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Cells are of only two kinds: bacteria, with DNA segregated by surface membrane motors, dating back approximately 3.5Gy; and eukaryotes, which evolved from bacteria, possibly as recently as 800-850My ago. The last common ancestor of eukaryotes was a sexual phagotrophic protozoan with mitochondria, one or two centrioles and cilia. Conversion of bacteria (=prokaryotes) into a eukaryote involved approximately 60 major innovations. Numerous contradictory ideas about eukaryogenesis fail to explain fundamental features of eukaryotic cell biology or conflict with phylogeny. Data are best explained by the intracellular coevolutionary theory, with three basic tenets: (1) the eukaryotic cytoskeleton and endomembrane system originated through cooperatively enabling the evolution of phagotrophy; (2) phagocytosis internalised DNA-membrane attachments, unavoidably disrupting bacterial division; recovery entailed the evolution of the nucleus and mitotic cycle; (3) the symbiogenetic origin of mitochondria immediately followed the perfection of phagotrophy and intracellular digestion, contributing greater energy efficiency and group II introns as precursors of spliceosomal introns. Eukaryotes plus their archaebacterial sisters form the clade neomura, which evolved from a radically modified derivative of an actinobacterial posibacterium that had replaced the ancestral eubacterial murein peptidoglycan by N-linked glycoproteins, radically modified its DNA-handling enzymes, and evolved cotranslational protein secretion, but not the isoprenoid-ether lipids of archaebacteria. I focus on this phylogenetic background and on explaining how in response to novel phagotrophic selective pressures and ensuing genome internalisation this prekaryote evolved efficient digestion of prey proteins by retrotranslocation and 26S proteasomes, then internal digestion by phagocytosis, lysosomes, and peroxisomes, and eukaryotic vesicle trafficking and intracellular compartmentation.

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It is argued here that the anaerobic protozoan zooflagellate Parabasalia, Carpediemonas and Eopharyngia (diplomonads, enteromonads, retortamonads) constitute a holophyletic group, for which the existing name Trichozoa is adopted as a new subphylum. Ancestrally, Trichozoa probably had hydrogenosomes, stacked Golgi dictyosomes, three anterior centrioles and one posterior centriole: the typical tetrakont pattern. It is also argued that the closest relatives of Trichozoa are Anaeromonada (Trimastix, oxymonads), and the two groups are classified as subphyla of a revised phylum Metamonada. Returning Parabasalia and Anaeromonadea to Metamonada, as in Grassé's original classification, simplifies classification of the kingdom Protozoa by reducing the number of phyla within infrakingdom Excavata from five to four. Percolozoa (Heterolobosea plus Percolatea classis nov.) and Metamonada are probably both ancestrally quadriciliate with a kinetid of four centrioles attached to the nucleus; the few biciliates among them are probably secondarily derived. Metamonada ancestrally probably had two divergent centriole pairs, whereas, in Percolozoa, all four centrioles are parallel. It is suggested that Discicristata (Percolozoa, Euglenozoa) are holophyletic, ancestrally with two parallel centrioles. In the phylum Loukozoa, Malawimonadea classis nov. is established for Malawimonas (with a new family and order also) and Diphyllatea classis nov., for Diphylleida (Diphylleia, Collodictyon), is transferred back to Apusozoa. A new class, order and family are established for the anaerobic, biciliate, tricentriolar Carpediemonas, transferring it from Loukozoa to Trichozoa because of its triply flanged cilia; like Retortamonas, it may be secondarily biciliate--its unique combination of putative hydrogenosomes and flanged cilia agree with molecular evidence that Carpediemonas is sister to Eopharyngia, diverging before their ancestor lost hydrogenosomes and acquired a cytopharynx. Removal of anaeromonads and Carpediemonas makes Loukozoa more homogeneous, being basically biciliate, aerobic and free-living, in contrast to Metamonada. A new taxon-rich rRNA tree supports holophyly of Discicristata and Trichozoa strongly, holophyly of Metamonada and Excavata and paraphyly of Loukozoa weakly. Mitochondria were probably transformed into hydrogenosomes independently in the ancestors of lyromonad Percolozoa and Metamonada and further reduced in the ancestral eopharyngian. Evidence is briefly discussed that Metamonada and all other excavates share a photosynthetic ancestry with Euglenozoa and are secondarily non-photosynthetic, as predicted by the cabozoan hypothesis for a single secondary symbiogenetic acquisition of green algal plastids by the last common ancestor of Euglenozoa and Cercozoa. Excavata plus core Rhizaria (Cercozoa, Retaria) probably form an ancestrally photophagotrophic clade. The origin from a benthic loukozoan ancestor of the characteristic cellular features of Percolozoa and Euglenozoa through divergent adaptations for feeding on or close to surfaces is also discussed.

