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Europ. J. Protistol. 39, 338–348 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp

Protist phylogeny and the high-level classification of

Thomas Cavalier-Smith

Department of , University of Oxford, South Parks Road, Oxford, OX1 3PS, UK; E-mail: [email protected]

Received 1 September 2003; 29 September 2003. Accepted: 29 September 2003

Protist large-scale phylogeny is briefly reviewed and a revised higher classification of the Pro- tozoa into 11 phyla presented. Complementary fusions reveal a fundamental bifurcation among eu- karyotes between two major : the ancestrally uniciliate (often unicentriolar) unikonts and the an- cestrally biciliate , which undergo ciliary transformation by converting a younger anterior into a dissimilar older posterior cilium. Unikonts comprise the ancestrally unikont protozoan and the (kingdom Animalia, phylum , their sisters or ancestors; and kingdom Fungi). They share a derived triple-gene fusion, absent from bikonts. Bikonts contrastingly share a derived gene fusion between and and include and all other , comprising the protozoan infrakingdoms [phyla and Re- taria (Radiozoa, )] and (phyla Loukozoa, Metamonada, , ), plus the kingdom Plantae [Viridaeplantae, Rhodophyta (sisters); Glaucophyta], the chromalveolate , and the protozoan phylum (Thecomonadea, Diphylleida). Chromalveolates comprise kingdom (, Heterokonta, Haptophyta) and the protozoan infrakingdom Alveolata [phyla Cilio- phora and Miozoa (= Protalveolata, Dinozoa, )], which diverged from a common ancestor that enslaved a red alga and evolved novel -targeting machinery via the rough ER and the enslaved algal plasma membrane (periplastid membrane). The branching order of the five groups is uncertain: Plantae may be sisters of or ancestral to chromalveolates (jointly designated corti- cates as they share cortical alveoli); Rhizaria and Excavata (jointly cabozoa) are probably sisters if the formerly green algal plastid of euglenoids and chlorarachneans (Cercozoa) was enslaved in a single event in their common ancestor. Apusozoa may be sisters of Excavata and may be sisters to Haptophyta.

Key words: Protozoa; protist; classification; phylogeny; bikonts; unikonts.

Protists are polyphyletic

Eukaryotes comprise the kingdom Proto- plasmodial in the trophic state, are derived from zoa and four derived kingdoms: Animalia, Fungi, bilateral ancestors by the loss of gut and Plantae, and Chromista (Cavalier-Smith 1998). nervous system (Okamura et al. 2002) and that the Protists (unicellular ) can no longer be anaerobic evolved from aerobic fila- considered a (e.g. a kingdom) as they are mentous zygomycotine fungi (Keeling 2003) as found in all five kingdoms and are undoubtedly postulated (Cavalier-Smith 2000a). Such phyloge- polyphyletic. This is most strikingly shown by the netically deceptive by simplification and recent evidence that , most of which are character loss has occurred not only in such para-

0932-4739/03/39/04-338 $ 15.00/0 Phylogeny and classification of Protozoa 339 sites but also in free-living protists. Molecular evi- the resolution to group together taxa that really are dence now implies that all known groups of anaer- related, sometimes group together those that are obic, apparently amitochondrial, protists evolved not and can be profoundly misleading about where by the loss of mitochondrial , cy- the of a subtree really lies. tochromes, and oxidative phosphorylation and the Despite these problems, the overall evidence conversion of mitochondria into double-mem- now allows us to group protists into a relatively braned of different function: hy- small number of phyla (25, counting all four fungal drogenosomes or (Roger 1999; Silber- phyla as including protists (Cavalier-Smith 1998), man et al. 2002) – these retain their ancestral mito- of a total of 48 eukaryotic phyla), most now estab- chondrial mechanisms of membrane heredity and lished with reasonable confidence as monophyletic protein targeting: (Cavalier-Smith 2004a; Williams - mainly holophyletic, though one is certainly pa- et al. 2002; Embley et al. 2003; van der Giezen et al. raphyletic (i.e. , one of the three 2003). Likewise it is almost certain that all non-cil- phyla containing protists: Cavalier-Smith 1998) iated protists {e.g. many amoebae (Cavalier-Smith and two probably are (Archemycota, Loukozoa). et al. 2004), , (Cavalier- The present paper summarises this evidence and Smith and Chao 2003a), Myxozoa, microsporidia, outlines my current interpretation of relationships } evolved ultimately from ciliated an- among the 11 phyla of the necessarily paraphyletic cestors by losing cilia (= eukaryotic flagella) and kingdom Protozoa and the four derived (holo- (= basal bodies). phyletic) kingdoms (Fig. 1). In this revised system for kingdom Protozoa (see appendix) the phyla Choanozoa and Amoebozoa (both I suspect holo- A new systematic synthesis phyletic, but either or both may be paraphyletic as shown on some trees: resolution is insufficient to These new insights have come not just from decide either way – Cavalier-Smith and Chao molecular sequence studies but by integrating 2003a; Cavalier-Smith et al. 2004) are grouped as them with numerous other lines of evidence, ge- the undoubtedly paraphyletic subkingdom Sarco- netic, structural and biochemical. The classical mastigota, from which and fungi indepen- view developed over two centuries that reliance on dently evolved (Cavalier-Smith 2000a; King et al. a single line of evidence or character is often very 2003). The other nine protozoan phyla (all but misleading for phylogeny and is at last Loukozoa probably holophyletic) constitute the penetrating the previously over-dogmatic and paraphyletic subkingdom Biciliata from which the over-self-confident of molecular systematics. holophyletic kingdoms Plantae and Chromista There really ought to be no such field, for good evolved. Biciliata comprise three probably holo- systematics should be fully integrative of all avail- phyletic infrakingdoms (Alveolata, Rhizaria, Ex- able evidence. ‘Molecular systematics’ that con- cavata) plus the phylum Apusozoa, which may centrates on trees from just one molecule and ig- have affinities with Excavata or Rhizaria or be nores other evidence is poor systematics. Al- more deeply branching. though there have long been conclusive theoretical reasons for thinking that sequence trees can some- be profoundly misleading and all too easily Bikonts, unikonts misinterpreted (Felsenstein 1978) and that riboso- and the eukaryotic root mal rRNA is certainly not a molecular clock (Cav- alier-Smith 1980), the recent spread of a more criti- Plantae, Chromista, and Biciliata are all clearly cal approach to protist molecular phylogenies ancestrally biciliate and together constitute a clade owes much to the balanced and integrative per- designated the bikonts (Cavalier-Smith 2002). A spective of André Adoutte (Baroin et al. 1988; major shared derived character for all three groups Philippe and Adoute 1996) and critical analyses by (not yet clearly demonstrated for Rhizaria) is cil- his former collaborators (Philippe and Adoutte iary transformation in which the anterior 1998; Philippe 2000; Philippe and Germot 2000; cilium/ and its associated are always Philippe et al. 2000a; Philippe et al. 2000b; Lopez the first formed; in the next cycle they undergo et al. 2002). It is now widely, but by no means uni- often marked changes in structure and function to versally, accepted that single-gene trees often lack become the corresponding posterior organelles 340 T. Cavalier-Smith

