The twilight of Sarcodina: a molecular perspective on the polyphyletic origin of amoeboid protists Текст научной статьи по специальности «Биологические науки»

The twilight of Sarcodina: a molecular perspective on the polyphyletic origin of amoeboid protists 1

Jan Pawlowski

Department of Zoology and Animal Biology, University of Geneva

“All known eukaryotes are the sarcodines” (Kirill A. Mikrjukov)

Summary

For convenience, the traditional classifications placed all amoeboid protists in the group Sarcodina, subdivided based on the type of pseudopodia into Rhizopoda and Actinopoda. Early molecular phylogenies provided evidence for the polyphyly of Sarcodina, but the relationships between different amoeboid taxa remained largely unresolved. It was only recently that a more congruent view of the phylogeny of amoeboid protists emerged from multigene analyses. According to this view, the majority of amoeboid protists are placed in the supergroups Amoebozoa and Rhizaria. Amoebozoa include almost all lobose amoebae and myce-tozoans, while Rhizaria comprise the majority of protists bearing filopodia, reticulopodia and axopodia. Both supergroups also contain a variety of free-living flagellates and some parasitic lineages. The few amoeboid protists that did not find their place within these two supergroups have been placed by molecular phylogenies either among excavates (Heterolobosea), opisthokonts (Nuclearia), and stramenopiles (Actinophryida, some Filosea), or as independent lineages (Centrohelida, Breviata). Although the molecular data clearly indicate that the pseu-dopodial structures have been developed several times in the history of eukaryotes, they also show that a large diversification of amoeboid protists occurred only twice, in lineages leading to Amoebozoa and Rhizaria. The evolution of different types of pseudopodia within these supergroups constitutes a new challenge for future phylogenomic studies of amoeboid protists.

Introduction

Reconstructing the tree of life is one of the main objectives of evolutionary biology since Ernst Haeckel’s legendary “Staumbaum des Lebens” (1866). Yet, neither Haeckel nor his direct successors could produce an accurate representation of the tree of life, because of limited access to the phylogenetic characters shared by all living organisms available at that epoch. The reconstruction of the evolution-

ary history of protists constituted a particularly difficult case. For years the macrosystem of protists was based on gross morphological and life cycle features. The application of electron microscopy to study the ultrastructure of the protistan cell provided a new set of characters that could resolve some particular taxonomic problems but offered no resolution of the global phylogeny of eukaryotes (Patterson, 1994). For more than a century the higher ranks in classification of protists were based on convenience (Corliss, 1984)

1 Materials presented on the V European Congress of Protistology (July 23-27, 2007, St. Petersburg, Russia).

© 2008 by Russia, Protistology

or “matter of taste” (Margulis and Schwartz, 1988) rather than on testable phylogenetic hypotheses.

This situation has radically changed with the advent of molecular phylogenies, which offer the possibility to compare homologous DNA sequences across the whole living world. Early phylogenetic reconstructions based on ribosomal DNA sequences have totally modified the systematics of eukaryotes (Cavalier-Smith, 1993, 1998). However, after an enthusiastic period of the pioneering molecular studies, it became obvious that the first ribosomal phy-logenies of eukaryotes were strongly biased by long branch attraction artefacts (Philippe and Adoutte, 1998; Philippe and Germot, 2000). New methods were developed to correct these biases (Philippe et al., 2000) and the single gene phylogenies were progressively replaced by multigene and phylogenomic approaches (Delsuc et al., 2005). Based on these approaches, novel phylogenetic groupings have emerged (Cavalier-Smith, 2002; Baldauf, 2003; Simpson and Roger, 2004; Keeling et al., 2005; Burki et al., 2007), providing the basis for a new macrosystem of eukaryotes (Adl et al., 2005).

Placement of amoeboid protists in this emerging macrosystem has been considerably delayed mainly due to the lack of sequence data for most amoeboid taxa and biased analyses of existing data (Philippe and Adoutte, 1998; Pawlowski et al., 1996; Amaral-Zettler et al., 1997). Amoeboid protists were globally underrepresented in early molecular trees, particularly in multigene phylogenies (Baldauf et al., 2000). Their molecular database began to grow rapidly only recently (Amaral-Zettler et al., 2000; Bolivar et al., 2001; Fahrni et al., 2003; Cavalier-Smith and Chao 2003; Nikolaev et al., 2004, Kudryavstev et al., 2005; Fiore-Donno et al., 2007; Brown et al., 2007; Tekle et al., 2007, and many others). Although in many cases these data are still limited to SSU rDNA sequences, their analyses combined with studies of a few available protein coding genes revealed new phylogenetic relationships among amoeboid protists and prompted their grouping in two new supergroups of eukaryotes: Amoebozoa and Rhizaria. This paper presents a brief review of molecular studies that led to the formation of these new supergroups, discussing the current position of all taxa traditionally included in Sarcodina.

The concept of Sarcodina

In traditional, morphology-based classifications, the protists were subdivided into four major types: amoebae, ciliates, flagellates and sporozoa. This system, founded by Bütschli (1880), remained in use,

with some modifications, for more than a century (Levine et al., 1980; Lee et al., 1985). Its different variants consequently included the amoeboid protists in the class or phylum Sarcodina (Schmarda, 1871). Although the term “sarcode” was originally applied by Dujardin (1835) to designate the material forming the protoplasm of all protists, it soon came to designate mainly the protoplasm of amoeboid protists. According to the classical definition, the Sarcodina included all protists possessing pseudopodia or locomotive protoplasmic flow, with flagella usually restricted to developmental or other temporary stages (Levine et al., 1980).

Depending on the type of pseudopodia, the Sarcodina were further subdivided into the Rhizopodea von Siebold 1845 and Actinopodea Calkins 1901 (Levine et al., 1980). The superclass Rhizopodea comprised mainly protists having lobopodia, filo-podia and reticulopodia, and included eight classes: Lobosea, Acarpomyxea, Acrasea, Eumycetozoea, Plasmodiophorea, Filosea, Granuloreticulosea and Xenophyophorea. The superclass Actinopodea was composed of all axopodia-bearing protists and included four classes: Acantharea, Polycystinea, Phaeodarea and Heliozoea (Table 1). This dichotomy of Sarcodina, introduced by Calkins (1901) and postulated already by Haeckel (1894), was not followed by all taxonomists (Jepps, 1956); nevertheless, the taxonomic composition of Sarcodina varied little from one classification to the other (Kudo, 1954; Honigberg et al., 1964; Levine et al., 1980).