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Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes (30 encoding plastid proteins) and a red or blue phycobiliprotein antenna pigment, and the chromobiotes (heterokonts and haptophytes), which lost phycobilins and evolved the brown carotenoid fucoxanthin that colours brown seaweeds, diatoms and haptophytes. Chromobiotes transferred the 30 genes to the nucleus and lost the NM genome and nuclear-pore complexes, but retained its membrane as the periplastid reticulum (PPR), putatively the phospholipid factory of the periplastid space (former algal cytoplasm), as did the ancestral alveolate independently. The chlorarachnean NM has three minute chromosomes bearing approximately 300 genes riddled with pygmy introns. I propose that the periplastid membrane (PPM, the former algal plasma membrane) of chromalveolates, and possibly chlorarachneans, grows by fusion of vesicles emanating from the NM envelope or PPR. Dinoflagellates and euglenoids independently lost the PPM and PPR (after diverging from Sporozoa and chlorarachneans, respectively) and evolved triple chloroplast envelopes comprising the original plant double envelope and an extra outermost membrane, the EM, derived from the perialgal vacuole. In all metaalgae most chloroplast proteins are coded by nuclear genes and enter the chloroplast by using bipartite targeting sequences--an upstream signal sequence for entering the ER and a downstream chloroplast transit sequence. I present a new theory for the four-fold diversification of the chloroplast OM protein translocon following its insertion into the PPM to facilitate protein translocation across it (of both periplastid and plastid proteins). I discuss evidence from genome sequencing and other sources on the contrasting modes of protein targeting, cellular integration, and evolution of these two major lineages of eukaryote "cells within cells". They also provide powerful evidence for natural selection's effectiveness in eliminating most functionless DNA and therefore of a universally useful non-genic function for nuclear non-coding DNA, i.e. most DNA in the biosphere, and dramatic examples of genomic reduction. I briefly argue that chloroplast replacement in dinoflagellates, which happened at least twice, may have been evolutionarily easier than secondary symbiogenesis because parts of the chromalveolate protein-targeting machinery could have helped enslave the foreign plastids.

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Nucleomorphs of cryptomonad and chlorarachnean algae are the relict, miniaturised nuclei of formerly independent red and green algae enslaved by separate eukaryote hosts over 500 million years ago. The complete 551 kb genome sequence of a cryptomonad nucleomorph confirms that cryptomonads are eukaryote-eukaryote chimeras and greatly illuminates the symbiogenetic event that created the kingdom Chromista and their alveolate protozoan sisters. Nucleomorph membranes may, like plasma membranes, be more enduring after secondary symbiogenesis than are their genomes. Partial sequences of chlorarachnean nucleomorphs indicate that genomic streamlining is limited by the mutational difficulty of removing useless introns. Nucleomorph miniaturisation emphasises that selection can dramatically reduce eukaryote genome size and eliminate most non-functional nuclear non-coding DNA. Given the differential scaling of nuclear and nucleomorph genomes with cell size, it follows that most non-coding nuclear DNA must have a bulk-sequence-independent function related to cell volume.

