Protist Evolution and Advanced article

Phylogeny . Introduction
Article Contents

Jan Pawlowski, University of Geneva, Geneva, Switzerland . Ribosomal Tree of Eukaryotes

. Phylogenomic Adjustments

. Metagenetic Explosion

. Characteristics of Major Eukaryotic Supergroups

. Future Challenges

Online posting date: 17th March 2014

The beginning of this century brought spectacular chan- foraminifera, whose sponge-like tests pave the deep-sea
ges in our understanding of eukaryote phylogeny, espe- bottom, or the laminarian seaweeds, whose plant-like thalli
cially the early evolution of microeukaryotic lineages become several metres long and form dense forests in
commonly called protists. Phylogenomic studies based on coastal regions. Moreover, aggregative multicellularity is
analysis of more than 200 genes produced a relatively
commonly found in some protist groups (e.g. cellular slime
moulds).
well-resolved tree of eukaryotes. Metagenetic studies
From an evolutionary perspective, protists are a para-
using environmental deoxyribonucleic acid (eDNA) phyletic group, that is, a group that shares its ancestor with
revealed an extraordinary diversity of protist lineages, other groups. In fact, the last common ancestor of protists
highlighting their great ecological and evolutionary is also the ancestor of all other eukaryotes. For con-
importance. A new higher level classification of eukar- venience, the traditional ‘five kingdoms’ system is used to
yotes combining the results of microscopic and various separate typically microscopic and unicellular protists
‘omics’ studies has been proposed. In this classification, from macroscopic and multicellular animals, plants and
the majority of eukaryotes are placed in seven mono- fungi. However, this separation has no other justification
phyletic supergroups: Amoebozoa, Opisthokonta, than our anthropocentric perspective. As shown by mod-
Archaeplastida, Alveolata, Stramenopiles, Rhizaria and ern phylogenetic analyses, the three dominant kingdoms
are in reality just small branches amidst a vast radiation of
Excavata. Phylogenetic relationships between these
eukaryotes. Genetic distance between the ancestor of ani-
supergroups and 11 independent eukaryotic lineages are
mals and fungi is as large as the distance between two
relatively well established. However, there is no consensus distant groups of amoebas. For these reasons the ‘five
concerning the position of the root of eukaryotic tree. kingdoms’ system was abandoned and replaced by a much
Further single-cell microscopic and genomic studies are more objective and phylogenetically coherent system of
also necessary for exploring the extraordinary diversity of eukaryotic supergroups. The new system is widely accepted
protistan phyla revealed by the eDNA surveys. now and its historical development and current inter-
pretation are presented here.

Introduction Ribosomal Tree of Eukaryotes

Protists is an informal term describing all eukaryotes other
The first molecular trees of eukaryotes were based exclu-
than animals, plants and fungi. The majority of protists,
sively on analyses of ribosomal genes. This was mainly due
also called ‘microeukaryotes’, ‘microbial eukaryotes’ or
to the exceptional conservation of these genes across all
‘unicellular eukaryotes’, are unicellular and microscopic.
eukaryotic phyla. From a practical point of view, riboso-
However, small size and their single-celled nature
mal genes represented by thousands of identical or nearly
can hardly define all protists. Some of them can reach
identical copies in every single cell were the easiest to
extraordinary dimensions, such as xenophyophorean
amplify and to sequence. Therefore, it was not surprising
that in the early days of molecular systematics, all phylo-
eLS subject area: Microbiology genetic studies of prokaryotes and eukaryotes were based
on ribosomal genes (Pace et al., 1986; Sogin, 1991).
How to cite: As prokaryotes phylogeny was based on the small sub-
Pawlowski, Jan (March 2014) Protist Evolution and Phylogeny. In: eLS.
unit 16S ribosomal ribonucleic acid (rRNA) gene, eukar-
John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0001935.pub2
yotic phylogenetists predominantly used the small subunit
18S rRNA gene. Indeed, this gene still constitutes a gold

