Phylogeny of Lobose Amoebae Based on Actin and Small-Subunit Ribosomal RNA Genes

José F. Fahrni*,, Ignacio Bolivar*, Cédric Berney*, Elena Nassonova{dagger}, Alexey Smirnov{ddagger} and Jan Pawlowski*

* Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland
{dagger} Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, St. Petersburg, Russia
{ddagger} Department of Invertebrate Zoology, St. Petersburg State University, St. Petersburg, Russia

Correspondence: E-mail: jose.fahrni{at}zoo.unige.ch.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

Key Words: actin • Amoebozoa • Lobosea • molecular phylogeny • small-subunit ribosomal RNA


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Despite their obvious ecological and medical importance (Anderson 1997; Szenasi et al. 1998; Butler and Rogerson 2000; Finlay et al. 2000), the origin and evolutionary history of lobose amoebae remain enigmatic. For convenience, earlier protist classifications placed all amoebae possessing lobose pseudopodia in the class Lobosea, belonging to the superclass or phylum Rhizopoda (Levine et al. 1980; Bovee 1985). However, based on ultrastructure and life cycle studies, the amoebae and amoeboflagellates with discoidal mitochondrial cristae and without typical dictyosomes were excluded from the Lobosea and placed in the class Heterolobosea (Page and Blanton 1985; Page 1987). The distinction of both classes was confirmed later by analysis of small-subunit ribosomal RNA (SSU rRNA) sequences (Clark and Cross 1988; Hinkle and Sogin 1993). Furthermore, the position of pelobionts—the free-living amitochondriate amoebae—is debated. For example, this group was considered either as a separate phylum (Margulis 1974; Margulis et al. 1990), as a separate class within the Rhizopoda (Page 1987), as an order within the Lobosea (Bovee 1985), or placed in the phylum Archamoebae, among early diverging amitochondriate eukaryotes (Cavalier-Smith 1987, 1993; Corliss 1994). Another group of amitochondriate amoebae, the entamoebids, viewed by some as the model of primitive eukaryotes (Bakker-Grunwald and Wöstmann 1993) was transferred from Lobosea to the Archamoebae (Cavalier-Smith 1987) or later placed in a separate phylum, the Entamoebia (Cavalier-Smith 1993). Early SSU rRNA–based phylogenies suggested independent origins for pelobionts, entamoebids, and other lobose amoebae (Sogin 1991; Hinkle et al. 1994; Sims, Rogerson, and Aitken 1999), supporting their separation into different classes or phyla. Based on ultrastructural data and following ribosomal RNA phylogenies, recent protist classifications widely accept the polyphyly of lobose amoebae, splitting them into at least three taxonomic groups (Hausmann and Hülsmann 1996; Lee, Leedale, and Bradbury 2000).

A recent opposite view proposes that all lobose amoebae, with the exception of Heterolobosea, are monophyletic (Cavalier-Smith 1998). This view is based on molecular evidence that the pelobionts and entamoebids have lost their mitochondria secondarily (Clark and Roger 1995) and that they group together with lobose amoebae in some revised ribosomal RNA phylogenies (discussed in Cavalier-Smith and Chao 1996, and Cavalier-Smith 2000, and demonstrated later by Bolivar et al. 2001, and Milyutina et al. 2001). The phylum Amoebozoa Lühe, 1913 was emended to group together the naked and testate lobose amoebae, the pelobionts, the entamoebids, and the Mycetozoa (Cavalier-Smith 1998). The latter group was included into Amoebozoa based on analysis of actin and actin-related proteins (Kelleher, Atkinson, and Pollard 1995; Bhattacharya and Weber 1997; Schafer and Schroer 1999). This was recently confirmed by the combined analysis of nuclear (Baldauf et al. 2000) and mitochondrial (Forget et al. 2002) protein sequences of the lobosean Acanthamoeba and the slime molds Dictyostelium and Physarum. A common origin for Entamoeba, Mastigamoeba, and Dictyostelium was also inferred from combined analysis of EF-1a and EF-2 sequences (Arisue et al. 2002) and is strongly supported by the analysis of 123 genes obtained from EST libraries (Bapteste et al. 2002). However, none of these studies includes representatives of typical free-living, lobose amoebae (order Euamoebida).