Fig. 1. The eukaryotic based on a synthesis of ultrastructural, cell biological and molecular evi- dence, showing the four major symbiogenetic events. Taxa outside the four derived kingdoms belong to the basal kingdom Protozoa (unlabelled). The ancestral is held to have been a phagotrophic uniciliate, unicentrio- lar aerobic that arose from a neomuran bacterial ancestor by the simultaneous origin of the cytoskele- ton, , nucleus, and cilium, coupled with the overlapping symbiogenetic origin of mitochon- dria from an intracellular α-proteobacterium (Cavalier-Smith 2000b, 2002). Unikonts are ancestrally heterotrophic if arose symbiogenetically in an early bikont, as is almost certain. Chromalveolates are holophyletic, ancestrally photophagotrophic, and evolved by the single enslavement of a red alga ® by a bikont host to form a eu- karyote-eukaryote chimaera. There are almost equally strong protein-targeting arguments for the single secondary origin of the cabozoan (G) in a common ancestor of Cercozoa and Euglenozoa (Cavalier-Smith 1999), but the idea remains controversial as compelling sequence evidence is unavailable to disprove the alternative possi- bility that euglenoid and chlorarachnean were separately implanted as shown by the asterisks. Whether Apusozoa are really ancestrally heterotrophic, early divergent bikonts, as shown, is also uncertain. It is unclear whether the duplication within phosphofructokinase is a synapomorphy for unikonts or its loss one for bikonts (Stechmann and Cavalier-Smith 2003a).