The concept of Sarcodina and its classification was widely accepted, firstly because it was extremely convenient, and secondly because there were no data available to construct an alternative system. The inadequacy of the traditional system was strongly criticized based on results of ultrastructural studies (Patterson, 1994). However, the distinction of the major lineages of eukaryotes based on common patterns of cell organization was of little help in resolving the relations among amoeboid protists (Patterson, 1999). It is therefore not surprising that the amoeboid groups were classified in alphabetic order (Lee et al., 2000), with some classical amoeboid macrotaxa remaining in use, albeit always with clear indication of their polyphyletic character (Hausmann et al., 2003).

Early eukaryotic phylogenies

The first molecular phylogenies, mainly based on the SSU (18S) rDNA sequences, unfortunately brought more confusion than resolution to the phy-logeny of amoeboid protists. Only very few lobose amoebae were sequenced, displaying a patchy dis-

Table 1. Classification of amoeboid protists: comparison oftraditional (Levine et al., 1980) and modern (Adl et al., 2005) versions

Levine et al. 1980 Adl et al. 2005

Subphylum Sarcodina AMOEBOZOA

Superclass Rhizopodea • Tubulinea

Class Lobosea • Flabellinea

Subclass Gymnamoebia • Stereomyxida

Order Amoebida • Acanthamoebidae

Order Schizopyrenida • Entamoebida

Order Pelobiontida • Mastigamoebidae

Subclass Testacealobosia • Pelomyxa

Order Arcellinida • Eu mycetozoa

Order Trichosida

Class Acarpomyxea RHIZARIA

Order Stereomyxida • Cercozoa

Order Leptomyxida • Haplosporidia

Class Acrasea • Foraminifera

Class Eumycetozoea • Gromia

Subclass Protosteliia • Radiolaria

Subclass Dictyosteliia

Subclass Myxogastria OPISTHOKONTA

Class Plasmodiophorea • Nucleariida

Class Filosea

Order Achonchulinida CHROMALVEOLATA

Order Gromiida • Actinophryidae (Actinophrys)

Class Granuloreticulosea

Order Athalamida EXCAVATA

Order Monothalamida* • Heterolobosea

Order Foraminiferida

Class Xenophyophorea Incertae sedis EUKARYOTA

Superclass Actinopodea • Centrohelida

Class Acantharea

Class Polycystinea

Class Phaeodarea

Class Heliozoea

Order Actinophryida

Order Desmothoracida

Order Taxopodida

Order Centrohelida

tribution across the phylogenetic tree, strongly sug- Filosea, Granuloreticulosea and Actinopoda) re-

gesting a polyphyletic origin (Clark and Cross, 1988; mained unclear for a much longer time. The first

Sogin, 1991; Hinkle et al., 1994). The basal position analyses of filosean SSU rDNA showed that the eu-

of pelobionts and entamoebids led to their placement glyphid testate amoebae and the photosynthetic

in a separate phylum, Archamoebae, postulated to chlorarachniophytes with reticulate pseudopodia

be early branching amitochondriate eukarytotes formed a clade (Bhattacharya et al., 1995). This clade

(Cavalier-Smith, 1991). When it was demonstrat- was later enlarged to include also the plasmodio-

ed that the entamoebids lost their mitochondria phorids and some free-living flagellates (Cavalier-

secondarily (Clark and Roger, 1995), the phylum Smith and Chao, 1996/1997), leading to the creation

Archamoebae was abandoned and almost all lobose of the phylum Cercozoa (Cavalier-Smith, 1998).

amoebae, with exception of the Heterolobosea, were On the other hand, early phylogenetic analyses of

placed in the phylum Amoebozoa (Cavalier-Smith, Foraminifera (Pawlowski et al., 1994, 1996) and

1998). Radiolaria (Amaral-Zettler et al., 1997) showed that

The position of the remaining Sarcodina (i.e. these groups branched separately in the SSU trees,

suggesting that they may represent independent phyla (Cavalier-Smith, 1998). It was only after the publication of actin-based evidence for the possible relationships between Cercozoa and Foraminifera (Keeling, 2001) that the infrakingdom Rhizaria was proposed (Cavalier-Smith, 2002).

There are several reasons explaining why it was so difficult to find the correct phylogenetic position for most amoeboid protists in early eukaryotic phy-logenies. Firstly, as mentioned above, the early ribo-somal trees suffered from a general lack of molecular data representing amoeboid taxa. Secondly, many amoeboid protists show an extreme heterogeneity of substitution rates in ribosomal genes, often leading to a misinterpretation of their phylogenetic position. The exemplary case is the spectacular acceleration of the stem lineage leading to foraminifera and the extreme variations of rDNA rates between planktonic and benthic foraminiferal groups (Pawlowski et al., 1996; Pawlowski et al., 1997; Pawlowski and Berney,

2003). Unusually rapid evolutionary rates are also observed in Polycystinea (Amaral-Zettler et al., 1997), as well as in most pelobionts, entamoebids and my-cetozoans (Bolivar et al., 2001; Edgcomb et al., 2002). Their fast evolving rRNA genes are often much longer than the usual (Hinkle et al., 1994; Pawlowski et al., 1996; Milyutina et al., 2001) and contain lineage-specific insertions (Habura et al., 2004), long introns (Lundblad et al., 2004) or additional variable regions that further complicate their accurate phylogenetic placement.

The problem of rate heterogeneity of ribosomal genes was partially resolved with the introduction of new evolutionary models correcting for among-site heterogeneity and invariable sites (GTR + G + I), as well as by progressive replacement of distance and parsimony methods by probabilistic approaches that are much less sensitive to rate variations (Lewis,

2001). However, a veritable revolution in the global view of eukaryotic phylogeny was brought about by the use of multigene dataset that permitted challenging the phylogenetic hypotheses based solely on the SSU sequences (Baldauf et al., 2000).

Phylogenomics and a new classification of eukaryotes

The first large-scale analysis of 123 genes from EST libraries of Dictyostelium, Mastigamoeba and Entamoeba provided well supported evidence for the monophyly of the Conosa (Bapteste et al., 2002). Monophyly of Amoebozoa and Rhizaria was later suggested by combined SSU and actin sequence data (Fahrni et al., 2003; Nikolaev et al., 2004). Several

multigene studies demonstrated a relationship between Amoebozoa and Opisthokonts, unifying them into the super-assemblage of Unikonts (Cavalier-Smith, 2002; Keeling et al., 2005). On the other hand, the analysis of 85 genes from the chlorarachniophyte Bigellowiella and the foraminiferan Reticulomyxa confirmed the monophyly of Rhizaria (Burki and Pawlowski, 2006). The analysis of 16 genes indicated a relationship between Rhizaria and Stramenopiles (Hackett et al., 2007). Further analyses of larger sampling of taxa and genes confirmed the close relationship of Stramenopiles, Alveolates and Rhizaria, suggesting the existence of a new super-assemblage of eukaryotes (SAR), which comprises the largest diversity of protists (Burki et al., 2007).