eLS & 2014, John Wiley & Sons, Ltd. www.els.net 1

 Protist Evolution and Phylogeny

standard for eukaryotic species identification and phylo- them are under different names or are subdivided into
genetic inferences at a lower taxonomic level (Pawlowski smaller groups. These trees were principally based on
et al., 2012). The Protist Ribosomal Reference (PR2) analysis of four proteins (actin, a-tubulin, b-tubulin, and
database http://ssu-rrna.org/ comprises currently approxi- EF1), combined with analysis of ribosomal genes (Baldauf
mately 130 000 18S ribosomal deoxyribonucleic acid et al., 2000). Further development of multigene phylo-
(rDNA) sequences that represent all eukaryotic phyla genies led to a reduction in the number of supergroups,
(Guillou et al., 2012). In addition, thousands of 18S obtained by introducing larger assemblages, such as Uni-
sequences generated by high-throughput environmental konts (grouping Opisthokonts with Amoebozoa) and
DNA surveys are submitted yearly to DNA databases. Chromalveolates (grouping Alveolates, Stramenopiles,
Although ribosomal genes remain a reference for Haptophytes and Cryptophytes).
studying protist diversity, their current use for inferring The support for these groups in early multigene phylo-
eukaryotes phylogeny is much more limited. This is mainly genies composed usually of less than 10 genes was relatively
due to artefacts caused by an excessive heterogeneity of weak, leading to a critical evaluation of the new eukaryotic
evolutionary rates across eukaryotic phyla. Early riboso- system (Parfrey et al., 2006). Nevertheless, with the devel-
mal trees of eukaryotes have a characteristic ladder struc- opment of new phylogenomic tools, the analysis of large
ture with longer branches at the bottom and shorter datasets became much more accurate and the detection and
branches in the crown of the tree (Sogin, 1991). As most of correction of various biases more efficient (Rodrguez-
the amitochondrial parasitic lineages have been found at Ezpeleta et al., 2007). Moreover, the database of protistan
the base of the tree, it gave rise to the Archeozoa hypoth- genomes grew rapidly. The major progress was obtained by
esis, suggesting that early eukaryotes, such as diplomo- analysis of transcriptomic sequences developed within the
nads, trichomonads and microsporidians, originated Protist EST Program (PEP) and other similar projects. The
before the event of mitochondrial endosymbiosis (Cava- access to EST data allowed analysis of more than 100 genes
lier-Smith, 1993). Although this hypothesis was very and obtaining much more robust phylogenies (Burki et al.,
appealing, it did not last for long because several studies 2008).
demonstrated that most of these putative early eukaryotic Among several issues raised by the development of
lineages contain residual mitochondrial genes, suggesting eukaryotic phylogenomics, the most controversial one was
that they have lost mitochondria due to their parasitic the Chromalveolate hypothesis suggesting a unique origin
mode of life. Moreover, the genomic studies showed that of red algal secondary endosymbiosis (Keeling, 2009).
some of them (e.g. microsporidia) were misplaced due to From a phylogenetic perspective, this hypothesis was
rapid evolutionary rates of their ribosomal genes. Hence, confirmed by the robust clade of Stramenopiles and Ale-
the group of Archeozoa was finally abandoned, remaining volates. However, the clustering of these two groups with
the most famous example of long-branch attraction arte- Haptophytes and Cryptophytes was never strongly sup-
fact in eukaryotic phylogeny (Philippe et al., 2000). ported. A serious doubt about the existence of Chro-
Yet, not all phylogenetic relationships inferred from 18S malveolates appeared when it became evident that the
rRNA gene were false. In fact, most of the currently sister group to Stramenopiles and Alveolates is Rhizaria,
recognised eukaryotic supergroups have been already which is known to comprise no phototrophes, except for
present in ribosomal phylogenies (Cavalier-Smith, 1998, Chlorarachniophytes bearing chloroplasts of green algal
2002). Some of them, such as Alveolates and Stramenopiles origin (Burki et al., 2007). The introduction of Strameno-
(Heterokonta), have been established very early based on piles+Alveolates+Rhizaria (SAR) and the growing sup-
ultrastructural and molecular evidence. Ribosomal trees port for this assemblage with an increasing number of
also show evidence for close relationships between animals analysed genes were difficult to reconcile with the Chro-
and fungi and a common origin of green and red algae. malveolate hypothesis.
However, these relationships were not strongly supported. Another negative evidence for Chromalveolates
The most problematic groups were amoeboid protists, hypothesis came from the phylogenomic analyses of
whose 18S rRNA genes evolved at highly variable rates; Haptophytes and Cryptophytes. In early studies, both
lobose amoebae, for example, were scattered across groups were grouped together with other orphan protistan
eukaryotic ribosomal phylogenies, whereas foraminifera lineages, such as Telonemia and Centroheliozoa, in new
were consistently placed at the base of the trees far from putative supergroups called cryptomonads, centrohelids,
other amoeboid protists. telonemids, haptophytes (Burki et al., 2009) or Hacrobia
(Okamoto et al., 2009). However, there was no strong
support for these groupings and further analyses based on
Phylogenomic Adjustments more than 200 genes showed that Cryptophytes, Hapto-
phytes, Centrohelids, Telonemids and some other proti-
The currently accepted structure of the eukaryotic tree stan lineages branch independently in the eukaryotic tree
appeared with the advent of multigene phylogenies. Early (Burki et al., 2012). The Chromalveolate hypothesis was
consensus multigene trees, such as the ones published by finally abandoned and the current structure of the eukar-
Simpson and Roger (2002) and Baldauf (2003), comprise yotic tree comprises 4–7 supergroups and a few micro-
almost all currently recognised supergroups, albeit some of kingdoms, as illustrated in Figure 1.