To test further the relationships among Amoebozoa, we obtained 10 new actin sequences and eight new SSU rRNA sequences of naked, lobose amoebae. Phylogenetic analyses using several evolutionary models support the hypothesis that all lobose amoebae are closely related and reveal the existence of two well-defined clades within Amoebozoa.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Cultures and Sequencing
Most amoebae strains were obtained from the Culture Collection of Algae and Protozoa (Windermere, United Kingdom). The strain numbers, food, and culture mediums for each species are available in table 1 of the Supplementary Material online (www.molbiolevol.org). The DNA was extracted either with guanidinium buffer (Chomczynski and Sacchi 1987) or with NaOH (Wang, Qi, and Cutler 1993). Total RNA was extracted as described previously (Bolivar et al. 2001). The actin gene was amplified by RT-PCR with primers ActN2 (5'-AACTGGGAYGAYATGGA3') and 1354R (5'-GGACCAGATTCATCATAYTC-3'); the N-terminal part of the molecule was obtained by 5'-RACE. The SSU rRNA gene was amplified by PCR or RT-PCR in two overlapping fragments, using primer pairs sA (5'-CYGGTYGATCCTGCCAGT-3') – s14r (5'-AAGTTTCAGCCTTGCGACCA-3') and s12.2 (5'-GATYAGATACCGTCGTAGTC-3') – sB (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). Amplification, purification, cloning, and sequencing were performed as described previously (Pawlowski et al. 1999). The new sequences reported in this paper were deposited in the GenBank/EMBL database under accession numbers AY294143 to AY294160 (see table 2 in the Supplementary Material online for the species names, taxonomic position, and accession numbers of all actin and SSU rRNA sequences used in our analyses).

Actin Analysis
The actin protein sequences were manually aligned using the Genetic Data Environment (GDE) software (Larsen et al. 1993). Only complete sequences were selected among already available data, and a total of 364 amino acid positions were used in the phylogenetic analyses. An evolutionary tree of actin was inferred from the amino acid sequences with the maximum-likelihood (ML) method (Felsenstein 1981) using the JTT substitution matrix (Jones, Taylor, and Thornton 1992) and taking into account a proportion of invariable sites and a gamma-shaped distribution of the rates of substitution among variable sites with eight rate categories. All necessary parameters were estimated from the data using Tree-Puzzle version 5.0 (Strimmer and von Haeseler 1996), and the tree topology was constructed with the ProML program of the PHYLIP version 3.6a3 package (Felsenstein 2002), using the -R option with 10 input order jumbles and global rearrangements. The reliability of internal branches was assessed using the bootstrap method (Felsenstein 1985), with 100 replicates, based on a distance analysis using the program Fitch of PHYLIP. For each data resampling, JTT + G + I corrected distances were calculated by Tree-Puzzle with the utility PuzzleBoot, using the parameters estimated above. ML analyses were also carried out with the ProtML program of the Molphy version 2.3 package (Adachi and Hasegawa 1996). Trees were inferred using the local rearrangement search option, starting from a distance topology obtained with the NJdist program included in the same package. Bootstrap probabilities were estimated with the RELL method (Kishino, Miyata, and Hasegawa 1990; Hasegawa and Kishino 1994). In addition, a quartet-puzzling tree was obtained with Tree-Puzzle (using the JTT + G + I model with the parameters estimated above).

SSU rRNA Analysis
The SSU rRNA sequences were manually aligned using the GDE software, as above, following secondary structure models (Neefs et al. 1993; Wuyts et al. 2000). Already available sequences were selected so that most major taxonomic groups of eukaryotes were represented, and the sampling more or less matched the one for actin; highly diverging lineages such as Foraminifera and Microsporidia were omitted. A total of 1,150 unambiguously aligned positions were used in the phylogenetic analyses. Evolutionary trees were inferred utilizing the ML method, the neighbor-joining (NJ) method (Saitou and Nei 1987), and the maximum-parsimony (MP) method, using PAUP* (Swofford 1998). The reliability of internal branches was assessed with 100, 1,000, and 500 bootstrap replicates for ML, NJ, and MP analyses, respectively. ML analyses were performed with the GTR model of substitution (Lanave et al. 1984; Rodriguez et al. 1990), taking into account a proportion of invariable sites and a gamma-shaped distribution of the rates of substitution among variable sites, with eight rate categories. All necessary parameters were estimated from the data using Modeltest (Posada and Crandall 1998). Starting trees of ML searches were obtained via NJ and swapped with the tree-bisection-reconnection algorithm. NJ analyses were performed with ML-corrected distances using the same parameters. The most parsimonious trees for each MP bootstrap replicate were determined using a heuristic search procedure with 10 random-addition-sequence replicates and tree-bisection-reconnection branch-swapping. The transversions cost was set to twice the transitions cost. ML and NJ analyses using simpler models (see table 3 in Supplementary Material online) were performed with Phylo_win (Galtier, Gouy, and Gautier 1996).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Sequence Data
Ten new actin sequences of Amoeba proteus, Chaos carolinense, Dermamoeba algensis, Glaeseria mira, Hartmannella cantabrigiensis, Mayorella sp., Pelomyxa palustris, Platyamoeba placida, Thecamoeba similis, and Vannella ebro were obtained. No introns or peculiar structural patterns were found.