A new view of global phylogeny of eukaryotes is emerging based on the rapidly growing genomic database (Embley and Martin, 2006). According to this view, the majority of eukaryotes are distributed between several major divisions, called supergroups or “kingdoms”. The number of these supergroups is progressively decreasing, from eight (Baldauf, 2000) to six (Simpson and Roger, 2004), five (Keeling et al., 2005) and four (Burki et al., 2007). Six of these supergroups form the backbone for a new classification of eukaryotes (Adl et al., 2005). The majority of amoeboid protists is placed in the supergroups of Amoebozoa and Rhizaria (Table 1). Only five amoeboid lineages branch outside these supergroups, either within excavates (Heterolobosea), stramenopiles (Actinophryida), opisthokonts (Nucleariidae), or independently (Centrohelida, Breviatea). The composition and phylogeny of the two amoeboid supergroups as well as the phylogenetic position of the remaining amoeboid taxa are discussed below.

Amoebozoa

The taxon Amoebozoa (Lühe, 1913) was emended by Cavalier-Smith (1998) to unify in a single taxonomic group the naked and testate lobose amoebae, pelobionts, entamoebids and mycetozoans as well as some flagellates (Multicilia). In Cavalier-Smith’s (1998) classification, the Amebozoa had the rank of a phylum subdivided into the subphyla Lobosa and Conosa. The subphylum Lobosa included all protists traditionally classified in the class Lobosea (Page, 1987). The subphylum Conosa included the Archamoebae, previously classified among the Archeozoa (Cavalier-Smith, 1983), and Mycetozoa, shown to be related to lobose amoebae, by analysis of actin and actin-related proteins (Kelleher et al., 1995; Bhattacharya and Weber, 1997) as well as by the fusion between cox1 and cox2 genes and the similar

order of ribosomal protein genes in the mitochondrial genomes of Acanthamoeba and Dictyostelium (Iwamoto et al., 1998; Gray et al., 1999).

The first molecular evidence for the mono-phyly of Amoebozoa was presented independently by Bolivar et al. (2001) and Milyutina et al. (2001). The monophyletic grouping of gymnamoebae, my-cetozoans, entamoebids, and pelobionts in the SSU rDNA trees was later confirmed by analyses of actin and SSU rDNA with a much larger taxon sampling of loboseans (Fahrni et al., 2003). In the meantime the monophyly of Dictyostelium discoideum and two amitochondriate amoebae (Entamoeba histolytica and Mastigamoeba balamuthi) was inferred from combined analysis of the SSU rDNA and translation elongation factors (EF-1alpha and EF-2) sequences (Arisue et al., 2002), as well as the analysis of 123 genes from EST libraries of these species (Bapteste et al., 2002). Later, the close relationship between Dictyostelium and Entamoeba was demonstrated by comparison of their completely sequenced genomes (Song et al., 2005). Simultaneously, the monophyly of Amoebozoa was suggested by myosin II phylogeny (Richards and Cavalier-Smith, 2005).

The composition of the Amoebozoa has hardly changed since its emendation (Cavalier-Smith, 1998). The only significant modification was to include the uniciliate zooflagellate Phalansterium soli-tarium of unclear phylogenetic position (Cavalier-Smith et al., 2004). The analyses of SSU rDNA sequences obtained from Arcellinida (Nikolaev et al., 2005), Cochliopodium (Kudryavtsev et al., 2005) and Multicilia (Nikolaev et al., 2006) confirmed their placement among Amoebozoa. Very recently, the amoebozoan origin has also been confirmed for the genus Trichosphaerium (Pawlowski and Fahrni, 2007; Tekle et al., 2008), representing the last unsequenced order of Lobosea. On the other hand, the recent multigene analysis of Corallomyxa tenera sp. n. (Tekle et al., 2007) refuted its placement within the Amoebozoa, suggesting that the order Stereomyxida, which traditionally included Corallomyxa and Stereomyxa (Levine et al., 1980), may belong to the supergroup Rhizaria instead of Amoebozoa.

While it is reasonable to think that the composition of Amoebozoa is now well established, the relationships within this supergroup still comprise many unresolved or controversial points (Fig. 1). The initial division of Amoebozoa into Lobosa and Conosa (Cavalier-Smith 1998) can be retained only if the root is placed between these two groups, as has been proposed by Nikolaev et al. (2006) and if Conosa is emended to include some loboseans, such as Filamoeba, Acramoeba, and Multicilia. Such

rooting is interesting from the point of view of the evolution of flagellated amoebae because it leads to a considerable reduction in the number of flagella losses by separating most aflagellate lobose amoebae from ancestrally flagellated Conosa. However, most analyses using various outgroups place the root between Tubulinea and other amoebozoans (Fahrni et al., 2003; Cavalier-Smith et al., 2004; Nikolaev et al., 2005; Smirnov et al., 2005). Such rooting would make Lobosa paraphyletic, which was used as an argument to abandon this taxon in recent classifications (Smirnov et al., 2005).

The majority of taxa traditionally included in the Lobosa are now grouped into two classes: Tubulinea and Flabellinea (Smirnov et al., 2005). The Tubulinea represent a strongly supported clade grouping the orders Tubulinida (Amoebidae and Hartmanellidae), Leptomyxida, Arcellinida, and some species of unknown affinities (Echinamoeba spp. and Hartmannella vermiformis). The synapo-morphies for this clade are tubular pseudopodia and monoaxial cytoplasmic flow (Smirnov et al., 2005). The Flabellinea, defined by the flattened shape of the cells and polyaxial cytoplasmic flow or cytoplasmic flow without pronounced axis (Smirnov et al., 2005), include Vannellida and Dactylopodida and correspond to the order Glycostylida (Cavalier-Smith et al., 2004). This clade, revealed by Peglar et al. (2003), is almost always recovered by molecular phylogenies, albeit with weaker support than Tubulinea.

Among other monophyletic groupings within Amoebozoa, the clade Acanthamoeba + Balamuthia, characterized by a trilaminated cytoplasmic MTOC, consistently appeared in all molecular phylogenies since Stothard et al. (1998). There is also a relatively good support for the Archamoebae, the taxon composed of amitochondriate amoebae, including Pelomyxa, entamoebids and mastigamoebids (Ca-valier-Smith, 1998). Weak evidence for a sister group relation between entamoebids and mastigamoebids shown in the SSU rDNA analyses (Silberman et al., 1999; Bolivar et al., 2001; Milyutina et al., 2001, and others) was later reinforced by multigene analyses (Arisue et al., 2002; Bapteste et al., 2002).