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Eukaryotes and archaebacteria form the clade neomura and are sisters, as shown decisively by genes fragmented only in archaebacteria and by many sequence trees. This sisterhood refutes all theories that eukaryotes originated by merging an archaebacterium and an alpha-proteobacterium, which also fail to account for numerous features shared specifically by eukaryotes and actinobacteria. I revise the phagotrophy theory of eukaryote origins by arguing that the essentially autogenous origins of most eukaryotic cell properties (phagotrophy, endomembrane system including peroxisomes, cytoskeleton, nucleus, mitosis and sex) partially overlapped and were synergistic with the symbiogenetic origin of mitochondria from an alpha-proteobacterium. These radical innovations occurred in a derivative of the neomuran common ancestor, which itself had evolved immediately prior to the divergence of eukaryotes and archaebacteria by drastic alterations to its eubacterial ancestor, an actinobacterial posibacterium able to make sterols, by replacing murein peptidoglycan by N-linked glycoproteins and a multitude of other shared neomuran novelties. The conversion of the rigid neomuran wall into a flexible surface coat and the associated origin of phagotrophy were instrumental in the evolution of the endomembrane system, cytoskeleton, nuclear organization and division and sexual life-cycles. Cilia evolved not by symbiogenesis but by autogenous specialization of the cytoskeleton. I argue that the ancestral eukaryote was uniciliate with a single centriole (unikont) and a simple centrosomal cone of microtubules, as in the aerobic amoebozoan zooflagellate Phalansterium. I infer the root of the eukaryote tree at the divergence between opisthokonts (animals, Choanozoa, fungi) with a single posterior cilium and all other eukaryotes, designated 'anterokonts' because of the ancestral presence of an anterior cilium. Anterokonts comprise the Amoebozoa, which may be ancestrally unikont, and a vast ancestrally biciliate clade, named 'bikonts'. The apparently conflicting rRNA and protein trees can be reconciled with each other and this ultrastructural interpretation if long-branch distortions, some mechanistically explicable, are allowed for. Bikonts comprise two groups: corticoflagellates, with a younger anterior cilium, no centrosomal cone and ancestrally a semi-rigid cell cortex with a microtubular band on either side of the posterior mature centriole; and Rhizaria [a new infrakingdom comprising Cercozoa (now including Ascetosporea classis nov.), Retaria phylum nov., Heliozoa and Apusozoa phylum nov.], having a centrosomal cone or radiating microtubules and two microtubular roots and a soft surface, frequently with reticulopodia. Corticoflagellates comprise photokaryotes (Plantae and chromalveolates, both ancestrally with cortical alveoli) and Excavata (a new protozoan infrakingdom comprising Loukozoa, Discicristata and Archezoa, ancestrally with three microtubular roots). All basal eukaryotic radiations were of mitochondrial aerobes; hydrogenosomes evolved polyphyletically from mitochondria long afterwards, the persistence of their double envelope long after their genomes disappeared being a striking instance of membrane heredity. I discuss the relationship between the 13 protozoan phyla recognized here and revise higher protozoan classification by updating as subkingdoms Lankester's 1878 division of Protozoa into Corticata (Excavata, Alveolata; with prominent cortical microtubules and ancestrally localized cytostome--the Parabasalia probably secondarily internalized the cytoskeleton) and Gymnomyxa [infrakingdoms Sarcomastigota (Choanozoa, Amoebozoa) and Rhizaria; both ancestrally with a non-cortical cytoskeleton of radiating singlet microtubules and a relatively soft cell surface with diffused feeding]. As the eukaryote root almost certainly lies within Gymnomyxa, probably among the Sarcomastigota, Corticata are derived. Following the single symbiogenetic origin of chloroplasts in a corticoflagellate host with cortical alveoli, this ancestral plant radiated rapidly into glaucophytes, green plants and red algae. Secondary symbiogeneses subsequently transferred plastids laterally into different hosts, making yet more complex cell chimaeras--probably only thrice: from a red alga to the corticoflagellate ancestor of chromalveolates (Chromista plus Alveolata), from green algae to a secondarily uniciliate cercozoan to form chlorarachneans and independently to a biciliate excavate to yield photosynthetic euglenoids. Tertiary symbiogenesis involving eukaryotic algal symbionts replaced peridinin-containing plastids in two or three dinoflagellate lineages, but yielded no major novel groups. The origin and well-resolved primary bifurcation of eukaryotes probably occurred in the Cryogenian Period, about 850 million years ago, much more recently than suggested by unwarranted backward extrapolations of molecular 'clocks' or dubious interpretations as 'eukaryotic' of earlier large microbial fossils or still more ancient steranes. The origin of chloroplasts and the symbiogenetic incorporation of a red alga into a corticoflagellate to create chromalveolates may both have occurred in a big bang after the Varangerian snowball Earth melted about 580 million years ago, thereby stimulating the ensuing Cambrian explosion of animals and protists in the form of simultaneous, poorly resolved opisthokont and anterokont radiations.