New SSU rRNA sequences of eight species of gymnamoebae (D. algensis, G. mira, H. cantabrigiensis, Mayorella sp., P. placida, Platyamoeba stenopodia, T. similis, and Vexillifera minutissima) were obtained. The size of these sequences varies from 1,893 base pairs in P. placida to 2,409 base pairs in T. similis. Size variations occur mainly in the 5' part of variable region V4, but expansions were observed in the variable region V2 for Mayorella sp. and in the variable regions V4, V7, and V8 for T. similis. The sequences also vary importantly in GC content, with a mean value of 42%, ranging from 33.5% in V. minutissima to 52.8% in P. stenopodia. However, these variations in the GC content occur mainly in the variable regions of the molecule and are smaller in the set of sites selected for phylogenetic analyses (42.5% to 47.9%, with a mean value of 45%).

Phylogenetic Analyses
Figure 1A shows the result of a ML analysis of 55 actin sequences of eukaryotes, including 13 lobose amoebae. The topology shown was obtained using the JTT substitution matrix, taking into account a proportion of invariable sites (10%) and a gamma-shaped distribution of the rates of substitution among variable sites, with eight rate categories (alpha = 0.55). Figure 1B shows the result of a ML analysis of 60 SSU rRNA sequences of eukaryotes, including 26 lobose amoebae. The topology shown was obtained using the GTR model of substitution, taking into account a proportion of invariable sites (7.92%) and a gamma-shaped distribution of the rates of substitution among variable sites, with eight rate categories (alpha = 0.42). Because the position of the root of the eukaryotic tree is still subject to debate (see e.g., Stechmann and Cavalier-Smith 2002), the trees are presented in an unrooted format, with a basal trifurcation. The general topology of both trees is congruent with previous large-scale actin and SSU rRNA phylogenies of eukaryotes, and all well-recognized high-level taxa are recovered with good statistical support. In both trees, all lobose amoebae and Mycetozoa cluster together at the exclusion of any other eukaryote in a clade called Amoebozoa. Although this group is not supported by bootstrap analysis, in either actin or SSU rRNA trees, it is recovered by several methods of tree reconstruction and models of substitution (see table 3 in Supplementary Material online). Moreover, sites 289, 321, 385, 515, 777, 1010, and 1051 of the SSU rRNA gene show an Amoebozoa-specific character configuration that was not found in any other eukaryote (see table 4 in Supplementary Material online), and sequences of the members of Amoebozoa are characterized by an insertion of one nucleotide in the short loop between stems 30 and 28 (sites 1060 to 1064 [see table 4 in Supplementary Material online]).



View larger version (39K):
[in this window]
[in a new window]
 
FIG. 1. Phylogenetic position of lobose amoebae and relationships among Amoebozoa, as inferred from actin (A) and SSU rRNA (B) sequences. Both phylogenies show that all lobose amoebae cluster together, and they distinguish two well-defined clades of Amoebozoa, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa. Because the position of the root of the eukaryotic tree is subject to debate, both trees are presented in an unrooted format, with a basal trifurcation. In both trees, branches are drawn to scale. (A) Actin tree inferred from a ML analysis of 55 amino acid sequences of eukaryotes with ProML, using the JTT + G + I model of substitution (see Materials and Methods). Numbers at nodes indicate bootstrap support values for 100 replicates of a Fitch-Margoliash analysis using ML-corrected distances (upper) and bootstrap support values estimated through the RELL method with ProtML (lower). Values under 50% were omitted. (B) SSU rRNA tree inferred from a ML analysis of 60 sequences of eukaryotes with PAUP*, using the GTR + G + I model of substitution (see Materials and Methods). Numbers at nodes indicate bootstrap support values of ML (upper) and NJ (lower) analyses, using the same model, after 100 and 1,000 replicates, respectively. Values under 50% were omitted

