydrogenase maturases with apparent organelle-targeting peptides, indicating that its MRO likely functions as a hydrogenosome. We also identified 38 genes encoding canonical mitochondrial proteins in S. marylandensis, many of which possess putative targeting peptides and are phylogenetically related to putative mitochondrial proteins of its heteroloboseid relative Naegleria gruberi. Several of these proteins, such as a branched-chain alpha keto acid dehydrogenase, likely function in pathways that have not been previously associated with the well-studied hydrogenosomes of parabasalids. Finally, morphological reconstructions based on transmission electron microscopy indicate that the S. marylandensis MROs form novel cup-like structures within the cells. Overall, these data suggest that Sawyeria marylandensis possesses a hydrogenosome of mitochondrial origin with a novel combination of biochemical and structural properties.

Phylogeny of the "forgotten" cellular slime mold, Fonticula alba, reveals a key evolutionary branch within Opisthokonta.

The shared ancestry between Fungi and animals has been unequivocally demonstrated by abundant molecular and morphological data for well over a decade. Along with the animals and Fungi, multiple protists have been placed in the supergroup Opisthokonta making it exceptionally diverse. In an effort to place the cellular slime mold Fonticula alba, an amoeboid protist with aggregative, multicellular fruiting, we sequenced five nuclear encoded genes; small subunit ribosomal RNA, actin, beta-tubulin, elongation factor 1-alpha, and the cytosolic isoform of heat shock protein 70 for phylogenetic analyses. Molecular trees demonstrate that Fonticula is an opisthokont that branches sister to filose amoebae in the genus Nuclearia. Fonticula plus Nuclearia are sister to Fungi. We propose a new name for this well-supported clade, Nucletmycea, incorporating Nuclearia, Fonticula, and Fungi. Fonticula represents the first example of a cellular slime mold morphology within Opisthokonta. Thus, there are four types of multicellularity in the supergroup-animal, fungal, colonial, and now aggregative. Our data indicate that multicellularity in Fonticula evolved independent of that found in the fungal and animal radiations. With the rapidly expanding sequence and genomic data becoming available from many opisthokont lineages, Fonticula may be fundamental to understanding opisthokont evolution as well as any possible commonalities involved with the evolution of multicellularity.

A wide diversity of previously undetected free-living relatives of diplomonads isolated from marine/saline habitats.

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free-living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas-like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small-subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad-retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation - it is likely that undersampling at the level of major lineages is still widespread for protists.

A morphologically simple species of Acrasis (Heterolobosea, Excavata), Acrasis helenhemmesae n. sp.

In the course of a large-scale global survey of mycetozoans, amoeboid organisms that form fruiting bodies, a new species of Acrasis was discovered from several subtropical locales in Hawaii, Australia, Bermuda, and South Africa. We isolated four strains from dead, still attached, plant material, and one strain from attached bark of a tree. Each isolate forms simple uniseriate multicellular fruiting bodies typically consisting of two bottle-shaped, basal stalk cells and a chain of <20 spores. The isolate from Bermuda often forms dichotomous simple branches, each consisting of <10 spores. Amoebae from these new isolates are limax with eruptive pseudopodial formation and display rapid locomotion-characters indicative of amoebae in the excavate taxon Heterolobosea. These isolates form simpler fruiting bodies than is typical of the well-known Acrasis rosea. Although in the original description, A. rosea is known to form uniseriate fruiting bodies similar to our isolates, A. rosea isolates typically form more complex fruiting structures along side simple ones, but never strictly simple ones. Nuclear-encoded 18S rRNA gene phylogenies demonstrate that our five isolates form a highly supported clade that is sister to A. rosea. Given the differences both in gene sequences and fruiting body morphology between our isolates and A. rosea, we propose the new species, Acrasis helenhemmesae n. sp.

Evolutionary position of breviate amoebae and the primary eukaryote divergence.

Integration of ultrastructural and molecular sequence data has revealed six supergroups of eukaryote organisms (excavates, Rhizaria, chromalveolates, Plantae, Amoebozoa and opisthokonts), and the root of the eukaryote evolutionary tree is suggested to lie between unikonts (Amoebozoa, opisthokonts) and bikonts (the other supergroups). However, some smaller lineages remain of uncertain affinity. One of these unassigned taxa is the anaerobic, free-living, amoeboid flagellate Breviata anathema, which is of key significance as it is unclear whether it is a unikont (i.e. possibly the deepest branching amoebozoan) or a bikont. To establish its evolutionary position, we sequenced thousands of Breviata genes and calculated trees using 78 protein sequences. Our trees and specific substitutions in the 18S RNA sequence indicate that Breviata is related to other Amoebozoa, thereby significantly increasing the cellular diversity of this phylum and establishing Breviata as a deep-branching unikont. We discuss the implications of these results for the ancestral state of Amoebozoa and eukaryotes generally, demonstrating that phylogenomics of phylogenetically 'nomadic' species can elucidate key questions in eukaryote evolution. Furthermore, mitochondrial genes among the Breviata ESTs demonstrate that Breviata probably contains a modified anaerobic mitochondrion. With these findings, remnants of mitochondria have been detected in all putatively deep-branching amitochondriate organisms.