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Mutation plays the primary role in evolution that Weismann mistakenly attributed to sex. Homologous recombination, as in sex, is important for population genetics--shuffling of minor variants, but relatively insignificant for large-scale evolution. Major evolutionary innovations depend much more on illegitimate recombination, which makes novel genes by gene duplication and by gene chimaerisation--essentially mutational forces. The machinery of recombination and sex evolved in two distinct bouts of quantum evolution separated by nearly 3 Gy of stasis; I discuss their nature and causes. The dominant selective force in the evolution of recombination and sex has been selection for replicational fidelity and viability; without the recombination machinery, accurate reproduction, stasis, resistance to radical deleterious evolutionary change and preservation of evolutionary innovations would be impossible. Recombination proteins betray in their phylogeny and domain structure a key role for gene duplication and chimaerisation in their own origin. They arose about 3.8 Gy ago to enable faithful replication and segregation of the first circular DNA genomes in precellular ancestors of Gram-negative eubacteria. Then they were recruited and modified by selfish genetic parasites (viruses; transposons) to help them spread from host to host. Bacteria differ fundamentally from eukaryotes in that gene transfer between cells, whether incidental to their absorptive feeding on DNA and virus infection or directly by plasmids, involves only genomic fragments. This was radically changed by the neomuran revolution about 850 million years ago when a posibacterium evolved into the thermophilic cenancestor of eukaryotes and archaebacteria (jointly called neomurans), radically modifying or substituting its DNA-handling enzymes (those responsible for transcription as well as for replication, repair and recombination) as a coadaptive consequence of the origin of core histones to stabilise its chromosome. Substitution of glycoprotein for peptidoglycan walls in the neomuran ancestor and the evolution of an endoskeleton and endomembrane system in eukaryotes alone required the origin of nuclei, mitosis and novel cell cycle controls and enabled them to evolve cell fusion and thereby the combination of whole genomes from different cells. Meiosis evolved because of resulting selection for periodic ploidy reduction, with incidental consequences for intrapopulation genetic exchange. Little modification was needed to recombination enzymes or to the ancient bacterial catalysts of homology search by spontaneous base pairing to mediate chromosome pairing. The key innovation was the origin of meiotic cohesins delaying centromere splitting to allow two successive divisions before reversion to vegetative growth and replication, necessarily yielding two-step meiosis. Also significant was the evolution of synaptonemal complexes to stabilise bivalents and of monopolins to orient sister centromeres to one spindle pole. The primary significance of sex was not to promote evolutionary change but to limit it by facilitating ploidy cycles to balance the conflicting selective forces acting on rapidly growing phagotrophic protozoa and starved dormant cysts subject to radiation and other damage.