 
Interestingly, both trees congruently show a division of the Amoebozoa in the same three groups. The first one contains most of the well-known typical gymnamoebae such as A. proteus, and corresponds to the "Gymnamoebia sensu stricto," as defined by Bolivar et al. (2001). The second one comprises all amitochondriate amoebae—that is, the pelobionts and the entamoebids—and corresponds to the Archamoebae, as defined by Cavalier-Smith (1998). The third group represents a very weakly supported and ill-resolved clustering of various other lobose amoebae plus the Mycetozoa. Table 3 in the Supplementary Material online indicates the support for these groups according to different methods of tree reconstruction and models of substitution.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Monophyly of Amoebozoa
Our analyses based on actin and SSU rRNA sequences show that lobose amoebae are all closely related to each other and support the idea that they constitute, together with pelobionts, entamoebids, and Mycetozoa, the phylum Amoebozoa (fig. 1). Although the existence of this phylum was already assessed, our results—including the first well-sampled protein data on lobose amoebae—significantly widen our knowledge about which organisms actually belong to the group. According to our data, the Amoebozoa clade includes all amoebae belonging to the subclass Gymnamoebia (Page 1987) plus the pelobionts, the entamoebids, and the slime molds. It could also include the testate lobose amoebae of the subclass Testacealobosia (Page 1987); however, no molecular data for this group exist at the moment.

Although the Amoebozoa clade is not supported by bootstrap analysis in either actin or SSU rRNA trees (fig. 1; see table 3 in Supplementary Material online), it is recovered in all ML analyses and also in SSU rRNA distance trees if a gamma correction is used and the divergent sequence of T. similis is excluded. Furthermore, eight diagnostic positions were found in the SSU rRNA alignment (see table 4 in Supplementary Material online). The lack of bootstrap support for the Amoebozoa in SSU rRNA phylogenies is probably due to a very low number of changes during the stem evolution of the group, coupled with the fact that there are many different rates of evolution among the different extant amoebozoan species. The lack of bootstrap support for the Amoebozoa in the actin phylogeny might be due to the apparently slow rate of actin evolution in amoebae and animals. In view of our analyses, previous SSU rRNA studies suggesting the polyphyly of lobose amoebae and/or an independent origin for pelobionts, entamoebids, and Mycetozoa (e.g., Sogin 1991; Hinkle et al. 1994; Cavalier-Smith 2000), were probably biased by the limited number of available amoebae sequences, coupled with the wide range of divergence between them (Bolivar et al. 2001).

High-level Relationships Among Amoebozoa
Among the three major groups distinguished within Amoebozoa in figure 1, two (the "Gymnamoebia sensu stricto" and the Archamoebae) are well supported by molecular, morphological, and ultrastructural data.

The "Gymnamoebia sensu stricto" comprise two families of the order Euamoebida—Amoebidae (A. proteus + C. carolinense) and Hartmannellidae (H. cantabrigiensis + G. mira + S. limax)—as well as members of the order Leptomyxida (Leptomyxa reticulata + Paraflabellula hoguae). The closest relatives to these taxa are Echinamoeba exundans and Hartmannella vermiformis (the generic status of the latter species is uncertain, as it is not closely related to other Hartmannellidae), but their relations to other "Gymnamoebia sensu stricto" are not well supported (fig. 1B). The close relationship between Leptomyxida and the clade E. exundans + H. vermiformis was already demonstrated by Amaral Zettler et al. (2000). Here, we confirm their relationship to the Amoebidae, as suggested in our previous study (Bolivar et al. 2001), and show a highly supported relationship (bootstrap values of 96% to 100%) between Amoebidae and Hartmannellidae.

The Archamoebae comprise all amitochondriate amoebae, including entamoebids, mastigamoebids, and Pelomyxa. The relationship between entamoebids and the pelobiont genus Mastigamoeba (but not Mastigamoeba invertens [e.g., Edgcomb et al. 2002]) was already suggested by SSU rRNA–based studies (Silberman et al. 1999; Edgcomb et al. 2002), and a strong support for the relationship between Entamoeba histolytica and Mastigamoeba balamuthi was inferred from a combined analysis of rRNA and protein data (Arisue et al. 2002; Bapteste et al. 2002). The classic pelobiont Pelomyxa was recently added to this group (Milyutina et al. 2001). The actin sequence of Pelomyxa presented in this study confirms that this genus belongs to Archamoebae, although its relationship to other pelobionts and entamoebids is not well resolved.

