Thomas Cavalier-Smith

I thoroughly discuss ciliary transition zone (TZ) evolution, highlighting many overlooked evolutionarily significant ultrastructural details. I establish fundamental principles of TZ ultrastructure and evolution throughout eukaryotes, inferring unrecognised ancestral TZ patterns for Fungi, opisthokonts, and Corticata (i.e., kingdoms Plantae and Chromista). Typical TZs have a dense transitional plate (TP), with a previously overlooked complex lattice as skeleton. I show most eukaryotes have centriole/TZ junction acorn-V filaments (whose ancestral function was arguably supporting central pair microtubule-nucleating sites; I discuss their role in centriole growth). Uniquely simple malawimonad TZs (without TP, simpler acorn) pinpoint the eukaryote tree's root between them and TP-bearers, highlighting novel superclades. I integrate TZ/ciliary evolution with the best multiprotein trees, naming newly recognised major eukaryote clades and revise megaclassification of basal kingdom Protozoa. Recent discovery of non-photosynthetic phagotrophic flagellates with genome-free plastids (Rhodelphis), the sister group to phylum Rhodophyta (red algae), illuminates plant and chromist early evolution. I show previously overlooked marked similarities in cell ultrastructure betweenRhodelphisandPicomonas, formerly considered an early diverging chromist. In both a nonagonal tube lies between their TP and an annular septum surrounding their 9+2 ciliary axoneme. Mitochondrial dense condensations and mitochondrion-linked smooth endomembrane cytoplasmic partitioning cisternae further support grouping Picomonadea and Rhodelphea as new plant phylum Pararhoda. As Pararhoda/Rhodophyta form a robust clade on site-heterogeneous multiprotein trees, I group Pararhoda and Rhodophyta as new infrakingdom Rhodaria of Plantae within subkingdom Biliphyta, which also includes Glaucophyta with fundamentally similar TZ, uniquely in eukaryotes. I explain how biliphyte TZs generated viridiplant stellate-structures.

Palaeontologically, eubacteria are > 3× older than neomura (eukaryotes, archaebacteria). Cell biology contrasts ancestral eubacterial murein peptidoglycan walls and derived neomuran N-linked glycoprotein coats/walls. Misinterpreting long stems connecting clade neomura to eubacteria on ribosomal sequence trees (plus misinterpreted protein paralogue trees) obscured this historical pattern. Universal multiprotein ribosomal protein (RP) trees, more accurate than rRNA trees, are taxonomically undersampled. To reduce contradictions with genically richer eukaryote trees and improve eubacterial phylogeny, we constructed site-heterogeneous and maximum-likelihood universal three-domain, two-domain, and single-domain trees for 143 eukaryotes (branching now congruent with 187-protein trees), 60 archaebacteria, and 151 taxonomically representative eubacteria, using 51 and 26 RPs. Site-heterogeneous trees greatly improve eubacterial phylogeny and higher classification, e.g. showing gracilicute monophyly, that many ‘rDNA-phyla’ belong in Proteobacteria, and reveal robust new phyla Synthermota and Aquithermota. Monoderm Posibacteria and Mollicutes (two separate wall losses) are both polyphyletic: multiple outer membrane losses in Endobacteria occurred separately from Actinobacteria; neither phylum is related to Chloroflexi, the most divergent prokaryotes, which originated photosynthesis (new model proposed). RP trees support an eozoan root for eukaryotes and are consistent with archaebacteria being their sisters and rooted between Filarchaeota (=Proteoarchaeota, including ‘Asgardia’) and Euryarchaeota sensu-lato (including ultrasimplified ‘DPANN’ whose long branches often distort trees). Two-domain trees group eukaryotes within Planctobacteria, and archaebacteria with Planctobacteria/Sphingobacteria. Integrated molecular/palaeontological evidence favours negibacterial ancestors for neomura and all life. Unique presence of key pre-neomuran characters favours Planctobacteria only as ancestral to neomura, which apparently arose by coevolutionary repercussions (explained here in detail, including RP replacement) of simultaneous outer membrane and murein loss. Planctobacterial C-1 methanotrophic enzymes are likely ancestral to archaebacterial methanogenesis and β-propeller-α-solenoid proteins to eukaryotic vesicle coats, nuclear-pore-complexes, and intraciliary transport. Planctobacterial chaperone-independent 4/5-protofilament microtubules and MamK actin-ancestors prepared for eukaryote intracellular motility, mitosis, cytokinesis, and phagocytosis. We refute numerous wrong ideas about the universal tree.