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Prokaryotes constitute a single kingdom, Bacteria, here divided into two new subkingdoms: Negibacteria, with a cell envelope of two distinct genetic membranes, and Unibacteria, comprising the new phyla Archaebacteria and Posibacteria, with only one. Other new bacterial taxa are established in a revised higher-level classification that recognizes only eight phyla and 29 classes. Morphological, palaeontological and molecular data are integrated into a unified picture of large-scale bacterial cell evolution despite occasional lateral gene transfers. Archaebacteria and eukaryotes comprise the clade neomura, with many common characters, notably obligately co-translational secretion of N-linked glycoproteins, signal recognition particle with 7S RNA and translation-arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin, prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs), exosomes and similar replication, repair, transcription and translation machinery. Eubacteria (posibacteria and negibacteria) are paraphyletic, neomura having arisen from Posibacteria within the new subphylum Actinobacteria (possibly from the new class Arabobacteria, from which eukaryotic cholesterol biosynthesis probably came). Replacement of eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are the keys to neomuran origins. All 19 common neomuran character suites probably arose essentially simultaneously during the radical modification of an actinobacterium. At least 11 were arguably adaptations to thermophily. Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft of glycoprotein, not flagellin; DNA-binding protein lob; specially modified tRNA; absence of Hsp90) were subsequent secondary adaptations to hyperthermophily and/or hyperacidity. The insertional origin of protein-spliced tRNA introns and an insertion in proton-pumping ATPase also support the origin of neomura from eubacteria. Molecular co-evolution between histones and DNA-handling proteins, and in novel protein initiation and secretion machineries, caused quantum evolutionary shifts in their properties in stem neomura. Proteasomes probably arose in the immediate common ancestor of neomura and Actinobacteria. Major gene losses (e.g. peptidoglycan synthesis, hsp90, secA) and genomic reduction were central to the origin of archaebacteria. Ancestral archaebacteria were probably heterotrophic, anaerobic, sulphur-dependent hyperthermoacidophiles; methanogenesis and halophily are secondarily derived. Multiple lateral gene transfers from eubacteria helped secondary archaebacterial adaptations to mesophily and genome re-expansion. The origin from a drastically altered actinobacterium of neomura, and the immediately subsequent simultaneous origins of archaebacteria and eukaryotes, are the most extreme and important cases of quantum evolution since cells began. All three strikingly exemplify De Beer's principle of mosaic evolution: the fact that, during major evolutionary transformations, some organismal characters are highly innovative and change remarkably swiftly, whereas others are largely static, remaining conservatively ancestral in nature. This phenotypic mosaicism creates character distributions among taxa that are puzzling to those mistakenly expecting uniform evolutionary rates among characters and lineages. The mixture of novel (neomuran or archaebacterial) and ancestral eubacteria-like characters in archaebacteria primarily reflects such vertical mosaic evolution, not chimaeric evolution by lateral gene transfer. No symbiogenesis occurred. Quantum evolution of the basic neomuran characters, and between sister paralogues in gene duplication trees, makes many sequence trees exaggerate greatly the apparent age of archaebacteria. Fossil evidence is compelling for the extreme antiquity of eubacteria [over 3500 million years (My)] but, like their eukaryote sisters, archaebacteria probably arose only 850 My ago. Negibacteria are the most ancient, radiating rapidly into six phyla. Evidence from molecular sequences, ultrastructure, evolution of photosynthesis, envelope structure and chemistry and motility mechanisms fits the view that the cenancestral cell was a photosynthetic negibacterium, specifically an anaerobic green non-sulphur bacterium, and that the universal tree is rooted at the divergence between sulphur and non-sulphur green bacteria. The negibacterial outer membrane was lost once only in the history of life, when Posibacteria arose about 2800 My ago after their ancestors diverged from Cyanobacteria.

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Chloroplasts originated from cyanobacteria only once, but have been laterally transferred to other lineages by symbiogenetic cell mergers. Such secondary symbiogenesis is rarer and chloroplast losses commoner than often assumed.