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 Protist Evolution and Phylogeny

Phylogenomic tree of eukaryotes

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Figure 1 Phylogenomic tree of eukaryotes. Modified after the analysis of 258 genes by Burki et al. (2012) and other recent publications.

Metagenetic Explosion high-throughput sequencing technologies removed these

limitations and we are currently witnessing an explosion of
In parallel to phylogenomic attempts to reinforce the sup- metagenetic data.
port for eukaryotic phylogenies, a particular effort was also Metagenetic studies showed right from the beginning
made to assess the diversity of eukaryotic phyla by meta- that the traditional view of protistan diversity is strongly
genetic studies. These studies consist of sequencing riboso- underestimated (Moreira and Lopez-Garcia, 2002). Most
mal gene fragments obtained by PCR amplification of of the huge diversity of protistan ribotypes revealed by
environmental DNA (eDNA) using more or less specific eDNA surveys could not be assigned to known taxa. As
eukaryotic primers. Early metagenetic studies were based on highlighted by some studies, this astonishing richness
the cloning of ribosomal amplicons, which strongly limited could partly be due to technical artefacts, such as chimera
the number of obtained sequences. The development of (Berney et al., 2004). Yet, a careful examination of eDNA

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 Protist Evolution and Phylogeny

ribotypes shows that many of them represent true eukar- A recent phylogenetic revision divided amoebozoans
yotic lineages that either are new to science or have not into five major groups: Tubulinea, Discosea, Variosea,
been sequenced yet. See also: Microbial Diversity Archamoebea and Mycetozoa (Smirnov et al., 2011). The
The upsurge of protist diversity played a pivotal role in first two groups comprise most of the free-living naked and
phylogenomics. The eukaryotic tree was enriched by hun- testate species, differing in pseudopodial morphology and
dreds of environmental lineages branching often at the type of cytoplasmic flow: monoaxial in Tubulinea and
base of protistan phyla (Bass and Cavalier-Smith, 2004). polyaxial or without a pronounced axis in Discosea.
Some of them (e.g. Rappemonads and Picozoa – formerly Archamoebae include the amitochondrial free-living
Picobiliphytes) could not be assigned to any supergroup pelobionts and parasitic entamoebids, encompassing the
(Not et al., 2007; Kim et al., 2011). Others formed new human parasite Entamoeba histolytica. In some phyloge-
highly diversified picoeukaryotic phyla within known netic trees, the archamoebae are related to Mycetozoa
supergroups, such as marine stramenopiles (MAST) and (slime moulds), which comprises Dictyostelia and Myx-
marine alvolates (MALV). Cryptic diversity was also ogastria; however, there is no support for this grouping
observed at lower taxonomic level, with practically all (Fiore-Donno et al., 2010). The protostelid mycetozoans
protistan species subdivided into a number of genetically have been shown to form a polyphyletic assemblage
distinct phylotypes or operational taxonomic units. (Shadwick et al., 2009). The phylogenetic position of
Variosea, which include flagellate species, and some other
enigmatic amoebozoans, such as Trichosphaerium,
remained unresolved. See also: Amoeba