Phylogeny and Classification of Novel Diversity in Sainouroidea (Cercozoa, Rhizaria) Sheds Light on a Highly Diverse and Divergent Clade.

Sainouroidea is a molecularly diverse clade of cercozoan flagellates and amoebae in the eukaryotic supergroup Rhizaria. Previous 18S rDNA environmental sequencing of globally collected fecal and soil samples revealed great diversity and high sequence divergence in the Sainouroidea. However, a very limited amount of this diversity has been observed or described. The two described genera of amoebae in this clade are Guttulinopsis, which displays aggregative multicellularity, and Rosculus, which does not. Although the identity of Guttulinopsis is straightforward due to the multicellular fruiting bodies they form, the same is not true for Rosculus, and the actual identity of the original isolate is unclear. Here we isolated amoebae with morphologies like that of Guttulinopsis and Rosculus from many environments and analyzed them using 18S rDNA sequencing, light microscopy, and transmission electron microscopy. We define a molecular species concept for Sainouroidea that resulted in the description of 4 novel genera and 12 novel species of naked amoebae. Aggregative fruiting is restricted to the genus Guttulinopsis, but other than this there is little morphological variation amongst these taxa. Taken together, simple identification of these amoebae is problematic and potentially unresolvable without the 18S rDNA sequence.

The glycolytic pathway of Trimastix pyriformis is an evolutionary mosaic.

BACKGROUND: Glycolysis and subsequent fermentation is the main energy source for many anaerobic organisms. The glycolytic pathway consists of ten enzymatic steps which appear to be universal amongst eukaryotes. However, it has been shown that the origins of these enzymes in specific eukaryote lineages can differ, and sometimes involve lateral gene transfer events. We have conducted an expressed sequence tag (EST) survey of the anaerobic flagellate Trimastix pyriformis to investigate the nature of the evolutionary origins of the glycolytic enzymes in this relatively unstudied organism. RESULTS: We have found genes in the Trimastix EST data that encode enzymes potentially catalyzing nine of the ten steps of the glycolytic conversion of glucose to pyruvate. Furthermore, we have found two different enzymes that in principle could catalyze the conversion of phosphoenol pyruvate (PEP) to pyruvate (or the reverse reaction) as part of the last step in glycolysis. Our phylogenetic analyses of all of these enzymes revealed at least four cases where the relationship of the Trimastix genes to homologs from other species is at odds with accepted organismal relationships. Although lateral gene transfer events likely account for these anomalies, with the data at hand we were not able to establish with confidence the bacterial donor lineage that gave rise to the respective Trimastix enzymes. CONCLUSION: A number of the glycolytic enzymes of Trimastix have been transferred laterally from bacteria instead of being inherited from the last common eukaryotic ancestor. Thus, despite widespread conservation of the glycolytic biochemical pathway across eukaryote diversity, in a number of protist lineages the enzymatic components of the pathway have been replaced by lateral gene transfer from disparate evolutionary sources. It remains unclear if these replacements result from selectively advantageous properties of the introduced enzymes or if they are neutral outcomes of a gene transfer 'ratchet' from food or endosymbiotic organisms or a combination of both processes.

The Earliest Stages of Mitochondrial Adaptation to Low Oxygen Revealed in a Novel Rhizarian.

Mitochondria exist on a functional and evolutionary continuum that includes anaerobic mitochondrion-related organelles (MROs), such as hydrogenosomes. Hydrogenosomes lack many classical mitochondrial features, including conspicuous cristae, mtDNA, the tricarboxylic acid (TCA) cycle, and ATP synthesis powered by an electron transport chain (ETC); instead, they produce ATP anaerobically, liberating H2 and CO2 gas in the process. However, our understanding of the evolutionary transformation from aerobic mitochondria to various MRO types remains incomplete. Here we describe a novel MRO from a cercomonad (Brevimastigomonas motovehiculus n. sp.; Rhizaria). We have sequenced its 30,608-bp mtDNA and characterized organelle function through a combination of transcriptomic, genomic, and cell biological approaches. B. motovehiculus MROs are metabolically versatile, retaining mitochondrial metabolic pathways, such as a TCA cycle and ETC-driven ATP synthesis, but also possessing hydrogenosomal-type pyruvate metabolism and substrate-level phosphorylation. Notably, the B. motovehiculus ETC is degenerate and appears to be losing cytochrome-based electron transport (complexes III and IV). Furthermore, the F1Fo ATP synthase (complex V) is unique, with the highly conserved Atpα subunit fragmented into four separate pieces. The B. motovehiculus MRO appears to be in the process of losing aerobic metabolic capacities. Our findings shed light on the transition between organelle types, specifically the early stages of mitochondrial adaptation to anaerobiosis.

Marine Isolates of Trimastix marina Form a Plesiomorphic Deep-branching Lineage within Preaxostyla, Separate from Other Known Trimastigids (Paratrimastix n. gen.).