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I attempt to sketch a unified picture of the origin of living organisms in their genetic, bioenergetic, and structural aspects. Only selection at a higher level than for individual selfish genes could power the cooperative macromolecular coevolution required for evolving the genetic code. The protein synthesis machinery is too complex to have evolved before membranes. Therefore a symbiosis of membranes, replicators, and catalysts probably mediated the origin of the code and the transition from a nucleic acid world of independent molecular replicators to a nucleic acid/protein/lipid world of reproducing organisms. Membranes initially functioned as supramolecular structures to which different replicators attached and were selected as a higher-level reproductive unit: the proto-organism. I discuss the roles of stereochemistry, gene divergence, codon capture, and selection in the code's origin. I argue that proteins were primarily structural not enzymatic and that the first biological membranes consisted of amphipathic peptidyl-tRNAs and prebiotic mixed lipids. The peptidyl-tRNAs functioned as genetically-specified lipid analogues with hydrophobic tails (ancestral signal peptides) and hydrophilic polynucleotide heads. Protoribosomes arose from two cooperating RNAs: peptidyl transferase (large subunit) and mRNA-binder (small subunit). Early proteins had a second key role: coupling energy flow to the phosphorylation of gene and peptide precursors, probably by lithophosphorylation by membrane-anchored kinases scavenging geothermal polyphosphate stocks. These key evolutionary steps probably occurred on the outer surface of an 'inside out-cell' or obcell, which evolved an unambiguous hydrophobic code with four prebiotic amino acids and proline, and initiation by isoleucine anticodon CAU; early proteins and nucleozymes were all membrane-attached. To improve replication, translation, and lithophosphorylation, hydrophilic substrate-binding and catalytic domains were later added to signal peptides, yielding a ten-acid doublet code. A primitive proto-ecology of molecular scavenging, parasitism, and predation evolved among obcells. I propose a new theory for the origin of the first cell: fusion of two cup-shaped obcells, or hemicells, to make a protocell with double envelope, internal genome and ribosomes, protocytosol, and periplasm. Only then did water-soluble enzymes, amino acid biosynthesis, and intermediary metabolism evolve in a concentrated autocatalytic internal cytosolic soup, causing 12 new amino acid assignments, termination, and rapid freezing of the 22-acid code. Anticodons were recruited sequentially: GNN, CNN, INN, and *UNN. CO2 fixation, photoreduction, and lipid synthesis probably evolved in the protocell before photophosphorylation. Signal recognition particles, chaperones, compartmented proteases, and peptidoglycan arose prior to the last common ancestor of life, a complex autotrophic, anaerobic green bacterium.

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Dinoflagellates are a trophically diverse group of protists with photosynthetic and non-photosynthetic members that appears to incorporate and lose endosymbionts relatively easily. To trace the gain and loss of plastids in dinoflagellates, we have sequenced the nuclear small subunit rRNA gene of 28 photosynthetic and four non-photosynthetic species, and produced phylogenetic trees with a total of 81 dinoflagellate sequences. Patterns of plastid gain, loss, and replacement were plotted onto this phylogeny. With the exception of the apparently early-diverging Syndiniales and Noctilucales, all non-photosynthetic dinoflagellates are very likely to have had photosynthetic ancestors with peridinin-containing plastids. The same is true for all dinoflagellates with plastids other than the peridinin-containing plastid: their ancestors have replaced one type of plastid for another, in some cases most likely through a non-photosynthetic intermediate. Eight independent instances of plastid loss and three of replacement can be inferred from existing data, but as more non-photosynthetic lineages are characterized these numbers will surely grow.

