All naked lobose amoebae were grouped in the class Lobosea, subclass Gymnamoebia, with four orders (Euamoebida, Leptomyxida, Acanthopodida and Loboreticulatida), while the testate lobose amoebae (order Arcellinida) were placed in the subclass Testacealobosia. This system was mainly aimed at resolving the low-level relationships among amoebae and provided practical tools for species identification. However, higher-level phylogenetic relationships within amoebae remained "unrecoverable from morphology" (Page 1987), and further development of Page's system (see Rogerson and Patternson 2002) did not improve the situaltion.
Early molecular studies based on small-subunit ribosomal RNA (SSU rRNA) gene sequences suggested independent origins for different lineages of lobose amoebae (Cavalier-Smith 2000; Hinkle et al. 1994; Silberman et al. 1999; Sims et al. 1999). However, as the number of available sequences increased, and with the use of more accurate methods of phylogenetic analyses, it became evident that independent branching of lobose amoebae in early phylogenies was an artifact of phylogenetic reconstructions related to the long branch attraction phenomenon. Several recent phylogenetic analyses, and including a large taxon sampling of eukaryotes, and using methods correcting for among-site rate heterogeneity, showed that all sequenced lobose amoebae form a clade that generally also includes mycetozoans (Bolivar et al. 2001; Cavalier-Smith et al. 2004; Fahrni et al. 2003; Milyutina et al. 2001). Although usually supported, the monophyly of lobose amoebae and mycetozoans was confirmed by phylogenetic analysis of actin (Fahrni et al, 2003), and multigene analyses of nuclear (Baldauf et al. 2000) and mitochondrial (Forget et al. 2002) protein sequences, as well as the analysis of 123 genes obtained from EST libraries of Entamoeba, Mastigamoeba, and Dictyostelium (Bapteste et al. 2002). However, none of these multigene studies contained representatives of all lineages of amoebae simultaneously, nor included a typical free-living lobose amoeba. In the high-level classification of Protozoa, all naked and testate lobose amoebae, together with entamoebids, pelobionts, and mycetozoans, were grouped in an emended phylum Amoebozoa (Cavalier-Smith 1998, 2002, 2003; Cavalier-Smith et al. 2004).
The molecular phylogeny of lobose amoebae is broadly congruent with the morphological system at the level of genera and some families, but strongly disagrees with it starting at the level of orders and subclasses. The first attempts to find a correlation between the molecular phylogeny and the morphological characters of amoebae at higher taxonomic level generally failed (Amaral Zettler et al. 2000; Bolivar et al. 2001; Peglar et al, 2003). Only two orders of the Gymnamoebia sensu Page (1987), Acanthopodida and Leptomyxida, seem to be supported by molecular data. The other typical naked lobose amoebae (Euamoebida sensu Page 1987) are split into several lineages, one of which includes the Leptomyxida and the Arcellinida. In a revised classification of the phylum Amoebozoa based on the SSU rRNA phylogeny. Cavalier-Smith et al. (2004) attempted to produce a system congruent with both molecular and morphological data. The authors grouped the majority of typical naked lobose amoebae in two classes: the Lobosea, with "ancestrally cylindrical and non-eruptive" pseudopods, but "sometimes flattened or eruptive" and "lacking glycostyles or scales" (e.g. the families Amoebidae and Hartmannellidae), plus the orders Leptomyxida and Arcellinida); and the Discosea, defined as "flattened amoebae with leading lamellipodium" and "usually with glycostyles or organic scales" (e.g. the families Vannellidae, Paramoebidae, Vexilliferidae and Thecamoebidae). A third class, Variosea, was created to accommodate the Acanthopodida and the genera Filamoeba, Gephyramoeba, and Phalansterium. The shape of the cell and the pattern of pseudopods seem to be reasonable features to distinguish the first two classes. However, the evolutionary origin and phylogenetic significance of the various surface structures in amoebae are unclear. For example, glycostyle-like structures recognized as "sucker-like elements" (Page 1985) or pentagonal "cup-shaped elements" are present on the cell surface of Hartmannella and Saccamoeba (Anderson et al. 1997; Page 1988), which belong to Lobosea (sensu Cavalier-Smith et al. 2004), while glycostyle-like elements are completely absent in the cell coat of Thecamoeba and Dermamoeba (Page and Blakey 1979), which belong to Discosea. The wide diversity of surface structures among gymnamoebae and the fact that most of these surface structures are genus-specific suggest that they may be independent acquirements within low-level taxa.