Characteristics of Major Eukaryotic

Supergroups Opisthokonta
The International Society of Protozoology has recently Metazoa and Fungi dominate this supergroup, which
published a new version of eukaryotic classification (Adl comprises relatively few protistan taxa. These taxa are of
et al., 2012). According to this paper, signed by 25 experts pivotal evolutionary importance for explaining the origin
in protist taxonomy, the eukaryotes are divided into five of multicellularity and therefore have been the subject of
supergroups: Amoebozoa, Opisthokonta, Excavata, SAR many phylogenomic studies. There is strong genomic
and Archaeplastida, plus some independent taxa. Based on evidence for Choanoflagellates being the sister group of
phylogenetic and phylogenomic analyses, Amoebozoa and Metazoa. A recent phylogenomic study (Suga et al., 2013)
Opisthokonta are combined into the assemblage of suggests that Capsaspora owczarzaki, an endosymbiontic
Amorphea, previously referred to as ‘Unikonta’, whereas amoeba of a pulmonate snail, is the closest relative to
the Archaeplastida, SAR, Cryptophyceae, Centrohelida, Metazoa+Choanoflagellates clade. However, genomic
Telonemia and Haptophyta are grouped into the assem- data for other unicellular opisthokonts branching at the
blage of Diaphoretickes. base of Metazoa, especially Ministeria and ichtyosporean
For reasons of clarity, the large assemblages (Amor- fish parasites, are lacking and their phylogenetic position is
phea, Diaphoretickes and SAR) are omitted here. The not well resolved (Steenkamp et al., 2006). Similarly, there
composition of the remaining seven supergroups is pre- is no genomic data for unicellular lineages branching at the
sented on the basis of the latest phylogenetic studies. The base of Fungi, such as Nuclearia, Fonticula and Rozellida,
independent lineages are grouped according to their phy- the last one forming a huge clade composed mainly of
logenetic position and described separately as incertae sedis environmental sequences (Lara et al., 2010).
eukaryotes. The major taxa of each supergroup, including
their catalogued and estimated diversity, are indicated in
Table 1. Archaeplastida
Amoebozoa This supergroup has been introduced for the three main
lineages of primary photosynthetic organisms: glauco-
As indicated by its name, this supergroup is composed phytes, rhodophytes (red algae) and chlorophytes (green
mainly of amoebae. However, only the amoebae bearing algae), the latter forming the group of Chloroplastida
lobopodia, traditionally classified as Lobosea, belong to together with vascular plants. The three groups harbour
this supergroup; filose and reticulose amoebae branch plastids uniquely derived from cyanobacterial endo-
either within Rhizaria or Stramenopiles. In addition to the symbiosis, according to single- or multigenes phylogeny of
lobosean amoebae, Amoebozoa comprise a few flagellates, plastid genes (Rodrı́guez-Ezpeleta et al., 2005). However,
including the uniciliate genus Phalansterium and the mul- phylogenetic support for the Archaeplastida is weak,
ticiliated species Multicilia marina. Another amoebo- especially if trees are based on nuclear genes (Deschamps
flagellate species, Breviata anathema, branches as sister and Moreira, 2009). In particular, the glaucophytes, which
group to Amoebozoa in phylogenomic analyses (Minge are a small group of freshwater algae, have a tendency to
et al., 2009) and is also included in this supergroup. branch separately (Burki et al., 2012).