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Molecular chaperones mediate the correct folding of nascent or denatured proteins and are found in both the organelles and cytoplasm of eukaryotic cells. Cryptomonad algae are unusual in possessing an extra cytoplasmic compartment (the periplastid space), the result of having engulfed and retained a photosynthetic eukaryote. Within the periplastid space is a diminutive nucleus (the nucleomorph) that encodes mostly genes for its own expression as well as a few needed by the plastid. Two plastid-encoded chaperones (GroEL and DnaK) and a nucleomorph-encoded chaperone (Cpn60) have been reported from the cryptomonad, Guillardia theta. Here we analyse G. theta nucleomorph genes for members of the cytosolic HSP70 and HSP90 families of molecular chaperones, a heat shock transcription factor (HSF), and all eight subunits of the group II chaperonin, CCT. These are presumably all active in the periplastid space, assisting in the maturation of polypeptides required by the cell; we propose a central role for them also in the structure and assembly of a putative relict mitotic apparatus. Curiously, none of the genes for co-chaperones of HSP70, HSP90, or CCT have been detected in the nucleomorph genome; they are either not needed or are encoded in the host nuclear genome and targeted back into the periplastid space. Endoplasmic reticulum (ER) homologs of HSP70 and HSP90 are also not present. Striking differences in the degree of conservation of the various nucleomorph-encoded molecular chaperones were observed. While the G. theta HSP70 and HSP90 homologs are well conserved, each of the eight CCT subunits (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) is remarkably divergent. Such differences are likely evidence for reduced/different functional constraints on the various molecular chaperones functioning in the periplastid space.

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Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.

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Chromophyte algae differ fundamentally from plants in possessing chloroplasts that contain chlorophyll c and that have a more complex bounding-membrane topology. Although chromophytes are known to be evolutionary chimaeras of a red alga and a non-photosynthetic host, which gave rise to their exceptional membrane complexity, their cell biology is poorly understood. Cryptomonads are the only chromophytes that still retain the enslaved red algal nucleus as a minute nucleomorph. Here we report complete sequences for all three nucleomorph chromosomes from the cryptomonad Guillardia theta. This tiny 551-kilobase eukaryotic genome is the most gene-dense known, with only 17 diminutive spliceosomal introns and 44 overlapping genes. Marked evolutionary compaction hundreds of millions of years ago eliminated nearly all the nucleomorph genes for metabolic functions, but left 30 for chloroplast-located proteins. To allow expression of these proteins, nucleomorphs retain hundreds of genetic-housekeeping genes. Nucleomorph DNA replication and periplastid protein synthesis require the import of many nuclear gene products across endoplasmic reticulum and periplastid membranes. The chromosomes have centromeres, but possibly only one loop domain, offering a means for studying eukaryotic chromosome replication, segregation and evolution.

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Complete chloroplast 23S rRNA and psbA genes from five peridinin-containing dinoflagellates (Heterocapsa pygmaea, Heterocapsa niei, Heterocapsa rotun-data, Amphidinium carterae, and Protoceratium reticulatum) were amplified by PCR and sequenced; partial sequences were obtained from Thoracosphaera heimii and Scrippsiella trochoidea. Comparison with chloroplast 23S rRNA and psbA genes of other organisms shows that dinoflagellate chloroplast genes are the most divergent and rapidly evolving of all. Quartet puzzling, maximum likelihood, maximum parsimony, neighbor joining, and LogDet trees were constructed. Intersite rate variation and invariant sites were allowed for with quartet puzzling and neighbor joining. All psbA and 23S rRNA trees showed peridinin-containing dinoflagellate chloroplasts as monophyletic. In psbA trees they are related to those of chromists and red algae. In 23S rRNA trees, dinoflagellates are always the sisters of Sporozoa (apicomplexans); maximum likelihood analysis of Heterocapsa triquetra 16S rRNA also groups the dinoflagellate and sporozoan sequences, but the other methods were inconsistent. Thus, dinoflagellate chloroplasts may actually be related to sporozoan plastids, but the possibility of reproducible long-branch artifacts cannot be strongly ruled out. The results for all three genes fit the idea that dinoflagellate chloroplasts originated from red algae by a secondary endosymbiosis, possibly the same one as for chromists and Sporozoa. The marked disagreement between 16S rRNA trees using different phylogenetic algorithms indicates that this is a rather poor molecule for elucidating overall chloroplast phylogeny. We discuss possible reasons why both plastid and mitochondrial genomes of alveolates (Dinozoa, Sporozoa and Ciliophora) have ultra-rapid substitution rates and a proneness to unique genomic rearrangements.