In the present study, we revise the classification of lobose amoebae by defining the morphological characters of the phylogenetic lineages that are congruent with available molecular data. As a framwork for a new system, we use a comprehensive SSU rRNA phylogeny of 52 amoebozoans, including all sequenced genera of lobose amoebae and selected sequences of Conosa. We focus our study on two lineages, called here Tubulinea and Flabellinea, containing the majority of the Gymnamoebia sensu Page 1987 and the testate lobose amoebae of the order Arcellinida. We analyze the systematic composion and morphological characters of every well-defined clade and compare them with both modern and ancient systems of amoebae in order to recover the shared morphological features of each molecular lineage and to evaluate their potential phylogenetic importance.
New high-rank phylogenetic classification of Amoebozoa. The genera for which no sequence data are available, but that are morphologically evidently related to sequenced species, are listed as "probably belonging" to the appropriate taxa.
Class Tubulinea classis nov.
Naked or testate amoebae producing tubular, subcylindrical pseudopodia or capable of altering the locomotive form from a flattened, expanded one to a subcylindrical one. Monoaxial flow of the cytoplasm in every pseudopodium or in the entire cell. No cytoplasmic MTOCs, no flagellate stage in the life cycle.
Order Tubulinida ord. nov.
Naked amoebae producing subcylindrical pseudopodia in locomotion (or the entire cell is monopodial and subcylindrical). No alteration of the locomotive form to a flattened expanded and branched one. No adhesive uroidal structures.
Families Amoebidae (Ehrenberg, 1838) Page, 1987 and Hartmannellidae (Volkonsky, 1931) Page, 1974; genera Amoeba, Chaos, Glaeseria, Hartmannella, Saccamoeba (and probably Cashia, Deuteramoeba, Hydramoeba, Nolandella, Parachaos, Polychaos, Trichamoeba).
Order Leptomyxida (Pussard and Pons, 1976) Page, 1987
Naked amoebae with a locomotive form altering from a flattened, expanded, and branched one to a subcylindrical, monopodial one. Uroidal structures of adhesive type.
Families Leptomyxidae (Pussard and Pons, 1976) Page, 1987 and Flabellulidae (Bovee, 1970) Page, 1987; genera Leptomyxa, Rhizamoeba, Paraflabellula (and probably Flabellula).
Order Arcellinida (Kent, 1880)
Testate amoebae with the cell enclosed into an organic or mineral test with a single main opening (e.g. genera Arcella, Centropyxis, Heleopera). Systematic composition according to Meisterfeld (2002).
Tubulinea incertae sedis:
Echinamoeba, "Hartmannella" vermiformis
Class Flabellinea classis nov.
Flattened naked amoebae, never producing tubular, subcylindrical pseudopodia and never altering the locomotive form. Cytoplasmic flow polyaxial or without a pronounced axis. No cytoplasmic MTOCs, no flagellate stage in the life cycle.
Order Dactylopodida ord. nov.
Naked amoebae capable of producing hyaline finger-shaped subpseudopodia (dactylopodia) formed from the frontal hyaline margin or frontal hyaline area.
Families Paramoebidae (Poche, 1913) Page, 1987 and Vexilliferidae Page, 1987; genera Korotnevella, Neoparamoeba, Vexillifera (and probably Paramoeba, Pseudoparamoeba).
Order Vannellida ord. nov.
Naked, flattened amoebae, fan-shaped, saptulate or lingulate; the frontal zone of the hyaloplasm occupies about half of the cell, and never forms and antero-lateral crescent; in the locomotive cell, the front edge is always smooth; they do not produce discrete pseudopodia or subpseudopodia.
Family Vannellidae Bovee, 1979; genera: Clydonella, Lingulamoeba, Platyamoeba, Vannella (and probably Pessonella).
Class Conosea Cavalier-Smith, 1998 stat nov.
Diagnosis and composition accoridng to Cavalier-Smith et al. (2004). Rank of the Cavalier-Smith's infraphylums must be decreased to infraclasses; rank of classes - to subclasses; lower level system without changes.
Amoebozoa incertae sedis:
Order Acanthpodida Page, 1976 (Family Acanthamoebidae Sawyer and Griffin, 1975); genera: Acanthamoeba, Protacanthamoeba, Balamuthia, Comandonia
Families Stereomyxidae Grell, 1966, Corallmyxidae Page, 1987; genera: Dermamoeba, Filamoeba, Gephyramoeba, Mayorella, Phalansterium, Thecamoeba, "Platyamoeba" stenopodia, Cochliopodium, Flamella, Gocevia, Janickia, Malamoeba, Malpighamoeba, Paradermamoeba, Paragocevia, Parvamoeba, Pellita, Pseudothecamoeba, Sappinia, Stygamoeba, Thecochaos, Trichosphaerium.