(Moestrup 2000). An independent shared derived order in the cenancestral eukaryote compared with character for bikonts is a fusion between the the ancestral state in where they are sepa- for thymidylate synthase (TS) and dihydrofolate rately translated but usually in the same operon reductase (DHFR) to encode a single bifunctional (Stechmann and Cavalier-Smith 2002; Stechmann chimaeric protein. This fusion appears to have and Cavalier Smith 2003a). Both genes are sepa- taken place in the common ancestor of the ances- rately translated in Sarcomastigota, animals and tral bikont after the two genes became inverted in Fungi; these three taxa are referred to as unikonts Phylogeny and classification of Protozoa 341 because it is argued that their common ancestor indels used to interpret protist phylogeny have probably had only a single centriole and cilium per proved unreliable, in particular deletions used to kinetid (Cavalier-Smith 2002); this unikont state imply early branching of Parabasalia (Bapteste and (Cavalier-Smith 2002) is found in most distinct Philippe 2002). amoebozoan clades – , mastigamoe- If Choanozoa and Amoebozoa are both holo- bids, and protostelids ( Cavalier- phyletic, the branching order for the four unikont Smith et al. 2004) and possibly ‘ in- groups in Figure 1 is almost certainly correct, with vertens’. Unikonty is argued to be the ancestral Amoebozoa being the immediate outgroup to state for Amoebozoa (Cavalier-Smith 2002; Cava- opisthokonts. Sequence phylogenies based on over lier-Smith et al. 2004) as only myxogastrids and 100 protein-coding genes strongly support mono- former protostelids arguably related to them have phyly of (mastigamoebids, Pelo- bicentriolar kinetids. The bicentriolar state of myxa, and ) and their rela- myxogastrids and many Choanozoa is develop- tionship with the cellular slime mould Dic- mentally different from that of bikonts, as their an- tyostelium (Bapteste et al. 2002) as well as the clos- terior cilium remains anterior in successive cell cy- er relationship of to animals than cles and does not transform into a posterior one: to Fungi (Philippe, Bapteste pers. comm.). A thus it is arguably not homologous to the bikont shared derived fusion between cytochrome oxi- state with true ciliary transformation. dase 1 and 2 mitochondrial genes (Lang et al. 1999) A second gene fusion involving the first three shows that and are enzymes of pyrimidine biosynthesis (carbamoyl part of an amoebozoan clade within which the eu- phosphate synthase, dihydroorotase, aspartate car- karyote root cannot lie. These and other data indi- bamoyltransferase) is apparently a shared derived cate that slime moulds and acan- character for unikonts, absent from bikonts and thamoebids evolved by independent ciliary losses the ancestral (Stechmann and Cava- from ciliated Amoebozoa. The circumscription lier-Smith 2003a). As this involved two simultane- and phylogeny of both Amoebozoa and ous fusion events, it is even less likely to ever have Choanozoa have been clarified by recent molecu- been reversed than the DHFR/TS fusion. If none lar studies. The filose amoebae and the of these gene fusions has ever been reversed during enigmatic microvillar turn out to be evolution, then they together indicate that the root Choanozoa - phylogenetically distant from each of the eukaryote tree cannot lie within unikonts or other and the previously established Choanozoa bikonts, but must lie between these two clades (choanoflagellates, Corallochytrium, Ichthyo- (Stechmann and Cavalier-Smith 2003a). Earlier sporea) (Cavalier-Smith and Chao 2003a). The not structural and molecular evidence had supported obviously amoeboid uniciliate Phalansterium is an the idea that Animalia, Choanozoa and Fungi to- amoebozoan with affinities to /Fil- gether form a clade, the opisthokonts, charac- and (probably more distantly) acan- terised ancestrally by a single posterior cilium with thamoebids (Cavalier-Smith et al. 2004). The basal a bicentriolar kinetid and flat mitochondrial cristae branching order within bikonts is much less clear (Cavalier-Smith 1987). Extant Choanozoa are ei- than for unikonts. ther a clade that is sister to Animalia or a para- phyletic group ancestral to them (Cavalier-Smith and Chao 2003a; Rokas et al. 2003); a sister rela- The chromalveolate clade tionship between animals and choanoflagellates at least is supported by the probable gene fusion that Mechanisms of protein targeting to organelles generated the receptor tyrosine kinases from a cy- are remarkably conservative features of cells, as toplasmic kinase and calcium-binding epidermal Blobel and I argued when first discussing their ori- growth factor in their common ancestor after it di- gin 23 years ago (Blobel 1980; Cavalier-Smith verged from Amoebozoa (King and Carroll 2001). 1980). This led me to establish the kingdom The clade is also supported by a Chromista for all eukaryotes with plastids located shared derived 11–17 amino acid insertion in pro- within a periplastid membrane within the of tein synthesis elongation factor EF1-α absent from the rough and their putative prokaryotes and all other eukaryotes (Baldauf aplastidic descendants (Cavalier-Smith 1986). It is 1999). This character is quite strong, though some now clear that the mechanisms of protein targeting 342 T. Cavalier-Smith of nuclear-coded genes of all three major chromist mitochondrial cristae allowed to be groups (, Heterokonta, Haptophyta) recognised as a chromist even before molecular ev- are indeed homologous, all using bipartite (or tri- idence proved it. Such evidence is still lacking for partite for intrathylakoid ) N-terminal tar- kathablepharids, recently placed in Cryptista be- geting sequences. Increasing evidence reviewed cause of their single-scroll ejectisomes (Cavalier- elsewhere (Cavalier-Smith 2003b) shows that these Smith 2004b); their tubular cristae do not contra- mechanisms are also fundamentally similar in the dict such an assignment because this is the ances- two groups with plastids { tral state for chromalveolates, the flat cristae of and Sporozoa (with Apicomonadea constituting cryptomonads being secondarily derived. Many Apicomplexa), now grouped with protalveolates in aplastidic were recognised as such be- the phylum Miozoa (Cavalier-Smith 1999)} and cause of their tripartite (rarely bipartite) tubular that and chromists together form a clade hairs on the anterior younger cilium, but several of ancestrally photosynthetic eukaryotes created heterokonts have lost such hairs (notably Caecitel- by a single ancient enslavement of a red alga. Con- lus and Opalinata) or cilia altogether. Unless there catenated chloroplast gene trees (Yoon et al. 2002) is other ultrastructural evidence (as for Caecitellus) strongly support the of chromists and the chromist (rather than protozoan) affinity of Chromobiota (Haptophyta plus Heterokonta). such taxa is likely to be obscured in the absence of The shared derived of a nuclear gene sequences. Molecular data are needed to con- glyceraldehyde-3-phosphate dehydrogenase gene firm suggestions that actinophryid ‘heliozoa’ are and the acquisition of a bipartite targeting sequence related to pedinellid heterokonts. Centrohelid He- by one copy, allowing it to replace the original red liozoa are almost certainly not directly related to algal chloroplast protein, strongly supports chro- actinophryids or desmothoracids: recent rRNA malveolate monophyly (Fast et al. 2001). trees weakly suggest a relationship with Hapto- Several chromalveolate clades have lost photo- phyta (Cavalier-Smith and Chao 2003b). Although synthesis while retaining (e.g. most an alternative even weaker relationship with the Sporozoa, , some pedinellids, some di- thecomonad cannot be ruled out, I atoms, some chrysomonads), whereas others have now place Heliozoa (Centrohelea only) within apparently lost plastids altogether (e.g. Ciliophora, Chromobiota (Table 1), on the hypothesis that , , Goniomonas). The com- their microtubular are related to those plete loss of plastids has caused some colourless of the haptonema and that a common ancestor chromists to be treated in the past as protozoa. The with used long filiform - retention of cryptophyte-like ejectisomes and flat supported cell extensions to trap prey.