The Eumycetozoa are another possible mono-phyletic clade, grouping Dictyostelia + Myxogastria and some Protostelia. The monophyly of this group, represented by Dictyostelium, Physarum and Planoprotostelium, is strongly supported in the EF1A phylogenies (Baldauf and Doolittle, 1997; Arisue et al., 2002). However, because the EF1A trees usually include very few other amoebozoans, these analyses are of weak relevance for the question of mycetozo-an monophyly. In the SSU phylogenies, the dictyo-

Fig. 1. Hypothetical consensus tree of Amoebozoa. Two possible positions of the root are indicated by arrows.

stelids and myxogastrids branch either separately or in a weakly supported clade (Silberman et al., 1999; Bolivar et al., 2001). This can be explained by the extremely divergent SSU rDNA sequence of P. poly-cephalum as well as by its strong nucleotide composition bias (Baldauf and Doolittle, 1997). Addition of new sequences of myxogastrids breaking the long stem branch allows the recovery of the monophy-ly of Eumycetozoa in some (Nikolaev et al., 2006) but not all (Tekle et al., 2008) SSU analyses. Recent

SSU and EF1A phylogenies show that the clade of Eumycetozoa also includes some Protostelida (Ceratiomyxa), but most of them branch separately within Conosea (Brown et al., 2007; Fiore-Donno et al., subm.).

A hypothetical consensus tree of Amoebozoa based on the currently available molecular data is illustrated in Fig 1. The tree is presented with a basal dichotomy as suggested by Nikolaev et al. (2006) in unrooted form with possible positions of the root in-

dicated by an arrow. Five well-supported clades in the published molecular trees, as well as the Thecamoebida clade - thecamoebids s.s. (Thecamoeba, Sappinia, Stenamoeba) and Dermamoeba suggested by unpublished analysis of myosin II sequences (Berney, pers. comm.), are represented by triangles. Other taxa that could not be confidently placed in any of these clades are represented by separate branches; the environmental sequences of uncultured amoebozoans are not included. The clade of Conosea, grouping Archamoebae, Mycetozoa and several independently branching lineages (Smirnov et al., 2005; Tekle et al., 2008; Fiore-Donno et al., subm.), is indicated. However, the support for this clade is not very strong and the position of some genera (Phalansterium) varies depending on the type of analyses.

The main weakness of the present amoebozoan phylogeny is its almost exclusively single-gene (SSU rDNA) character. Actin is the only protein gene available for larger taxon sampling of Amoebozoa (Fahrni et al., 2003; Nikolaev et al., 2005, 2006; Tekle et al., 2008). However, the phylogenetic information of this relatively conserved gene is of limited value for inferring intra-group relationships (Fahrni et al.,

2003). The work in progress on myosin II provides a promising insight into their phylogeny (Berney and Cavalier-Smith, 2007; Berney, pers. comm.,). Among other molecular markers, the EF1A was extensively used in the study of Mycetozoa (Baldauf and Doolittle, 1997; Fiore-Donno et al., 2005; Schaap et al., 2006; Fiore-Donno et al., subm.), but there are almost no EF1A sequences for other amoebozoans. The EST and genome projects have focused mainly on Conosea (Entamoeba, Mastigamoeba, Dictyostelium, Physarum), whereas only two species of lobose amoebae Acanthamoeba castellanii and H. vermiformis were analyzed (Table 2).

Rhizaria

The term Rhizaria was introduced by Cavalier-Smith (2002) to define a group of protists characterized by “commonly root-like reticulose or filose pseudopodia”. The group was given a taxonomic status of infrakingdom and included four phyla: Apusozoa, Cercozoa, Retaria and Heliozoa (Centrohelida). The diagnosis of this infrakingdom proposes that the members of the Rhizaria are “ancestrally and typically bikonts with tubular mitochondrial cristae, the centrioles ancestrally with a single root of a microtubular band or fan, and the extrusomes are generally in the form of kinetocysts”. The initial description of Rhizaria was rather intuitive and based on actin evidence for relationship between Cercozoa and Foraminifera (Keeling, 2001), as well as the weakly supported branching of included phyla in some SSU-based trees. In fact, the Apusozoa as well as the Heliozoa (Centrohelida) were later removed from Rhizaria, and in the following classification, the Rhizaria comprises only two phyla: Cercozoa and Retaria, composed of the subphyla Filosa + Endomyxa, and Radiozoa + Foraminifera, respectively (Cavalier-Smith, 2003). The Apusozoa were considered as incertae sedis of the subkingdom Biciliata, while the Heliozoa (Centrohelida) were included in the kingdom Chromista (Cavalier-Smith,

2003).

Rhizaria were shown for the first time to be a strongly supported supergroup in combined analysis of SSU and actin genes, which included the representatives of all main groups of “Actinopoda” (Nikolaev et al., 2004). The authors of this study provided a multigene evidence for the polyphyly of “Heliozoa”, suggested previously by ultrastructural studies (Smith and Patterson, 1986; Mikryukov et al., 2000)

Table 2. Currently available molecular data for Amoebozoa and Rhizaria

Group Genera (species) Genes References

AMOEBOZOA

Tubulinida Amoeba spp. SSU, actin Bolivar et al. 2001, Fahrni et al. 2003

Chaos spp. SSU, actin Bolivar et al. 2001, Fahrni et al. 2003

Hartmannella cantabrigiensis SSU, actin Fahrni et al. 2003

Hartmannella abertawensis SSU Kuiper et al. 2006

Glaeseria mira SSU, actin Fahrni et al. 2003

Nolandella spp. SSU, actin Tekle et al. 2008

Saccamoeba limax SSU Amaral Zettler et al. 2000

Leptomyxida Leptomyxa reticulata SSU Amaral Zettler et al. 2000

Rhizamoeba saxonica SSU Smirnov et al. 2007a

other Leptomyxida SSU Amaral Zettler et al. 2000, Tekle et al. 2008

Arcellinida Arcella spp. SSU Nikolaev et al. 2005, Tekle et al. 2008

Heleopera sphagni SSU, actin Nikolaev et al. 2005, Lara et al. 2008

other Arcellinida SSU Nikolaev et al. 2005, Lara et al. 2008

Dactylopodida Korotnevella sp. SSU Peglar et al. 2003

Vexillifera spp. SSU Fahrni et al. 2003, Peglar et al. 2003

Neoparamoeba spp. SSU Dykova et al. 2005b, Moran et al. 2007, Wong

Pseudoparamoeba pagei SSU et al. 2004 Wong et al. 2004

Vannellida Vannella spp. SSU, actin SSU Fahrni et al. 2003 Dykova et al.2005a, Smirnov et al. 2007b,