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 Protist Evolution and Phylogeny

Table 1 Current classification of eukaryotes, following Adl et al. (2012), including major phyla and their representative genera.
Catalogued diversity according to Pawlowski et al. (2012)
Supergroups Major taxa Representative genera Catalogued diversity
Amoebozoa Tubulinea+Arcellinida Amoeba, Arcella 1100
Discosea Vannella, Paramoeba 180
Variosea Phalansterium, Multicilia 20
Mycetozoa Dictyostelium, Physarum 1062
Archamoebae Pelomyxa, Entamoeba 4248
Breviatea Breviata 120.5
Opisthokonta Metazoa Trichoplax, Drosophila 1 200 000
Fungi Chytridium, Saccharomyces 377 200
Choanoflagellida Diaphanoeca, Monosiga 250
Mesomycetozoa Capsaspora, Ministeria 470.5
Archaeplastida Chlorophyta (Streptophyta) Chlamydomonas, Ulva 9000
Streptophyta Chara, Pinus, Rosa 350 000
Rhodophyta Porphyridium, Bangia 5000
Glaucophyta Cyanophora 50.5
Alveolata Ciliophora Paramecium, Tetrahymena 8000
Dinophyceae Alexandrium, Symbiodinium 2280
Apicomplexa Plasmodium, Toxoplasma 6000
Perkinsea Perkinsus ?
Syndiniales Amoebophrya ?
Chromerida Chromera 10.5
Stramenopiles Bacillariophyta Thalassiosira, Nitschia 20 000
Bicosoecida Bicosoeca, Cafeteria 72
Chrysophyceae-Synurophyceae Chromulina, Ochromonas Synura 1200
Dictyochophyceae Dictyocha 15
Eustigmatales Vischeria 15
Hyphochytriales Hyphochytridium 25
Labyrinthulomycetes Labyrinthula, Amphitrema 40
Oomycetes Saprolegnia, Phytophthora 676
Opalinata Opalina 400
Pelagophyceae Aureococcus 12
Phaeophyceae Fucus, Laminaria 1750
Phaeothamniophyceae Phaeothamnion 25
Pinguiophyceae Pinguiochrysis 5
Raphidophyceae Goniostomum 20
Xanthophyceae Botrydium, Vaucheria 6000.5
Rhizaria Cercozoa Cercomonas, Euglypha 600
Foraminifera Ammonia, Allogromia 12 000
Acantharea Acanthometra 160
Polycystinea Collozoum, Saturnalis 850
Taxopodida Sticholonche 1
Ascetosporea Bonamia, Marteilia 43
Phytomyxea Plasmodiophora 41
Vampyrellida Arachnula, Vampyrella ?
Gromida Gromia 90.5
Excavata Euglenozoa Euglena, Trypanosoma 1520
Fornicata Giardia, Hexamita 146
Heterolobosea Naegleria, Vahlkampfia 80
Jakobida Jakoba, Andalucia 10
Malawimonadidae Malawimonas 3
Parabasalia Trichomonas, Trichonympha 4660.5
Incertaea sedis Apusomonadidae Apusomonas, Ancyromonas 12
Centrohelida Raphidiophrys 150
Cryptophyta Cryptomonas, Rhodomonas 70
(continued )

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 Protist Evolution and Phylogeny

Table 1 Continued
Supergroups Major taxa Representative genera Catalogued diversity
Haptophyta Emiliania, Coccolithus 350
Katablepharidophyta Roombia 9
Picozoa Picomonas 1
Telonemia Telonema 2

Alveolata metagenetic studies (Logares et al., 2012). See also:

Diatoms
This supergroup is composed of three major phyla: Cili-
ates, Dinoflagellates and Apicomplexa. Ciliates are mainly
free-living protists, characterised by the presence of Rhizaria
abundant cilia on their body surface, nuclear dualism and a Contrary to others, the definition of this supergroup is
conjugation stage during the sexual phase of the life cycle. based exclusively on molecular data (Cavalier-Smith,
They are one of the few classical protistan taxa, whose 2002). Its monophyly is well supported by multigene phy-
monophyly has never been questioned by molecular stu- logenies (Nikolaev et al., 2004; Burki et al., 2010) and by
dies. There is also good evidence for the monophyly of some molecular signatures, such as an amino acid insertion
dinoflagellates, characterised by two flagella in the motile in the polyubiquitin gene (Bass et al., 2005). Although any
stage, although this group is much more heterogeneous. ultrastructural characters that would unite the main rhi-
Some dinoflagellates are phototrophes, whereas others are zarian taxa are not known, most of the organisms included
parasitic (e.g. Alexandrium) or endosymbiotic (e.g. Sym- here possess filose or reticulose pseudopodia, the feature
biodinium). Closely related to dinoflagellates are Apicom- after which the supergroup is named. At present, the Rhi-
plexa, including among others the malarian parasite zaria are composed of two major assemblages (Cercozoa
Plasmodium, a scourge of humanity. All apicomplexans are and Retaria) and a number of independent lineages. The
obligate parasites characterised by the presence of an apical Cercozoa form a diverse clade composed of biciliated
complex that helps with the attachment and initial pene- heterotrophic taxa, such as cercomonads, the phototrophic
tration of the host. In addition to these three major groups, chlorarachniophytes with reticulate pseudopodia, and the
the Alveolates comprise some independent lineages, such euglyphid filose amoebae with silicate outer shells (Cava-
as the parasitic Syndiniales, whose huge diversity was lier-Smith and Chao, 2003). They also comprise taxa that
revealed by metagenetic studies; the Perkinsinidae, which have silica endoskeleton, such as ebriids and phaodarians,
are well-known parasites of shellfish; and the phototrophic the latter one traditionally classified with Radiolaria (Polet
Chromeridae, recently isolated from corals. See also: et al. (2004)). The Retaria is a monophyletic group that
Ciliophora; Gymnodinium and Related Dinoflagellates comprises two of the most important microfossil groups:
Foraminifera and Polycystinea. It also includes the radi-
olarian class Acantharea and the enigmatic Taxopodida,
Stramenopiles represented by a single formally described species Sticho-
lonche zanclea and possibly many more species revealed by
This supergroup is possibly the largest and most diverse metagenetic studies. The characteristic feature of Retaria is
group of eukaryotes and is also often referred to as a particular type of b-tubulin, which may be responsible for
Heterokonta. It is characterised by the presence of typically the unusually active pseudopodial network and the extra-
two morphologically different cilia, one of them bearing ordinary diversity of foraminiferal and radiolarian skele-
two rows of tripartite hair (mastigonemes). Like Alveo- tons. In phylogenetic trees of Rhizaria, Retaria and
lates, the Stramenopiles have been introduced at the dawn Cercozoa are separated by some independent taxa,
of molecular phylogenetic studies. They are composed of including marine filose Gromiida, amoeboid Vampyr-
several algal groups containing plastids resulting from ellida, the plant parasites Phytomyxea and Ascetosporean
secondary endosymbiosis, such as diatoms (Bacillar- parasites of shellfish and other invertebrates. These inde-
iophyta), brown algae (Phaeophyceae) and golden algae pendently branching groups are often combined in the
(Chrysophyceae). The Stramenopiles also comprise a wide assemblage Endomyxa (Bass et al., 2005), but phyloge-
range of heterotrophic groups, including parasitic Oomy- nomic analyses do not confirm its monophyly (Sierra et al.,
cetes (water moulds and downy mildews, previously clas- 2013).
sified as Fungi) and endocommensal opalinids as well
as free-living bicosoecids, amoeboid labyrinthulids, am- Excavata
phitremids and actinophryid heliozoans. Moreover, this
supergroup also includes a highly diversified assemblage of Most organisms belonging to this supergroup possess a
marine uncultured picoeukaryotes (MAST), revealed by conspicuous ‘excavated’ ventral feeding groove. In spite of