Table 1. Classification of the kingdom Chromista Cavalier-Smith 1981 emend.

Subkingdom 1. Cryptista Cavalier-Smith 1989 emend. (3 classes) Phylum Cryptista Cavalier-Smith 1986 (, goniomonads, kathablepharids) Subkingdom 2. Chromobiota Cavalier-Smith 1989 emend. Infrakingdom 1. Heterokonta Cavalier-Smith 1986 (19 classes) Phylum 1. Cavalier-Smith 1986 (13 essentially algal classes: with plastids) Phylum 2. Cavalier-Smith 1998 ( with double transitional helix) Subphylum 1. Bigyromonada Cavalier-Smith 1998 (1 : Developayella) Subphylum 2. Pseudofungi Cavalier-Smith 1986 (2 classes: , Hyphochytrea) Subphylum 3. Opalinata Wenyon 1926 stat. nov. Cavalier-Smith 1997 (3 classes) Phylum 3. Sagenista Cavalier-Smith 1995 (3 classes: Labyrinthulea, Bicoecea, Wobblia etc.) Infrakingdom 2. infrakingd. nov. Diagnosis: ancestrally biciliate and feeding on prey by one or more microtubular-supported filiform extensions. Phylum 1. Haptophyta Cavalier-Smith 1986 (2 classes: Pavlovophyceae, ) Phylum 2. Heliozoa Haeckel 1866 stat. nov. Margulis 1974 emend. (class Centrohelea only)

For fuller details, including the rest of the 25 classes see Cavalier-Smith 2004b Phylogeny and classification of Protozoa 343

