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Protist Phylogeny and The High-Level Classification of Protozoa - Cavalier-Smith 2003
Protist Phylogeny and The High-Level Classification of Protozoa - Cavalier-Smith 2003
Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK;
E-mail: tom.cavalier-smith@zoo.ox.ac.uk
Protist large-scale phylogeny is briefly reviewed and a revised higher classification of the kingdom Pro-
tozoa into 11 phyla presented. Complementary gene fusions reveal a fundamental bifurcation among eu-
karyotes between two major clades: the ancestrally uniciliate (often unicentriolar) unikonts and the an-
cestrally biciliate bikonts, which undergo ciliary transformation by converting a younger anterior cilium
into a dissimilar older posterior cilium. Unikonts comprise the ancestrally unikont protozoan phylum
Amoebozoa and the opisthokonts (kingdom Animalia, phylum Choanozoa, 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 dihydrofolate reductase and thymidylate synthase and include
plants and all other protists, comprising the protozoan infrakingdoms Rhizaria [phyla Cercozoa and Re-
taria (Radiozoa, Foraminifera)] and Excavata (phyla Loukozoa, Metamonada, Euglenozoa, Percolozoa),
plus the kingdom Plantae [Viridaeplantae, Rhodophyta (sisters); Glaucophyta], the chromalveolate clade,
and the protozoan phylum Apusozoa (Thecomonadea, Diphylleida). Chromalveolates comprise kingdom
Chromista (Cryptista, Heterokonta, Haptophyta) and the protozoan infrakingdom Alveolata [phyla Cilio-
phora and Miozoa (= Protalveolata, Dinozoa, Apicomplexa)], which diverged from a common ancestor
that enslaved a red alga and evolved novel plastid protein-targeting machinery via the host rough ER
and the enslaved algal plasma membrane (periplastid membrane). The branching order of the five bikont
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 centrohelid heliozoa may be
sisters to Haptophyta.
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 genomes, cy- the root 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 organelles of different function: hy- small number of phyla (25, counting all four fungal
drogenosomes or mitosomes (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. Chlorophyta, one of the three plant
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), yeasts, Corallochytrium (Cavalier- The present paper summarises this evidence and
Smith and Chao 2003a), Myxozoa, microsporidia, outlines my current interpretation of relationships
Blastocystis} 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-
centrioles (= 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 animals 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 systematics is at last Loukozoa probably holophyletic) constitute the
penetrating the previously over-dogmatic and paraphyletic subkingdom Biciliata from which the
over-self-confident field 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-
times 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/centriole and its associated roots are always
Philippe et al. 2000a; Philippe et al. 2000b; Lopez the first formed; in the next cell 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 phylogenetic tree 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 eukaryote is held to have been a phagotrophic uniciliate, unicentrio-
lar aerobic zooflagellate that arose from a neomuran bacterial ancestor by the simultaneous origin of the cytoskele-
ton, endomembrane system, 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 chloroplasts 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 chloroplast (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 plastids 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 genes the ancestral state in bacteria 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 – Phalansterium, mastigamoe- If Choanozoa and Amoebozoa are both holo-
bids, Multicilia and protostelids (sensu Cavalier- phyletic, the branching order for the four unikont
Smith et al. 2004) and possibly ‘Mastigamoeba 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 Archamoebae (mastigamoebids, Pelo-
bicentriolar kinetids. The bicentriolar state of myxa, Entamoeba and Endolimax) 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 choanoflagellates 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 Dictyostelium and Acanthamoeba 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 dictyostelid slime moulds and acan-
character for unikonts, absent from bikonts and thamoebids evolved by independent ciliary losses
the ancestral prokaryotes (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 Nuclearia and the
of these gene fusions has ever been reversed during enigmatic microvillar Ministeria 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 Gephyramoeba/Fil-
gether form a clade, the opisthokonts, charac- amoeba 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 opisthokont 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 lumen of
tein synthesis elongation factor EF1-α absent from the rough endoplasmic reticulum 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 Goniomonas to be
groups (cryptomonads, 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 proteins) 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 alveolate groups with plastids {dinoflagellates 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 heterokonts were recognised as such be-
the phylum Miozoa (Cavalier-Smith 1999)} and cause of their tripartite (rarely bipartite) tubular
that alveolates 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 monophyly 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 gene duplication 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 leucoplasts (e.g. most an alternative even weaker relationship with the
Sporozoa, Chilomonas, some pedinellids, some di- thecomonad Ancyromonas 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
Pseudofungi, Sagenista, Goniomonas). The com- their microtubular axonemes 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 haptophytes used long filiform microtubule-
retention of cryptophyte-like ejectisomes and flat supported cell extensions to trap prey.
For fuller details, including the rest of the 25 classes see Cavalier-Smith 2004b
Phylogeny and classification of Protozoa 343
very early in the group’s history. Several rRNA Smith 1999). If this were correct it would mean
and protein trees weakly suggest that jakobids are that Rhizaria and Excavata together form a clade
related to Euglenozoa, whereas Malawimonas 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 cyanobacteria-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 test 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 jakobid loukozoan Reclinomonas is notable zoa, any more than it can within Plantae or chro-
for the most primitive mitochondrial genome 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 virus 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 organelle protein-targeting machinery dur- olates sister to Glaucophyta alone (less likely but
ing symbiogenesis 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
chlorophyll a/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
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search and NERC for fellowship support. emend. (Jakobea, Malawimonas): their evolutionary
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