Multigene phylogeny and cell evolution of chromist infrakingdom Rhizaria: contrasting cell organisation of sister phyla Cercozoa and Retaria.

Infrakingdom Rhizaria is one of four major subgroups with distinct cell body plans that comprise eukaryotic kingdom Chromista. Unlike other chromists, Rhizaria are mostly heterotrophic flagellates, amoebae or amoeboflagellates, commonly with reticulose (net-like) or filose (thread-like) feeding pseudopodia; uniquely for eukaryotes, cilia have proximal ciliary transition-zone hub-lattices. They comprise predominantly flagellate phylum Cercozoa and reticulopodial phylum Retaria, whose exact phylogenetic relationship has been uncertain. Given even less clear relationships amongst cercozoan classes, we sequenced partial transcriptomes of seven Cercozoa representing five classes and endomyxan retarian Filoreta marina to establish 187-gene multiprotein phylogenies. Ectoreta (retarian infraphyla Foraminifera, Radiozoa) branch within classical Cercozoa as sister to reticulose Endomyxa. This supports recent transfer of subphylum Endomyxa from Cercozoa to Retaria alongside subphylum Ectoreta which embraces classical retarians where capsules or tests subdivide cells into organelle-containing endoplasm and anastomosing pseudopodial net-like ectoplasm. Cercozoa are more homogeneously filose, often with filose pseudopodia and/or posterior ciliary gliding motility: zooflagellate Helkesimastix and amoeboid Guttulinopsis form a strongly supported clade, order Helkesida. Cercomonads are polyphyletic (Cercomonadida sister to glissomonads; Paracercomonadida deeper). Thecofilosea are a clade, whereas Imbricatea may not be; Sarcomonadea may be paraphyletic. Helkesea and Metromonadea are successively deeper outgroups within cercozoan subphylum Monadofilosa; subphylum Reticulofilosa (paraphyletic on site-heterogeneous trees) branches earliest, Granofilosea before Chlorarachnea. Our multiprotein trees confirm that Rhizaria are sisters of infrakingdom Halvaria (Alveolata, Heterokonta) within chromist subkingdom Harosa (= SAR); they further support holophyly of chromist subkingdom Hacrobia, and are consistent with holophyly of Chromista as sister of kingdom Plantae. Site-heterogeneous rDNA trees group Kraken with environmental DNA clade 'eSarcomonad', not Paracercomonadida. Ectoretan fossil dates evidence ultrarapid episodic stem sequence evolution. We discuss early rhizarian cell evolution and multigene tree coevolutionary patterns, gene-paralogue evidence for chromist monophyly, and integrate this with fossil evidence for the age of Rhizaria and eukaryote cells, and revise rhizarian classification.

187-gene phylogeny of protozoan phylum Amoebozoa reveals a new class (Cutosea) of deep-branching, ultrastructurally unique, enveloped marine Lobosa and clarifies amoeba evolution.

Monophyly of protozoan phylum Amoebozoa, and subdivision into subphyla Conosa and Lobosa each with different cytoskeletons, are well established. However early diversification of non-ciliate lobose amoebae (Lobosa) is poorly understood. To clarify it we used recently available transcriptomes to construct a 187-gene amoebozoan tree for 30 species, the most comprehensive yet. This robustly places new genus Atrichosa (formerly lumped with Trichosphaerium) within lobosan class Tubulinea, not Discosea as previously supposed. We identified an earliest diverging lobosan clade comprising marine amoebae armoured by porose scaliform cell-envelopes, here made a novel class Cutosea with two pseudopodially distinct new families. Cutosea comprise Sapocribrum, ATCC PRA-29 misidentified as 'Pessonella', plus from other evidence Squamamoeba. We confirm that Acanthamoeba and ATCC 50982 misidentified as Stereomyxa ramosa are closely related. Discosea have a strongly supported major subclade comprising Thecamoebida plus Glycostylida (suborders Dactylopodina, Stygamoebina; Vannellina) phylogenetically distinct from Centramoebida. Stygamoeba is sister to Dactylopodina. Himatismenida are either sister to Centramoebida or deeper branching. Discosea usually appear holophyletic (rarely paraphyletic). Paramoeba transcriptomes include prokinetoplastid Perkinsela-like endosymbiont sequences. Cunea, misidentified as Mayorella, is closer to Paramoeba than Vexillifera within holophyletic Dactylopodina. Taxon-rich site-heterogeneous rDNA trees confirm cutosan distinctiveness, allow improved conosan taxonomy, and reveal previous dictyostelid tree misrooting.

Multiple origins of Heliozoa from flagellate ancestors: New cryptist subphylum Corbihelia, superclass Corbistoma, and monophyly of Haptista, Cryptista, Hacrobia and Chromista.