Infrakingdom Rhizaria revised acterised not by one or more universally shared derived characters but by an array of characters Removing and thecomonads from partially overlapping in distribution but missing Rhizaria makes the infrakingdom more homoge- from many members of the group. In this respect neous structurally and confined to the phyla Cer- and in the monophyly of the group often not being cozoa and (Radiozoa plus Foraminifera) supported by single-gene trees excavates resemble plus and order Desmothoracida, pro- Chromista and are encountering similar scepticism visionally left within Rhizaria. Else- from some with naïve expectations of what single- where I suggest that Foraminifera evolved from gene trees can resolve. Multiple losses of the feed- (Cavalier-Smith and Chao ing groove and ciliary vanes have impeded under- 2003b): I think their common ancestor was reticu- standing of the group. As presently constituted it lopodial and that the multiply porous central cap- embraces four phyla: Euglenozoa and Percolozoa sules of polycystine radiolaria and multiply porous (grouped together as superphylum on walls of foraminifera are evolutionarily related. account of their flat, typically discoid cristae and The triply porous central capsule of Phaeodarea is parallel centrioles) plus the basal biciliate Louko- more different; unlike Polycystinea and Acan- zoa and the highly derived, secondarily amito- tharia there is no evidence that they ever had do- chondrial, ancestrally tetrakont Metamonada decagonal axonemes or can sequester (, Parabasalia, Carpediemonadea, sulphate, so I exclude them from a revised Radio- and Eopharyngia) (Cavalier-Smith 2003a). zoa. A shared single amino acid insertion in polyu- It appears that the ancestral percolozoan and biquitin convincingly unites Cercozoa and independently evolved a quadriciliate Foraminifera (Archibald et al. 2003): I predict it condition (Cavalier-Smith 2003a); it is likely that, will be in Radiozoa and Phaeodarea too; it is ab- as in secondarily quadriciliate green such as sent from centrohelids and thecomonads (Herden, Pyramimonas, their ciliary transformation is Chao, Cavalier-Smith, unpublished), here exclud- spread over two cell cycles – a complex derived ed from Rhizaria. Cercozoa have proved to be a state. Though excavates sometimes appear holo- major protozoan phylum that probably includes phyletic (Cavalier-Smith 2003a), their unity is ob- the majority of of previously uncer- scured on many rRNA trees by dramatic variation tain phylogenetic position, several distinct groups in this molecule’s evolutionary rate. This also leads of filose testate rhizopods, reticulose protozoa the longest branch excavates to attract artefactually such as gymnophryids, , the parasitic archaebacterial outgroup sequences and thus mis- plasmodiophorids and (Cavalier- leadingly show the root within Metamonada. It Smith and Chao 2003c). Unpublished studies appears that secondary loss of mitochondria and using phylum-specific PCR primers to amplify their often leads to dramatically in- DNA extracted directly from environmental sam- creased evolutionary rates of cytosolic ribosomal ples suggest that Cercozoa are even more diverse rRNA and proteins, as in , mi- than this and may be one of the most speciose pro- crosporidia, Archamoebae and to spurious group- tozoan phyla (Bass and Cavalier-Smith). ing on the trees, especially when intra-site rate trees support the monophyly of Rhizaria (Keeling variation is not allowed for and taxon sampling is 2001) as more weakly do some rRNA trees (Cava- too sparse. Metamonad unity has also been ob- lier-Smith and Chao 2003c). scured by secondary losses of Golgi stacking (in- dependently in Eopharyngia and ) and by the presence of in some but Infrakingdom Excavata (probably) only mitosomes in others (e.g. Eo- pharyngia like ). Golgi stacking was also The excavate concept originated from shared lost in Percolozoa within Heterolobosea, the ly- ciliary structures (lateral vanes) and ciliary root romonad subclade of which became anaerobic/hy- patterns that were most pronounced in members drogenosomal independently of Metamonada of the group possessing a ventral feeding groove (O’Kelly et al. 2003). (Simpson and Patterson 1999, 2001). Excavates are Loukozoa are probably paraphyletic; the classes a good example of a polythetic taxon, i.e. one char- Jakobea and Malawimonadea apparently diverged 344 T. Cavalier-Smith very early in the group’s history. Several rRNA Smith 1999). If this were correct it would mean and protein trees weakly suggest that are that Rhizaria and Excavata together form a clade related to Euglenozoa, whereas may (cabozoa), that their last common ancestor was a be sister to Metamonada. I earlier added Diphyllei- photophagotroph and that all aplastidic cabozoa da to Loukozoa on the grounds that both are an- have lost plastids (roughly 14 separate losses!). cestrally biciliate taxa with ventral feeding grooves There are growing hints from -like (Cavalier-Smith 2002), even though diphylleids genes found in Percolozoa, trypanosomes and lack ciliary vanes and their ciliary microtubular even metamonads that the common ancestor of roots supporting the groove rims are less like those discicristates and metamonads (presumably a of jakobids and Malawimonas than the latter are to loukozoan) may have had a plastid, but there is each other. More recently, to make Loukozoa currently no comparable evidence for or against more homogeneous I transferred Diphyllatea to this for any aplastidic rhizarian (Cavalier-Smith Apusozoa as a second class (Cavalier-Smith 2003a, b). It is important to whether Rhizaria 2003a). Numerous protein gene sequences are and Excavata are sisters and seek plastid-related needed to test this relationship and establish the genes in aplastidic Rhizaria and Loukozoa. If the position of Apusozoa, as single-gene trees lack the cabozoan hypothesis turns out to be correct it will requisite resolution. The ventral groove makes it greatly simplify eukaryote phylogeny: a single en- possible that Apusozoa are sisters to excavates, but slavement of a green alga by the ancestral cabozoan rRNA and Hsp90 trees weakly suggest they may would add further compelling evidence that the be more distant. root of the eukaryote tree cannot lie within cabo- The loukozoan is notable zoa, any more than it can within Plantae or chro- for the most primitive mitochondrial malveolates. It would also show that bikonts com- known, in particular retaining proteobacterial prise but three major groups (Plantae, chromalve- RNA polymerase genes lost from most other eu- olates, cabozoa), each characterised by the ances- karyotes following the acquisition from a of a tral enslavement of a foreign cell. If Apusozoa are simpler nuclear-coded RNA polymerase (Lang sisters of Excavata or Rhizaria they also would be et al. 1999). The apparently derived position of cabozoa – but if not they could be the only primi- Reclinomonas within bikonts on concatenated mi- tively non-photosynthetic bikonts. tochondrial gene trees (Lang et al. 2002) does not support the a priori plausible suggestion based solely on this primitiveness that Loukozoa might The corticate hypothesis be very early diverging eukaryotes (Cavalier-Smith 1999, 2000a). If the viral polymerase was acquired If there are indeed only three or four bikont by the ancestral eukaryote, multiple independent groups, what is their branching order? Long ago I losses of the then redundant proteobacterial poly- postulated that cortical alveoli of Glaucophyta merase are entirely plausible. Multiple indepen- (kingdom Plantae) and alveolates are homologous dent losses of many other mitochondrial and (Cavalier-Smith 1982). If this is correct and they chloroplast genes have certainly occurred. evolved in the common ancestor of Plantae and chromalveolates, they would be a shared derived character (albeit multiply lost) for a clade compris- The cabozoan hypothesis ing both groups (hence designated corticates: Cav- alier-Smith 2003b) or alternatively a reason for The same considerations of economy in the ori- considering Plantae paraphyletic with chromalve- gin of protein-targeting machinery dur- olates sister to Glaucophyta alone (less likely but ing that led to the now widely ac- not impossible). If corticates and cabozoa both cepted (but initially ignored or opposed) ideas of turn out to be clades we should have established monophyly of Plantae, Chromista, Chromobiota the large-scale structure of the bikont part of the and chromalveolates led me to suggest that chlo- eukaryotic tree and rendered it highly probable rarachnean algae (phylum Cercozoa) and eu- that all bikont groups (i.e. most protozoan phyla) glenoids (Euglenozoa) obtained their green were ancestrally photosynthetic and that organelle /b plastids by a single enslavement of loss has been even more important in protist evo- a green alga by their common ancestor (Cavalier- lution than often thought. Phylogeny and classification of Protozoa 345