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Membrane heredity was central to the unique symbiogenetic origin from cyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae, glaucophytes) and to subsequent lateral transfers of plastids to form even more complex photosynthetic chimeras. Each symbiogenesis integrated disparate genomes and several radically different genetic membranes into a more complex cell. The common ancestor of Plantae evolved transit machinery for plastid protein import. In later secondary symbiogeneses, signal sequences were added to target proteins across host perialgal membranes: independently into green algal plastids (euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists). Conservatism and innovation during early plastid diversification are discussed.

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Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these "cells within cells" lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)(7)AAG(6)A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

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Photosynthetic dinoflagellates are important aquatic primary producers and notorious causes of toxic 'red tides'. Typical dinoflagellate chloroplasts differ from all other plastids in having a combination of three envelope membranes and peridinin-chlorophyll a/c light-harvesting pigments. Despite evidence of a dinoflagellete satellite DNA containing chloroplast genes, previous attempts to obtain chloroplast gene sequences have been uniformly unsuccessful. Here we show that the dinoflagellate chloroplast DNA genome structure is unique. Complete sequences of chloroplast ribosomal RNA genes and seven chloroplast protein genes from the dinoflagellate Heterocapsa triquetra reveal that each is located alone on a separate minicircular chromosome: 'one gene-one circle'. The genes are the most divergent known from chloroplast genomes. Each circle has an unusual tripartite non-coding region (putative replicon origin), which is highly conserved among the nine circles through extensive gene conversion, but is very divergent between species. Several other dinoflagellate species have minicircular chloroplast genes, indicating that this type of genomic organization may have evolved in ancestral peridinean dinoflagellates. Phylogenetic analysis indicates that dinoflagellate chloroplasts are related to chromistan and red algal chloroplasts and supports their origin by secondary symbiogenesis.

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DNA can be divided functionally into three categories: (1) genes--which code for proteins or specify non-messenger RNAs; (2) semons--short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA--which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether 'junk' or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago. In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively. The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection. The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.

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The biggest unsolved problems in chloroplast evolution are the origins of dinoflagellate and euglenoid chloroplasts,which have envelopes of three membranes not two like plants and chromists, and of the sporozoan plastid, bounded by four smooth membranes. I review evidence that all three of these protozoan plastid types originated by secondary symbiogenesis from eukaryotic symbionts. Instead of separate symbiogenetic events, I argue that dinoflagellate and sporozoan plastids are directly related and that the common ancestor of dinoflagellates and Sporozoa was photosynthetic. I suggest that the last common ancestor of all Alveolata was photosynthetic and acquired its chlorophyll c-containing plastids in the same endosymbiogenetic event as those of Chromista. Chromistaand Alveolata are postulated to be a clade designated chrornalveolates. I propose that euglenoids obtained their plastids from the same(possibly ulvophycean) green alga as chlorarachneans and that Discicristata (Euglenozoa plus Percolozoa) and Cercozoa (the group including chlorarachneans) form a clade designated cabozoa (protozoa with chlorophyll a + b). If both theories are correct, there were only two secondary symbiogenetic events (witnessed by the chlorarachnean and cryptomonad nucleormorphs) in the history of life, not seven as commonly assumed. This greatly reduces the postulated number of independent origins of chloroplast protein-targeting machinery and of gene transfers from endosymbiont to host nuclei. I discuss the membrane and plastid losses and innovations in protein targeting implied by these theories, the comparative evidence for them, and their implications for eukaryote megaphylogeny. The principle of evolutionary conservatism leads to a novel theory for the function of periplastid vesicles in membrane biogenesis ofchlorarachneans and chromists and of the key steps in secondary symbiogenesis. Protozoan classification is also slightly revised by abandoning the probably polyphyletic infrakingdom Actinopoda, grouping Foraminifera and Radiolaria as a new infrakingdom Retaria,placing Heliozoa within a revised infrakingdom Sarcomastigota, establishing a new flagellate phylum Loukozoa for Jakobea plus Anaeromonadea within an emended subkingdom Eozoa, and ranking Archezoa as an infrakingdom within Eozoa.

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