Trimastigids are free-living, anaerobic protists that are closely related to the symbiotic oxymonads, forming together the taxon Preaxostyla (Excavata: Metamonada). We isolated fourteen new strains morphologically corresponding to two species assigned to Trimastix (until now the only genus of trimastigids), Trimastix marina and Trimastix pyriformis. Unexpectedly, marine strains of Trimastix marina branch separately from freshwater strains of this morphospecies in SSU rRNA gene trees, and instead form the sister group of all other Preaxostyla. This position is confirmed by three-gene phylogenies. Ultrastructural examination of a marine isolate of Trimastix marina demonstrates a combination of trimastigid-like features (e.g. preaxostyle-like I fibre) and ancestral characters (e.g. absence of thickened flagellar vane margins), consistent with inclusion of marine T. marina within Preaxostyla, but also supporting its distinctiveness from 'freshwater T. marina' and its deep-branching position within Preaxostyla. Since these results indicate paraphyly of Trimastix as currently understood, we transfer the other better-studied trimastigids to Paratrimastix n. gen. and Paratrimastigidae n. fam. The freshwater form previously identified as T. marina is described as Paratrimastix eleionoma n. sp., and Trimastix pyriformis becomes Paratrimastix pyriformis n. comb. Because of its phylogenetic position, 'true' Trimastix is potentially important for understanding the evolution of mitochondrion-related organelles in metamonads.

Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: two new amitochondrial heterolobosean amoebae from anoxic environments.

Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.

Amoeba stages in the deepest branching heteroloboseans, including Pharyngomonas: evolutionary and systematic implications.

The taxon Heterolobosea (Excavata) is a major group of protists well known for its diversity of life stages. Most are amoebae capable of transforming into flagellates (amoeboflagellates), while others are known solely as flagellates or solely as amoebae. The deepest-branching heterolobosean taxon confirmed previously, Pharyngomonas, was generally assumed to be a pure flagellate, suggesting that the amoeba form arose later in the evolution of Heterolobosea sensu lato. Here we report that multiple isolates of Pharyngomonas are actually amoeboflagellates that also have cyst stages, with only amoebae transforming into cysts. The amoeba form of Pharyngomonas showed heterolobosean characteristics (e. g. eruptive movement), but also possessed unusual morphological features like slow-flowing crenulated hyaline crescents with conical subpseudopodia, finger-like projections and branching posterior extensions. Furthermore, phylogenetic analyses of 18S ribosomal RNA gene sequences that included two undescribed species of amoebae showed that Pharyngomonas is not the only deep-branching heterolobosean to possess an amoeba stage. These results suggest that possession of an amoeba stage was ancestral for Heterolobosea, unifying this taxon as a group of species with amoeba stages in their lifecycle or derived from organisms with such stages.

A contemporary evaluation of the acrasids (Acrasidae, Heterolobosea, Excavata).

Sorocarpic protists are organisms that individually aggregate and work together to form a fungus-like fruiting body (sorocarp). The amoeboid forms are often colloquially referred to as "cellular slime molds" or "acrasids". We argue the latter term should be used only to refer to members of Acrasidae in Heterolobosea. Here we study the diversity of two Acrasidae genera, Acrasis and the closely similar Pocheina, using a combination of morphological characteristics and small subunit rRNA gene sequences. A total of eight isolates of Acrasis and an example of Pocheina were examined. Acrasis/Pocheina form a well-supported monophyletic group that is the highly supported sister to a clade containing Allovahlkampfia and several other amoebae. Four molecular lineages of Acrasis were resolved, each of which is characterized by a distinctive fruiting body morphology. Each lineage represents a species, two of which are novel, Acrasis kona n. sp. and Acrasis takarsan n. sp. An isolate identified as Pocheina rosea is nested within the clade containing isolates of the taxon Acrasis rosea, into which P. rosea is tentatively subsumed. One member of the tightly knit allovahlkampfid clade was induced to form a simple sorocarp, leading us to include this clade in Acrasidae.

Aggregative multicellularity evolved independently in the eukaryotic supergroup Rhizaria.

Multicellular forms of life have evolved many times, independently giving rise to a diversity of organisms such as animals, plants, and fungi that together comprise the visible biosphere. Yet multicellular life is far more widespread among eukaryotes than just these three lineages. A particularly common form of multicellularity is a social aggregative fruiting lifestyle whereby individual cells associate to form a "fungus-like" sorocarp. This complex developmental process that requires the interaction of thousands of cells working in concert was made famous by the "cellular slime mold"Dictyostelium discoideum, which became an important model organism. Although sorocarpic protistan lineages have been identified in five of the major eukaryote groups, the ubiquitous and globally distributed species Guttulinopsis vulgaris has eluded proper classification. Here we demonstrate, by phylogenomic analyses of a 159-protein data set, that G. vulgaris is a member of Rhizaria and is thus the first member of this eukaryote supergroup known to be capable of aggregative multicellularity.