Clydonella sp. SSU Moran et al. 2007 Peglar et al. 2003

Lingulamoeba leei SSU Peglar et al. 2003

Pessonella sp. SSU, actin Tekle et al. 2008

Thecamoebida Thecamoeba similis SSU, actin Fahrni et al. 2003

Thecamoeba quadrilineata SSU Michel et al. 2006

Dermamoeba algensis SSU, actin Fahrni et al. 2003

Sappinia spp. SSU Michel et al. 2006, Brown et al. 2007

Stenamoeba ("Platyamoeba") SSU Fahrni et al. 2003, Smirnov et al. 2007b

stenopodia

Acanthamoebida Acanthamoeba castellanii genome mt genome in progress Burger et al. 1995

Acanthamoeba spp. SSU Stothard et al. 1998 and others

Protacanthamoeba SSU Dykova et al. 2005d

Balamuthia mandrillaris SSU Stothard et al. 1998

Entamoebida Entamoeba histolytica genome Loftus et al. 2005

other Entamoeba (4 sp) genome in progress

Mastigamoebida Mastigamoeba balamuthi SSU + EST EF1, EF2 Hinkle et al. 1994, Bapteste et al. 2002 Arisue et al. 2002

Mastigella SSU Edgcomb et al. 2002

Endolimax SSU Silbermann et al. 1999

Pelobiontida Pelomyxa SSU, actin Fahrni et al. 2003

Protostelia Planoprotostelium SSU, EF1 Baldauf & Doolittle 1997, Brown et al. 2007

Ceriatomyxa SSU, EF1 Fiore-Donno et al. (subm)

other Protostelia SSU, EF1 Fiore-Donno et al. (subm)

Protostelium SSU Brown et al. 2007

Dictyostelia Dictyostelium discoideum genome Eichinger et al. 2005

other Dictyostelium spp. genome in progress

Polysphondylium genome in progress

Acytostelium SSU, a-tubulin Schapp et al. 2006

Myxogastria Physarum polycephalum genome in progress

other Myxogastria SSU, EF1 SSU Fiore-Donno et al. 2005, Fiore-Donno et al. subm. Horton & Landweber 2000, Wikmark et al. 2007, Fiore-Donno et al. 2008

Incertae sedis Trichosphaerium sp. SSU, actin Tekle et al. 2008, Pawlowski & Fahrni 2007

Cochliopodium spp. SSU Kudryavtsev et al. 2005

Mayorella sp. SSU, actin Fahrni et al. 2003

Echinamoeba spp. SSU Amaral Zettler et al. 2000,

"Hartmannella" vermiformis EST Baumgartner et al. 2003 PEP

Filamoeba spp. SSU Amaral Zettler et al. 2000,

Acramoeba dendroida SSU Dykova et al. 2005c Amaral Zettler et al. 2000,

("Gephyramoeba" sp. ATCC) Multicilia marina SSU Smirnov et al. 2007a Nikolaev et al. 2006

Phalansterium solitarium SSU Cavalier-Smith et al. 2004

Vermistella antarctica SSU Moran et al. 2007

"Arachnula" sp. ATCC SSU, actin Tekle et al. 2008

RHIZARIA

Cercomonadida Cercomonas sp. EST PEP

other Cercomonadida SSU SSU LSU Zaman et al. 1999 Cavalier-Smith & Chao 2003 Moreira et al. 2007

Cryomonadida Cryothecomonas spp. SSU Kuhn et al. 2000

Heteromitida Heteromita globosa SSU LSU Cavalier-Smith and Chao 1996/1997 Markmann & Tautz 2005

other Heteromitida SSU Cavalier-Smith and Chao 2003

Thaumatomonadida Thaumatomonas seravini SSU Cavalier-Smith & Chao 2003

Thaumatomastix sp. LSU Moreira et al. 2007

others SSU Cavalier-Smith & Chao 2003

Euglyphida Euglypha rotunda SSU Bhattacharya et al. 1995

Paulinella chromatophora SSU Bhattacharya et al. 1995

Assulina muscorum SSU, LSU Wylezich et al. 2002, 2007

Cyphoderia spp. SSU Wylezich et al. 2002, Hegger, in prep

other Euglyphida SSU Wylezich et al. 2002, Lara et al. 2007

Chlorarachniophyta Bigellowiella natans EST genome PEP in progress

Chlorarachnion reptans SSU genome McFadden et al. 1994 in progress

Lotharella amoeboformis SSU,actin,pub genome Keeling 2001, Archibald et al. 2003 in progress

Gymnochlora stellata SSU Ishida et al. 1999

Phaeodarea Coelodendrum ramosissimum SSU Polet et al. 2004

Aulosphaera trigonopa SSU Polet et al. 2004

Aulacantha scolymantha SSU Polet et al. 2004

other Phaeodarea SSU Yuasa et al. 2006

Desmothoracida Hedriocystis spinifera SSU Nikolaev et al. 2004

Clathrulina elegans SSU Nikolaev et al. 2004

Ebriidea Ebria tripartita SSU Hoppenrath & Leander, 2006

Plasmodiophorida Plasmodiophora brassicae multigene Bulman et al. 2001,2006, 2007

other plasmodiophorids SSU actin,pub Bulman et al. 2001 Archibald & Keeling 2004

Phagomyxida Phagomyxa SSU Bulman et al. 2001

Haplosporidia Haplosporidium louisiana SSU, actin Flores et al. 1996, Reece et al. 2004

Haplosporidium spp. SSU Stokes and Burreson 1995

Minchinia teredinis SSU Stokes et al. 1995

other Haplosporidia SSU, actin Flores et al. 1996, Reece et al. 2004

Paramyxida Marteilia refringens SSU Berthe et al. 2000

Marteilioides chungmuensis SSU Itoh et al. 2003

Gromiida Gromia spp. SSU LSU actin, RPB1 Burki et al.2002, Aranda da Silva et al.2006 Pawlowski et al. 1994 Longet et al. 2004

Foraminifera Reticulomyxa filosa EST Burki et al. 2006, Burki & Pawlowski 2006

Quinqueloculina sp. EST Burki et al. 2007

Xenophyophorea SSU Pawlowski et al. 2003b

other foraminifera LSU SSU actin polyubiquitin RPB1 a-, b-tubulin Pawlowski et al. 1994, Moreira et al. 2007 Pawlowski et al. 1996, 2002, 2003a Habura et al. 2004 Flakowski et al. 2005, 2006 Archibald et al. 2003, Bass et al. 2005 Longet & Pawlowski 2007 Takishita et al. 2005