If the cabozoan or the corticate hypothesis (or Bapteste E., Brinkmann H., Lee J. A., Moore D. V., Sensen both) proves to be mistaken, such disproof would C. W., Gordon P., Duruflé L., Gaasterland T., Lopez P., Müller M. and Philippe H. (2002): The analysis of 100 probably in itself establish the correct branching genes supports the grouping of three highly divergent order of the four main bikont groups: excavates, amoebae: Dictyostelium, Entamoeba, and Mastigamoe- Rhizaria, Plantae and chromalveolates. We would ba. Proc. Natl Acad. Sci. U S A 99, 1414–1419. then probably have a well-corroborated, properly Baroin A., Perasso R., Qu L. H., Brugerolle G., Bachellerie J. P. and Adoutte A. (1988): Partial phylogeny of the rooted tree for all really major protist and eukary- unicellular eukaryotes based on rapid sequencing of a otic groups. portion of 28S ribosomal RNA. Proc. Natl Acad. Sci. U S A 85, 3474–3478. Blobel G. (1980): Intracellular protein topogenesis. Proc. Natl Acad. Sci. U S A 77, 1496–1500. Uncertainties Bourlat S. J., Nielsen C., Lockyer A. E., Littlewood D. T. and Telford M. J. (2003): is a Key things to be tested more thoroughly are the that eats molluscs. 424, 925–928. cabozoan hypothesis and the positions of Apuso- Cavalier-Smith T. (1980): Cell compartmentation and the zoa, centrohelids, Phaeodarea, and xenophyopho- origin of eukaryote membranous organelles. In: res. Telonemea, Holosea and Schizocladea remain Schwemmler W. and Schenk H. E. A. (eds): Endocyto- biology: Endosymbiosis and , a synthesis of virgin territory for , and there are recent research, pp. 893–916. de Gruyter, Berlin. numerous other obscure protists of uncertain Cavalier-Smith T. (1986): The kingdom Chromista: origin affinities that have not received the study they de- and systematics. In: Round F. E. and Chapman D. J. serve. Eventually they will probably slot into the (eds): Progress in Phycological Research Vol. 4, pp. 309–347. Biopress, Bristol. general scheme presented here. Some new classes Cavalier-Smith T. (1987): The origin of Fungi and pseudo- will almost certainly be characterised, but our enu- fungi. In: Rayner A. D. M., Brasier C. M. and Moore D. meration of eukaryote phyla may be close to com- (eds): Evolutionary Biology of the Fungi, pp. 339–353. pletion, though we may still be in for a few big sur- Cambridge University Press. Cavalier-Smith T. (1998): A revised six-kingdom system of prises, exemplified by the possibility that the ob- . Biol. Rev. Camb. Philos. Soc. 73, 203–266. scure worm Xenoturbella represents a new animal Cavalier-Smith T. (1999): Principles of protein and lipid tar- phylum (Bourlat et al. 2003). Nonetheless, I sus- geting in secondary symbiogenesis: euglenoid, dinoflag- pect that we are entering a period of consolidation ellate, and sporozoan plastid origins and the eukaryotic family tree. J. Euk. Microbiol. 46, 347–366. for high-level protist phylogeny and Cavalier-Smith T. 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(Anaeromonadea, Para- grants and the Canadian Institute for Advanced Re- basalia, , Eopharyngia) and Loukozoa search and NERC for fellowship support. emend. (Jakobea, Malawimonas): their evolutionary affinities and new higher taxa. Int. J. Syst. Evol. Micro- biol. 53, 1741–1758. Cavalier-Smith T. (2003b): Genomic reduction and evolu- References tion of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta- Archibald J. M., Longet D., Pawlowski J. and Keeling P. J. algae). Phil. Trans. Roy. Soc. B 358, 109–134. (2003): A novel polyubiquitin structure in Cercozoa Cavalier-Smith T. (2004a): The membranome and mem- and Foraminifera: evidence for a new eukaryotic super- brane heredity in development and evolution. In: Hirt group. Mol. Biol. Evol. 20, 62–66. R. P. and Horner D. S. (eds): Organelles, genomes and Baldauf S. L. (1999): A search for the origins of animals and eukaryote phylogeny. 