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 Protist Evolution and Phylogeny

this apparent synapomorphy, the monophyly of Excavates particular group of heliozoans with typical axopodia,
is not well supported even in multigenic trees (Hampl et al., whereas Telonema is a marine heterotrophic flagellate
2009). The Excavates comprise several groups of ami- genus with two flagella and very specific ultrastructural
tochondriate parasites, including diplomonads, retorta- features. Rappemonads are marine picoeukaryotes,
monads, oxymonads and parabasalids; some of them known only from environmental DNA sequences (Kim
possess hydrogenosomes or mitosomes instead of mito- et al., 2011).
chondria. Together with a few free-living taxa (e.g. Car-
pediemonas) they are classified into the group
Metamonada. Another large monophyletic assemblage of
Excavates is formed by Discoba. This grouping contains
free-living Jakobida, amoeboflagellate Heterolobosea and
Future Challenges
Euglenozoa that comprises Euglenida (some are photo-
Although the basal scaffold of eukaryotic phylogeny seems
trophic), heterotrophic Diplonemea and parasitic Kine-
to be solidified by now, there are still a lot of unresolved
toplastea (including the important human parasites
issues concerning the phylogenetic position of incertae
Trypanosoma and Leishmania). In addition, the Excavates
sedis microkingdoms. In many cases, phylogenetic rela-
also comprise a very unusual free-living genus Malawi-
tionships within the supergroups are not resolved either.
monas that usually branches separately from Metamonada
Compared with metazoans or plants, the number of
and Discoba in molecular phylogenies (Simpson et al.,
sequenced protistan genomes is relatively small, for
2006).
example, genomic data are available only for one species of
Rhizaria (Curtis et al., 2012). Furthermore, transcriptomic
Incertae sedis eukaryotes data are scarce or not available for many groups. Never-
theless, given the extraordinary progress of sequencing
Phylogenomic analyses reveal 11 orphan lineages that
technologies, we can expect that these limitations will be
cannot be confidently placed in any of the existing super-
rapidly overcome and the number of sequenced nonmodel
groups. As shown in Figure 1, these taxa can be grouped into
organisms will increase substantially.
three paraphyletic assemblages. One of these assemblages
A more difficult issue related to eukaryotic phylogeny
is composed of four lineages (Apusomonadidae, Ancyr-
might be the positioning of the root. This question has been
omonadidae, Collodictyonidae and Rigidifilida) that
recently addressed by analysing mitochondrial genes, but
branch close to Amoebozoa (Zhao et al., 2012). All of them
there is no unambiguous solution (Derelle and Lang,
are heterotrophic free-living protists with different
2012). Two major hypotheses have been formulated: One
morphologies. The Apusomonadidae and Ancyr-
proposes that the root is situated between Amorphea
omonadidae feature gliding cells with two unequal cilia and
(Amoebozoa+Opisthokontes) and other eukaryotes
short pseudopodia; Collodictyonidae have two or four
(Stechmann and Cavalier-Smith, 2003), the other points to
apical cilia; whereas Rigidifilida have branching pseudo-
the Excavates as the most basal group (Cavalier-Smith,
podia arising from a ventral depression (Yabuki et al.,
2010).
2013).
Further phylogenomic studies of known genera may
The second assemblage of incertae sedis protists includes
help in resolving this question, but we cannot exclude that
Cryptophyta, Katablepharidae and Picozoa. The first two
we are still missing some pivotal lineages. Therefore, it is
lineages group together, whereas the Picozoa branch close
very important to increase the effort of characterising new
to Glaucophyta. Cryptophytes are autotrophes with
protistan lineages. As shown by the recent study of Pico-
chloroplasts and residual nucleomorphs that issue from
zoa, a cultivation approach remains the most efficient way
red algal secondary symbiosis – Katablepharids are free-
to accurately describe hitherto unknown organisms (See-
swimming heterotrophs with two cilia inserted subapically
nivasan et al., 2013). However, this traditional approach is
(Okamoto et al., 2009). Picozoa, formerly known as Pico-
extremely time consuming and may not be appropriate for
biliphyta, are represented by many environmental
many uncultivable protists. More rapid single-cell micro-
sequences. Only recently the first picobiliphytes (Picomo-
scopic and genomic approaches are now available (Yoon
nas judraskeda) was successfully cultivated and formally
et al., 2011) and future studies will probably use these new
described; surprisingly, its detailed study showed no evi-
tools for exploring the extraordinarily diverse protistan
dence of autotrophy (Seenivasan et al., 2013). See also:
phyla.
Cryptomonads
The third assemblage branching close to SAR is com-
posed of Haptophyta, Centrohelida, Telonemia and Rap-
pemonads. The largest of these groups are Haptophytes,
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