Collodaria Collozoum inerme SSU, actin Polet et al. 2004, Nikolaev et al. 2004

Thalassicolla pellucida SSU, actin Polet et al. 2004, Nikolaev et al. 2004

Sphaerozoum punctatum SSU Amaral Zettler et al. 1997

Rhaphidozoum acuferum SSU Amaral Zettler et al. 1999

Nassellarida Pterocorys zancleus SSU Kunitomo et al. 2006

Eucyrtidium hexagonatum SSU Kunitomo et al. 2006

Spumellarida Dictyocoryne profunda SSU Takahashi et al. 2004

Spongodiscus biconcavus SSU Kunitomo et al. 2006

other Polycystinea SSU Amaral Zettler et al. 1997, 1998, 1999 Takahashi et al.2004, Kunitomo et al.2006

Acantharea Acanthometra sp. SSU Amaral Zettler & Caron 2000

other Acantharea SSU Amaral Zettler et al. 1997 Amaral Zettler & Caron 2000

Taxopodida Sticholonche zanclea SSU Nikolaev et al. 2004

Incertae sedis Gymnophrys cometa SSU Nikolaev et al. 2003

Lecythium sp. SSU, actin Nikolaev et al. 2003, Nikolaev et al. 2004

Massisteria marina SSU Atkins et al. 2000 Cavalier-Smith & Chao 2003

Pseudodifflugia gracilis SSU Wylezich et al. 2002

Dimorpha-like SSU LSU Cavalier-Smith & Chao 2003 Moreira et al. 2007

Spongomonas spp. SSU Cavalier-Smith & Chao 2003

Metopion fluens SSU Cavalier-Smith & Chao 2003

Metromonas simplex. SSU Bass and Cavalier-Smith 2004

Pseudopirsonia mucosa SSU Kühn et al. 2004

Cercobodo agilis SSU Bass et al. 2005

Aurigamonas solis SSU Vickerman et al. 2005

"Corallomyxa" tenera SSU, actin Tekle et al. 2007

and SSU analyses (Cavalier-Smith and Chao, 2003). The examined heliozoan taxa branched within stra-menopiles (Actinophryida), among cercozoans (Desmothoracida) or as an independent eukaryotic lineage (Centrohelida). This study also provided

further evidence for the polyphyly of “Radiolaria” confirming that Pheaodarea group within the core Cercozoa, as suggested by Polet et al. (2004). Acantharea and Polycystinea form a monophy-letic clade, as shown by Lopez-Garcia et al. (2002),

branching together with Taxopodida (Sticholonche), as a sister group to other Rhizaria.

The relationships within the Rhizaria have been extensively studied (Cavalier-Smith and Chao, 2003; Bass and Cavalier-Smith, 2004; Bass et al., 2005), but the branching order, in particular between the basal groups, remains unresolved. The supergroup has been divided into three phyla: Cercozoa, Foraminifera and Radiozoa (Cavalier-Smith, 2004). The phylum Cercozoa comprised two subphyla: Filosa and Endomyxa (Cavalier-Smith, 2003). Filosa correspond to core Cercozoa (Nikolaev et al., 2004) and include the filose testate amoebae (Euglyphida, Pseudodifflugia), the chlorarachnean algae (Bi-gellowiella, Lotharella) and a large array of various zooflagellates, which are often able to generate filo-podia (Cercomonas, Heteromita, Thaumatomonas, Proleptomonas, Massisteria, Cryothecomonas and others). Further studies showed that Cercozoa also include the radiolarians of the class Phaeodarea (Polet et al., 2004; Yuasa et al., 2006), the heliozo-ans of the class Desmothoracida (Nikolaev et al.,

2004) and the flagellates Ebriidae (Hoppenrath and Leander, 2006). The phylogenetic analyses usually recover the monophyly of the Filosa, albeit not always with strong support (Cavalier-Smith and Chao, 2003; Bass and Cavalier-Smith, 2004). It has been demonstrated that most Filosa possess a two amino acids insertion at the monomer-monomer junction of the polyubiquitin gene, with the exception of the chlora-rachniophytes (Lotharella, Bigelowiella) and some flagellates (Metopion, Helkesimastix and Cercobodo), which possess a single amino acid insertion (Bass et al., 2005).

Compared to a relatively well supported Filosa clade, the relationships between the cercozoan subphylum Endomyxa and other rhizarian phyla (Foraminifera and Radiozoa) are much more controversial. The Endomyxa have originally been defined as plasmodial endoparasites of other eukaryotes, and included the class Phytomyxea (Plasmodiophorida + Phagomyxida) and the class Ascetosporea (Haplosporida + Paramyxida) (Cavalier-Smith,

2002). Later, the group was extended to include the gromiids (class Gromiidea) (Cavalier-Smith and Chao, 2003) and some uncultivated environmental clades (Bass and Cavalier-Smith, 2004; Bass et al.,

2005). However, this definition of Endomyxa was based on non-exhaustive SSU analyses, which did not include the Foraminifera and Radiozoa. When these sequences are added, the SSU phylogenies give a much more complex image of relationships at the base of the Rhizaria.

Foraminifera either branch with Haplosporidia

and Gromida (Nikolaev et al., 2004; Berney et al., 2004) or as a sister group to the Polycystinea (Cavalier-Smith and Chao, 2003). The branching of Foraminifera with the Haplosporidia was considered as a long branch attraction artifact caused by the fast evolving stem lineage of foraminiferal SSU rDNA (Cavalier-Smith and Chao, 2003). However, even if the haplosporidian SSU sequences evolve faster than other Cercozoa, this is certainly not the case of Gromia. Its position close to Foraminifera has been suggested by some analyses of the SSU (Berney and Pawlowski, 2003), as well as the analyses of the largest subunit of the RNA polymerase II (Longet et al.,

2003, 2004) and actin (Longet et al., 2004; Flakowski et al., 2005, 2006), although the presence of two actin paralogues in Foraminifera complicates its interpretation in the latter case. A recent multigene analysis of the reticulate plasmodial protist Corallomyxa ten-era placed this new species in a clade that includes Gromia, Haplosporidia and Foraminifera, hence reinforcing the phylogenetic hypotheses of a close relationship among these taxa (Tekle et al., 2007)

An alternative hypothesis concerning the phylogenetic relationships at the base of the Rhizaria suggests that Foraminifera are closely related to the Radiozoa. Branching of both groups in some SSU rDNA trees led to the creation of an infrakingdom or phylum Retaria (Cavalier-Smith 1999). In fact, this relationship seems to be strongly influenced by rapid evolutionary rates in ribosomal genes of Foraminifera and some Radiozoa, particularly the Polycystinea. Foraminifera branch with the Radiozoa, as a sister group to the Polycystinea, in some SSU trees (Cavalier-Smith and Chao, 2003 a,b), and in combined analyses of SSU and LSU data with limited taxon sampling (Moreira et al., 2007). However, this relationship is absent from many other broadly sampled SSU trees (Berney et al., 2004; Nikolaev et al., 2004; Tekle et al., 2007).