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Appendix Superclass 2. Monadofilosa Cavalier-Smith 1997 stat. nov. 2003 Class 1. Sarcomonadea Cavalier-Smith 1993 emend. Classification of kingdom Protozoa** 2003 (e.g. Cercomonas, Heteromita, Bodomorpha, Proleptomonas, Allantion) and its 11 phyla and 64 or 65 classes Class 2. Cavalier-Smith 2003 (e.g. Cryp- todifflugia, Cryothecomonas) Subkingdom SARCOMASTIGOTA** Cavalier-Smith 1993 Class 3. Spongomonadea Cavalier-Smith 2000 (e.g. emend. Spongomonas, Rhipidodendron) Phylum 1. Amoebozoa Lühe 1913 stat. nov. Corliss 1984 Class 4. Cavalier-Smith 2003 (e.g. Thau- emend. Cavalier-Smith 1998 matomonas, , Allas, Gyromitus, Eug- Subphylum 1. Protamoebae*+ Cavalier-Smith 2004 lypha, , ) Class 1. Breviatea+ Cavalier-Smith 2004 (e.g. ‘Mastig- Subphylum 2. Cavalier-Smith 2002 emend. amoeba invertens’) 2003 Class 2. Lobosea Carpenter 1861 emend. (e.g. Amoe- Class 1. Engler and Prantl 1897 (e.g. ba, , Saccamoeba, , Echinamoeba, Phagomyxa, ) Leptomyxa, , Copromyxa, ) Class 2. Ascetosporea Sprague 1979 stat. nov. Cava- Class 3. + Cavalier-Smith 2004 (e.g. Vannel- lier-Smith 2002 (Minchinia, Haplosporidium, la, , Multicilia, , , Urosporidium, Bonamia, , Paramyxa, Para- , , Thecamoeba) marteilia, Claustrosporidium, Microcytos) Class 4. Variosea+ Cavalier-Smith 2004 (e.g. Pha- Class 3. Gromiidea Cavalier-Smith 2003 () lansterium, Acanthamoeba, Balamuthia, Gephyra- Phylum Retaria Cavalier-Smith 1999 moeba, Filamoeba, Stereomyxa) Subphylum 1. Radiozoa Cavalier-Smith 1987 emend. Subphylum 2. Cavalier-Smith 1998 (Ancestrally with axopodia having an open dodecagonal Infraphylum 1. Archamoebae Cavalier-Smith 1983 meshwork of and able to sequester stron- stat. nov. 1998 tium sulphate intracellularly) Class Archamoebea Cavalier-Smith 1983 stat. nov. Class 1. Haeckel 1881 stat. nov. Cavalier- (e.g. , Mastigina, Entamoeba, Mastigamoe- Smith 1993 (e.g. Acanthometra) ba, Phreatamoeba, Endolimax) Class 2. Sticholonchea Poche 1913 stat. nov. Petru- Infraphylum 2. De Bary stat. nov. shevskaya 1977 () Class 1. Stelamoebea*+ Cavalier-Smith 2004 (e.g. Class 3. Polycystinea Ehrenberg 1838 stat. nov. Cava- Protostelium, Schizoplasmodium, Cavostelium, lier-Smith 1993 (e.g. Collozoum) Planoprotostelium, Acytostelium, Dictyostelium) Subphylum 2. Foraminifera (D’Orbigny 1826) Eich- Class 2. Myxogastrea Fries 1829 stat. nov. Cavalier- wald 1830 stat. nov. Mikhalevich 1980 Smith 1993 emend. (e.g. Hyperamoeba, , Class 1. Athalamea Haeckel 1862 () Stemonitis, Physarum, Didymium) Class 2. Polythalamea Ehrenberg 1838 stat. nov. Phylum 2. Choanozoa Cavalier-Smith 1981 emend. 1998 Mikhalevich 1980 (e.g. , , Glo- Class 1. Choanoflagellatea Kent 1880 stat. nov. Cava- bigerina, Miliola) lier-Smith 1998 (e.g. Monosiga) Class 3. Schulze 1904 (e.g. Psammi- Class 2. Corallochytrea Cavalier-Smith 1995 na) Possibly also class Schizocladea Cedhagen and (Corallochytrium) Mattson 1992 (Schizocladus) Class 3. Ichthyosporea Cavalier-Smith 1998 (e.g. Rhizaria incertae sedis: Class Phaeodarea Haeckel 1879 (e.g. , ) Aulacantha, Castanella) Class 4. Cristidiscoidea Page 1987 stat. nov. Cavalier- Infrakingdom Excavata Cavalier-Smith 2002 Smith 1997 (e.g. Ministeria, Nuclearia) Phylum 1. Loukozoa* Cavalier-Smith 1999 emend. 2003 Subkingdom BICILIATA** subkingd. nov. Diagnosis: an- Class 1. Jakobea Cavalier-Smith 1999 (Reclinomonas, cestrally biciliate unicellular eukaryotes in which dihydrofo- , Jakoba) late reductase and thymidylate synthase genes, if present, are Class 2. Malawimonadea Cavalier-Smith 2003a fused into a single unit of ; lacking plastids in the (Malawimonas) with double envelopes and lacking plastids within Phylum 2. Metamonada Grassé 1952 stat. nov. emend. the rough endoplasmic reticulum or tubular ciliary hairs; mi- Cavalier-Smith 2003a tochondrial cristae usually tubular or discoid (i.e. all bikonts Subphylum 1. Anaeromonada Cavalier-Smith 1996/7 excluding Plantae and Chromista) Class Anaeromonadea Cavalier-Smith 1996/7 emend. Infrakingdom 1. Rhizaria Cavalier-Smith 2002 emend. 1999 (; Oxymonadida e.g. , Di- Phylum 1. Cercozoa Cavalier-Smith 1998 emend. 2002 nenympha) Subphylum 1. Filosa Cavalier-Smith 2003. Subphylum 2. Cavalier-Smith 1996/7 stat. Superclass 1. Reticulofilosa Cavalier-Smith 1997 stat. nov. emend. 2003* nov. 2003 Superclass 1. Parabasalia Honigberg 1973 stat. nov. Class 1. Chlorarachnea Hibberd and Norris 1984 Cavalier-Smith 2003 (e.g. Chlorarachnion, , Lotharella, Cryp- Class 1. Trichomonadea Kirby 1947 stat. nov. Mar- tochlora, Gymnochlora) gulis 1974 emend. Cavalier-Smith 2003 (e.g. Tri- Class 2. * Lankester 1885 em. Cava- chomonas, Lophomonas, Microjoenia, Spirotricho- lier-Smith 2003. (e.g. Pseudospora, Leucodictyon, nympha) , Massisteria, Dimorpha, Gymnophrys, Class 2. Trichonymphea Cavalier-Smith 2003a (e.g. Borkovia) ) 348 T. Cavalier-Smith