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.

In 1981 I established kingdom Chromista, distinguished from Plantae because of its more complex chloroplast-associated membrane topology and rigid tubular multipartite ciliary hairs. Plantae originated by converting a cyanobacterium to chloroplasts with Toc/Tic translocons; most evolved cell walls early, thereby losing phagotrophy. Chromists originated by enslaving a phagocytosed red alga, surrounding plastids by two extra membranes, placing them within the endomembrane system, necessitating novel protein import machineries. Early chromists retained phagotrophy, remaining naked and repeatedly reverted to heterotrophy by losing chloroplasts. Therefore, Chromista include secondary phagoheterotrophs (notably ciliates, many dinoflagellates, Opalozoa, Rhizaria, heliozoans) or walled osmotrophs (Pseudofungi, Labyrinthulea), formerly considered protozoa or fungi respectively, plus endoparasites (e.g. Sporozoa) and all chromophyte algae (other dinoflagellates, chromeroids, ochrophytes, haptophytes, cryptophytes). I discuss their origin, evolutionary diversification, and reasons for making chromists one kingdom despite highly divergent cytoskeletons and trophic modes, including improved explanations for periplastid/chloroplast protein targeting, derlin evolution, and ciliary/cytoskeletal diversification. I conjecture that transit-peptide-receptor-mediated ‘endocytosis’ from periplastid membranes generates periplastid vesicles that fuse with the arguably derlin-translocon-containing periplastid reticulum (putative red algal trans-Golgi network homologue; present in all chromophytes except dinoflagellates). I explain chromist origin from ancestral corticates and neokaryotes, reappraising tertiary symbiogenesis; a chromist cytoskeletal synapomorphy, a bypassing microtubule band dextral to both centrioles, favoured multiple axopodial origins. I revise chromist higher classification by transferring rhizarian subphylum Endomyxa from Cercozoa to Retaria; establishing retarian subphylum Ectoreta for Foraminifera plus Radiozoa, apicomonad subclasses, new dinozoan classes Myzodinea (grouping Colpovora gen. n., Psammosa), Endodinea, Sulcodinea, and subclass Karlodinia; and ranking heterokont Gyrista as phylum not superphylum.

Evolving multicellularity is easy, especially in phototrophs and osmotrophs whose multicells feed like unicells. Evolving animals was much harder and unique; probably only one pathway via benthic ‘zoophytes’ with pelagic ciliated larvae allowed trophic continuity from phagocytic protozoa to gut-endowed animals. Choanoflagellate protozoa produced sponges. Converting sponge flask cells mediating larval settling to synaptically controlled nematocysts arguably made Cnidaria. I replace Haeckel's gastraea theory by a sponge/coelenterate/bilaterian pathway: Placozoa, hydrozoan diploblasty and ctenophores were secondary; stem anthozoan developmental mutations arguably independently generated coelomate bilateria and ctenophores. I emphasize animal origin's conceptual aspects (selective, developmental) related to feeding modes, cell structure, phylogeny of related protozoa, sequence evidence, ecology and palaeontology. Epithelia and connective tissue could evolve only by compensating for dramatically lower feeding efficiency that differentiation into non-choanocytes entails. Consequentially, larger bodies enabled filtering more water for bacterial food and harbouring photosynthetic bacteria, together adding more food than cell differentiation sacrificed. A hypothetical presponge of sessile triploblastic sheets (connective tissue sandwiched between two choanocyte epithelia) evolved oogamy through selection for larger dispersive ciliated larvae to accelerate benthic trophic competence and overgrowing protozoan competitors. Extinct Vendozoa might be elaborations of this organismal grade with choanocyte-bearing epithelia, before poriferan water channels and cnidarian gut/nematocysts/synapses evolved. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.