The main difficulty in resolving the relationships between Radiozoa and other Rhizaria is the lack of radiolarian protein sequences. Only three actin sequences for two species of Polycystinea are available (Collozoum inerme and Thalassicolla pel-lucida). These three fast evolving sequences usually (Nikolaev et al., 2004; Longet et al., 2004), but not always (Flakowski et al., 2005), branch as sister groups to foraminiferan actin paralogue II. A recent analysis of broad taxon sampling of rhizarian actins shows that the polycystinean and foraminiferan paralogue

II actins form a clade with one paralogue of haplo-sporidian actins; the other haplosporidian paralogue groups with Plasmodiophoridae, as a sister group to the clade grouping Gromia, Corallomyxa and the

Fig. 2. Hypothetical consensus tree of Rhizaria.

foraminiferan paralogue I (Tekle et al., 2007). This suggests that two actin paralogues exist not only in Foraminifera but also in Haplosporidia. Whether this is the case also in Radiozoa is unknown. The unclear position of polycystinean actins and the apparent absence of amino acid insertion in polyubiquitin of Polycystinea and Acantharea (Bass et al., 2005) provide evidence against the taxonomic grouping of Radiozoa and Foraminifera; however, further protein coding genes analyses are necessary to test the Retaria hypothesis.

A hypothetical consensus tree of the Rhizaria is illustrated in Fig. 2. This supergroup has been tentatively divided into three major groups: the

core Cercozoa (Filosa), the clade Phytomyxea (Plasmodiophorida, Phagomyxida) + Ascetosporea (Haplosporida, Paramyxida) + Gromiida + Corallomyxa + Foraminifera, and the Radiozoa. At least nine clades have been distinguished among core Cercozoa, modified from the classification of Cavalier-Smith and Chao (2003). Many other taxa belonging to core Cercozoa have been presented as independent lineages. There is one monospecific lineage (Corallomyxa) next to Gromiida, but the number of lineages branching in this part of the rhizar-ian tree is certainly much larger (Bass, pers. comm.; Lecroq, work in progress). Finally, the Radiozoa are represented by five clades (Acantharea, Taxopodida,

Spumellarida, Nassellarida, Collodaria), in agreement with the polyphyly of the Polycystinea suggested by Kunitomo et al. (2006).

As in the case of Amoebozoa, the main weakness of rhizarian phylogeny is its basically single-gene character. Until now, the protein-coding gene data were mainly used to confirm the monophyly of the group. Apart from actin and ubiquitin, no protein sequences are available for most of the groups, particularly the Radiolaria. The EST data are currently available only for two Foraminifera (Reticulomyxa, Quinqueloculina), the chlorarachniophyte Bige-lowiella natans and two cercozoans Gymnophrys and Cercomonas. The sequencing of the genome of Bigelowiella is in progress and that of Reticulomyxa is in preparation (Table 2).

Other amoeboid lineages

There are at least five groups of amoeboid pro-tists, traditionally included in Sarcodina, that now branch outside the supergroups of Amoebozoa and Rhizaria. Among them, there are two orders of Heliozoa (Actinophryida and Centrohelida), one class of Lobosea (Heterolobosea), one genus of Filosea (Nuclearia) and the species Breviata anathema (ex. Mastigamoeba invertens) initially considered as Lobosea and then reclassified as the only representative of the new class Breviatea (Cavalier-Smith et al.,

2004). In molecular phylogenies, the Heterolobosea, Actinophryida and Nucleariidae have been placed within excavates, stramenopiles and opisthokonts, respectively, while the Centrohelida and Breviatea branch independently.

Heterolobosea

This group is characterized by eruptive flow of cytoplasm in the amoeboid stage, discoidal mitochondrial cristae and the absence of typical dictyo-somes. Based on these ultrastructural features, the Heterolobosea were removed from the Lobosea and placed in their own class (Page and Blanton, 1985; Page, 1987). The independent origin of Heterolobosea was confirmed by early ribosomal phylogenies (Clark and Cross, 1988). Further molecular studies increased the taxon sampling of Heterolobosea by including other Vahlkampfiidae (Hinkle and Sogin, 1993) and Acrasis (Keeling and Doolittle, 1996). They also revealed a weakly supported relationship between Heterolobosea and Euglenozoa in the SSU rDNA trees (Simpson, 2003), a cluster that is more strongly supported by analyses of combined protein data (Baldauf et al., 2000). Both Heterolobosea and

Euglenozoa share the unusual “discoidal” mitochondrial cristae and have been grouped in the taxon Discicristata, based on rDNA and protein sequence data (Keeling and Doolittle, 1996; Cavalier-Smith, 2002; Baldauf et al., 2003). However, the accuracy of this grouping was recently questioned by multigene phylogenies, which suggest that the Heterolobosea are closely related to jakobids possessing typical tubular or flattened cristae (Simpson et al., 2006).

Actinophryida

This order of heliozoans is composed of only two genera (Actinosphaerium and Actinophrys) that possess a typical heliozoan morphology. Their ultrastructure shows important differences compared to other heliozoans, and some cellular structures are similar to those found in pedinellids (Smith and Patterson, 1986; Mikrjukov and Patterson, 2001). The first molecular evidence confirming the affinities between actinophryids and stramenopiles was based on SSU and actin gene sequences (Nikolaev et al., 2004). However, because of an extremely fast evolving SSU sequence of Actinosphaerium eichornii and limited taxon sampling of stramenopile actin genes, these data could not firmly establish the position of the Actinophryida. A recent SSU phylogeny of Chromista suggested that actinophryids are sister group to Opalozoa (Cavalier-Smith and Chao, 2006), but the support for this relationship was very weak and could be the result of a long branch attraction artefact.

Nucleariidae

The nucleariids are filose amoebae traditionally classified within the Filosea (Levine et al., 1980). Unexpectedly, the first SSU-based molecular analyses found them to branch among the Opisthokonts (Amaral-Zettler et al., 2001). This position was confirmed by multigene analyses, including EF1a, actin, HSP70, a-tubulin, and ^-tubulin (Steenkamp et al., 2006). In all these analyses, the Nucleariidae appear as a sister group to the Fungi, and this relationship is confirmed by the presence in Nuclearia of a well conserved insertion in EF1A, typical for all Opisthokonta.