Superclass 2. Carpediemonadia Cavalier-Smith 2003a Smith 1993 (e.g. Noctiluca) Class Carpediemonadea Cavalier-Smith 2003a (Car- Class 2. Peridinea Ehrenberg 1830 stat. nov. pediemonas) Wettstein (e.g. , Prorocentrum, , Superclass 3. Eopharyngia Cavalier-Smith 1993 stat. , , Crypthecodinium, Symbiodini- nov. um, , ) Class 1. Trepomonadea Cavalier-Smith 1993 Subphylum 3 Apicomplexa Levine 1970 emend. stat. Subclass 1. Diplozoa Dangeard 1910 stat. nov. nov. Cavalier-Smith 1996 (diplomonads: Tre- Infraphylum 1. Apicomonada Cavalier-Smith 1993 pomonas, , , Giardia, stat. nov. ) Class 1. Apicomonadea Cavalier-Smith 1993 (e.g. Subclass 2. Enteromonadia Cavalier-Smith 1996 (En- Acrocoelus, , ) teromonas) Infraphylum 2. Sporozoa Leuckart 1879 stat. nov. Cav- Class 2. Retortamonadea Cavalier-Smith 1993 alier-Smith 1999 (e.g. , ) Class 1. Coccidea* Leuckart 1879 (e.g. , Superphylum 1. Discicristata Cavalier-Smith 1993 , Toxoplasma) Phylum 1. Percolozoa Cavalier-Smith 1991 stat. nov. Class 2. Gregarinea Dufour 1828 (e.g. Monocystis, Class 1. Heterolobosea Page and Blanton 1985 (e.g. Ophriocystis) , , , Lyromonas, Class 3. Hematozoa Vivier 1982 (e.g. , ) , ) Class 2. Percolatea Cavalier-Smith 2003a (Percolo- Phylum 2. Ciliophora Doflein 1901 ( and suctori- , ) ans) Phylum 2. Euglenozoa Cavalier-Smith 1981 Subphylum 1. Gerassimova and Subphylum 1. Plicostoma Cavalier-Smith 1998 Seravin 1976 Class 1. Euglenoidea Bütschli 1884 (e.g. , Class 1. Corliss 1974 (e.g. , , Rhabdomonas, , Astasia, , ) Eutreptia, ) Class 2. Heterotrichea Stein 1859 (e.g. , Folli- Class 2. Diplonemea Cavalier-Smith 1993 (Diplone- culina, ) ma, ) Subphylum 2. Lynn 1996 Subphylum 2. Saccostoma Cavalier-Smith 1998 8 Classes: Spirotrichea (e.g. Oxytricha, , Class 1. Kinetoplastea Honigberg 1963 stat. nov. Mar- Tintinnus, ), (e.g. , gulis 1974 (e.g. , Rhynchomonas, Ichthyobodo, Lacrymaria, Entodinium), (e.g. Dys- , , ) teria, Podophrya), (e.g. ), Nas- Class 2. Postgaardea Cavalier-Smith 1998 (, sophorea (e.g. ), (e.g. ), ) Plagiopylea, (e.g. , Infrakingdom Alveolata Cavalier-Smith 1991 , ) Phylum 1. Miozoa Cavalier-Smith 1987 stat. nov. 1999 Subphylum 1. Protalveolata* Cavalier-Smith 1991 em. Biciliata incertae sedes: Class 1. Colponemea Cavalier-Smith 1993 () 1. Phylum Apusozoa Cavalier-Smith 1996/7 stat nov. 2003 Class 2. Levine 1978 () emend. Subphylum 2. Dinozoa Cavalier-Smith 1981 em. Class 1. Diphyllatea Cavalier-Smith 2003a (Diphylleia, Infraphylum 1. Ellobiopsa infraph. nov. Diagnosis as ) for the class Ellobiopsea Class 2. Thecomonadea Cavalier-Smith 1993 stat. nov. Class Ellobiopsea Loeblich III 1970 (e.g. , 1995 (e.g. Amastigomonas, , Ancyromonas, Thalassomyces) Hemimastix, Spironema) Infraphylum 2. Dinoflagellata Butschli 1885 stat. nov. 2. Class Telonemea Cavalier-Smith 1993 (, Cavalier-Smith 1999 ) Superclass 1. Syndina Cavalier-Smith 1993 Class Syndinea Chatton 1920 stat. nov. Loeblich 1976 Protista incertae sedes (Protozoa or chromists): Class (e.g. Amoebophrya) Holosea Cavalier-Smith 1993 (Luffisphaera); Order Com- Superclass 2. Fensome et al. 1993 matiida Cavalier-Smith 1996/7 (Commation); Order Disco- Class 1. Noctilucea Haeckel 1866 stat. nov. Cavalier- celida Cavalier-Smith 1997 (Discocelis)

*Probably paraphyletic groups **Certainly paraphyletic groups +Diagnoses of these five new taxa are in Cavalier-Smith et al. (2004)