Heliozoan protists have radiating cell projections (axopodia) supported by microtubular axonemes nucleated by the centrosome and bearing granule-like extrusomes for catching prey. To clarify previously confused heliozoan phylogeny we sequenced partial transcriptomes of two tiny naked heliozoa, the endohelean Microheliella maris and centrohelid Oxnerella marina, and the cercozoan pseudoheliozoan Minimassisteria diva. Phylogenetic analysis of 187 genes confirms that all are chromists; but centrohelids (microtubules arranged as hexagons and triangles) are not sisters to Endohelea having axonemes in transnuclear cytoplasmic channels (triangular or square microtubular arrays). Centrohelids are strongly sister to haptophytes (together phylum Haptista); we explain the common origins of their axopodia and haptonema. Microheliella is sister to new superclass Corbistoma (zooflagellate Telonemea and Picomonadea, with asymmetric microfilamentous pharyngeal basket), showing that these axopodial protists evolved independently from zooflagellate ancestors. We group Corbistoma and Endohelea as new cryptist subphylum Corbihelia with dense fibrillar interorganellar connections; endohelean axopodia and Telonema cortex are ultrastructurally related. Differently sampled trees clarify why corticate multigene eukaryote phylogeny is problematic: long-branch artefacts probably distort deep multigene phylogeny of corticates (Plantae, Chromista); basal radiations may be contradictorily reconstructed because of their extreme closeness and the Bayesian star-tree paradox. Haptista and Hacrobia are holophyletic, and Chromista probably are.

Multigene phylogeny resolves deep branching of Amoebozoa.

Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo-flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR-Γ model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data.

Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa.

Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were previously argued to have evolved independently from the little-studied, largely flagellate, protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and the more primitive excavate flagellates that have ventral feeding grooves and the most primitive known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote-wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between homogeneous-model maximum-likelihood trees and evolutionarily more realistic site-heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 'podiate' clade, one of only three eukaryotic 'supergroups'. Our trees indicate that evolution of sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously) generated podiate eukaryotes from Malawimonas-like excavate flagellates.

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'.

Helkesimastix marina n. sp. (Cercozoa: Sainouroidea superfam. n.) a gliding zooflagellate of novel ultrastructure and unusual ciliary behaviour.

Unlike Helkesimastix faecicola and H. major, Helkesimastix marina is marine, ingests bacteria, is probably also a cannibal, and differs in cell cycle ciliary behaviour. Daughter kinetids have mirror symmetry; pre-division cilia beat asymmetrically. We sequenced its 18S rDNA and studied its ultrastructure to clarify its taxonomy. Helkesimastix (Helkesimastigidae fam. n.) differs unexpectedly radically from cercomonads, lacking their complex microtubular ciliary roots, grouping not with them but with Sainouridae within Pansomonadida. Longitudinal cortical microtubules emanate from a dense apical centrosomal plate, where a striated rhizoplast attaches the nucleus, and two very short subparallel centrioles attach by dense fibres. The marginally more posterior centriole, attached to the centrosomal plate by a dense forked fibre, bears the long 9+2 gliding posterior cilium and a microtubular root; the left-side, nucleus-attached, left centriole bears an immotile ciliary stump with abnormal axoneme of nine disorganized mainly singlet microtubules, unlike the sainourid anterior papilla. Both transitional regions have a proximal lattice, the posterior centriole with slender hub. Sainouroidea superfam. n. (Sainouridae; Helkesimastigidae) have homologous cytoskeletal geometry. Dorsal Golgi dictyosome and posterior microbody are attached to the nuclear envelope, which has slender micro-invaginations and probably a cortical lattice. Bacteria are digested posteriorly in association with numerous mitochondria with flat cristae.

Morphology and phylogeny of Sainouron acronematica sp. n. and the ultrastructural unity of Cercozoa.

Sainouron are soil zooflagellates of obscure taxonomy. We studied the ultrastructure of S. acronematica sp. n. and sequenced its extremely divergent 18S rDNA and that of Cholamonas cyrtodiopsidis (here grouped as new family Sainouridae) to clarify their phylogeny. Ultrastructurally similar, they weakly group together, deeply within Monadofilosa. Sainouron has three cytoplasmic microtubules; all organelles specifically link to them or the nucleus. Mature centrioles have fibrous rhizoplasts. The posterior centriole bearing the motile cilium (with cortical filaments) has a transitional hub-lattice; a dense spiral fibre links its thicker rhizoplast and triplets; its ciliary root has two microtubules: mt1, underlying the plasma membrane, initiates at the spiral fibre; mt2, laterally attached to mt1 and nucleus, initiates in the amorphous centrosomal region. The anterior younger cilium, an immotile stub with submembrane skeleton as in Cholamonas, lacks axoneme, microtubular root, rhizoplasts and spiral fibre, but becomes the posterior one every cell cycle. The nuclear envelope donates coated vesicles directly to the Golgi, which makes kinetocyst-type extrusomes, concentrated at the cell anterior for extrusion into phagosomes. Ciliary transition region proximal hub-lattices (postulated to contain centrin) and distal nonagonal fibres are cercozoan synapomorphies, found with slight structural variation in all flagellate Cercozoa, but not in outgroups.