Centrohelida

This group differs from other heliozoans by the presence of flat mitochondrial cristae and a centro-plast from which the axopodial microtubules arise (Febvre-Chevalier, 1990). The phylogenetic posi-

tion of the Centrohelida has been investigated by analyses of SSU (Cavalier-Smith and Chao, 2003), SSU + actin (Nikolaev et al., 2004), actin + a-tubu-lin + ß-tubulin (Sakaguchi et al., 2005; Takishita et al., 2005), SSU + LSU (Moreira et al., 2007), and a multigene analysis including actin + a-tubulin + ß-tubulin + EF2 + HSP70 + HSP90 genes (Sakaguchi et al., 2007). In spite of this considerable sequencing effort, the phylogenetic position of the Centrohelida remains unclear. There is no evidence for their affinity to any of the recognized eukaryotic supergroups. In a seven gene analysis, the Centrohelida are placed as a sistergroup to the clade uniting Chromalveolates and Plantae (Sakaguchi et al., 2007). A similar position was found in combined SSU and actin analysis (Nikolaev et al., 2004), while the SSU alone placed them as a sister group to Haptophytes (Cavalier-Smith and Chao, 2003) or as a sister group to the Rhodophyta (Sakaguchi et al., 2005). None of these positions is statistically supported. Most probably, the Centrohelida are an independent lineage of eukaryotes, a hypothesis that should be tested with larger scale multi-gene analyses and larger taxon sampling.

Breviatea

The class Breviatea was introduced by Cavalier-Smith et al. (2004) for the enigmatic free-living amoeboflagelate Mastigamoeba invertens. A lack of phylogenetic affinity between M. invertens and other mastigamoebids (Bolivar et al., 2001; Milyutina et al., 2001) suggested that the species was misidentified (Edgcomb et al., 2002; Cavalier-Smith et al., 2004). It has been recently redescribed as Breviata anathema, gen. n., sp. n., based on a light-microscopical and ultrastructural study (Walker et al., 2006). The phylogenetic position of B. anathema at the base of Amoebozoa in some SSU trees (Bolivar et al., 2001), and its putative single basal body prompted its inclusion in the Amoebozoa (Cavalier-Smith et al.,

2004). However, most SSU analyses placed B. anathema among Bikonts, often as a sister group to some Apusozoa (Cavalier-Smith and Chao, 2003; Simpson, 2003; Nikolaev et al., 2004; Walker et al., 2006), while RNA polymerase II trees placed it at the base of all other eukaryotes (Stiller et al., 1998; Longet et al., 2003).

Concluding remarks

The demise of the traditional system of Sarcodina is a logical consequence of the replacement of a “convenient” protist classification by a new macro-

system based on molecular phylogenetic hypotheses. There is certainly nothing unexpected in this taxonomic reshuffling. The polyphyly of sarcodinids was suggested by many protistologists from the pre-molecular era. The macrotaxa such as Sarcodina, Rhizopoda, Actinopoda and Heliozoa survived for more than a century mainly because there was no alternative to the convenient morphology-based system. The advances of molecular phylogenies not only confirmed their obsolete character but also provided an opportunity to replace them by new groupings of eukaryotes.

It is interesting to notice, however, that the traditional grouping of amoeboid protists based on their pseudopodial characteristics might be partially right. Although practically all traditional macrotaxa have been shown to be polyphyletic, many amoeboid groups persist in a slightly modified form. For example, the majority of lobosean amoebae remain grouped within the Amoebozoa, while the filosean and reticulosean amoebae are mainly found among the Rhizaria. Even if these supergroups also contain some flagellated species, many of them possess the capacity to develop pseudopodia. Although almost all other major eukaryotic groups, with the notable exception of plants and alveolates, include at least one amoeboid lineage. In most cases these lineages represent minor taxonomic groups of one or a few genera and their phylogenetic position is often difficult to establish.

The ability of protists to produce pseudopods is certainly a universal feature of eukaryotes. However, it seems that this ability was fully expressed only in Amoebozoa and Rhizaria, which contain the most diversified assemblage of amoeboid protists. Despite a certain criticism of the taxonomic status of these two supergroups (Parfrey et al., 2006), the rapidly accumulating phylogenomic data continuously reinforce the support for these, par excellence, amoeboid assemblages. The monophyly of Amoebozoa and Rhizaria seems indisputable, even if there are still some uncertainties concerning their final composition, in particular the phylogenetic position of some basal lineages and some yet unsequenced taxa.

Today, the principal challenge is to establish the phylogenetic relationships within Amoebozoa and Rhizaria and to understand the genomic basis for the development of different patterns of pseudopo-dial form and movement. This can be achieved only by substantially increasing the genomic data on major components of both supergroups. New EST and genome sequencing projects are necessary to search for genomic signatures that could help to infer the root of each supergroup and to determine the evo-

lutionary steps leading to the formation of different types of pseudopods. In particular, a more extensive study of actin, myosins, tubulins and other proteins involved in pseudopodial movement and their interactions may help to better understand the evolution of both amoeboid supergroups.

Another challenge is to describe more accurately the diversity of Amoebozoa and Rhizaria. The study of uncultured isolates and environmental samples showed an unexpectedly high diversity of some amoeboid groups, such as monothalamous foramin-ifera (Pawlowski et al., 2002, 2003), Cercozoa (Bass and Cavalier-Smith, 2004), Euglyphida (Lara et al., 2007) or Radiozoa (Not et al., 2007). However, many amoeboid groups, for example, the lobose amoebae and foraminifera, are rarely recovered from environmental sampling (Berney et al., 2004). This is partly due to the difficult amplification of their highly divergent ribosomal genes, and can be overcome by using specific PCR primers (Habura et al., 2004). More specific search for amoebozoans and rhizarians in environmental DNA surveys, as well as more extensive molecular studies of uncultured amoeboid taxa, will certainly reveal many new, undescribed species, which may change our view on their diversity and evolution.

Acknowledgements

This paper is dedicated to the memory of Kirill A. Mikrjukov, who performed an extensive research on the phylogeny of “heliozoa” and inspired my interest in the evolution of this group. I thank Cédric Berney, Fabien Burki, Anna Maria Fiore-Donno, José Fahrni, Alexander Kudryavtsev and Robert Peck for comments and discussion. The Swiss National Science Foundation is acknowledged for the generous support to my research, through grants 3100-064073.00 and 3100A0-112645, and SCOPES Joint Research Projects (7SUPJ062342 and IB73A0-111064).

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Address for correspondence: J. Pawlowski. University of Geneva, Department of Zoology, Geneva, Switzerland. E-mail: jan.pawlowski@zoo.unige.ch