The third group is a very weakly supported and ill-resolved clustering, composed of morphologically different amoeboid lineages. In addition to Mycetozoa, it also comprises amoebae belonging to the order Acanthopodida and various families of the order Euamoebida (Vannellidae, Thecamoebidae, Paramoebidae, and Vexilliferidae). The grouping of Acanthamoeba and Mycetozoa was considered as evidence for a common origin of all amoebae (Baldauf et al. 2000), but in view of our data, it represents only part of the Amoebozoa. The Vannellidae (P. placida, V. ebro, and V. anglica) and Thecamoebidae (T. similis) share some common morphological features (Smirnov 2001), but the relationships between other families are unclear. However, given the weak support for this cluster (see table 3 in Supplementary Material online), it is probable that with an increasing number of taxa, this group will prove paraphyletic and disappear, replaced by a series of independent lineages that might include the "Gymnamoebia sensu stricto" and/or the Archamoebae.

Our data are in opposition to the division of Amoebozoa into two groups: Lobosa and Conosa (Cavalier-Smith 1998). The independent branching of Mycetozoa and Archamoebae within loboseans in both actin and SSU rRNA trees makes the Lobosa paraphyletic and refutes the holophyly of Conosa (Archamoebae + Mycetozoa) suggested by Bapteste et al. (2002). Their analysis was apparently misleading because of an insufficient taxonomic sampling and, more particularly, the lack of protein data for the lobose amoebae, which, together with Mycetozoa, form the third group in our analyses. Although the relationships within this group are not well established, there is no indication of closeness between Mycetozoa and Archamoebae in either actin or SSU rRNA sequences.

Phylogenetic Position of Amoebozoa
The phylogenetic position of Amoebozoa is of crucial importance for inferring early events in eukaryotic evolution. Following a recent hypothesis (Stechmann and Cavalier-Smith 2002), the root of the eukaryotic tree lies between opisthokonts and bikonts, but the position of Amoebozoa in this work is still unclear. Our results are congruent with the idea that Amoebozoa are branching between opisthokonts and bikonts. In the actin tree (fig. 1A), the Amoebozoa are even included in the opisthokonts and appear as a sister-group to the Metazoa + Choanoflagellata clade. However, such a position is very suspicious, because several independent lines of evidence clearly support the monophyly of the opisthokonts at the exclusion of Amoebozoa (e.g., Baldauf and Palmer 1993). Thus, the topology shown in figure 1A might rather reflect slightly higher rates of actin evolution in fungi as compared with animals and Amoebozoa.

Given the available data, the possibility that the root of the eukaryotic tree lies within Amoebozoa, and that all other extant eukaryotes derive from an amoebozoan ancestor, cannot be excluded. A larger sampling of genes will be needed to test further both the holophyly of Amoebozoa and the position of the phylum in relation to opisthokonts and bikonts.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Table 1 in the online Supplementary Material contains strains data. Table 2 includes taxonomic position, species names, and GenBank accession numbers of the actin and SSU rRNA sequences used in this study. Table 3 shows phylogenetic groupings according to different methods of analysis and evolutionary models. Table 4 lists the selected sites in the SSU rRNA alignment defining the Amoebozoa and their position among other eukaryotes.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
The authors thank Y. Deng, M. Kiersnowska, R. Peck, F. Duborgel, and B. Barandun for kindly providing cultures of amoebae, as well as two anonymous reviewers for helpful, critical comments on an earlier version of this manuscript. This work was supported by the Swiss NSF grants 7SUPJ062343 and 31-064073.00 (J.P.).


    Footnotes
 
Manolo Gouy, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 

    Adachi, J., and M. Hasegawa. 1996. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28:1-150.

    Amaral Zettler, L. A., T. A. Nerad, C. J. O'Kelly, M. T. Peglar, P. M. Gillevet, J. D. Silberman, and M. L. Sogin. 2000. A molecular reassessment of the leptomyxid amoebae. Protist 151:275-282.[ISI][Medline]

    Anderson, O. 1997. Annual abundances, diversity and growth potential of gymnamoebae in a shallow freshwater pond. J. Eukaryot. Microbiol. 44:393-398.[ISI]

    Arisue, N., T. Hashimoto, J. A. Lee, D. V. Moore, P. Gordon, C. W. Sensen, T. Gaasterland, M. Hasegawa, and M. Müller. 2002. The phylogenetic position of the pelobiont Mastigamoeba balamuthi based on sequences of rDNA and translation elongation factors EF-1alpha and EF-2. J. Eukaryot. Microbiol. 49:1-10.[ISI][Medline]

    Bakker-Grunwald, T., and C. Wöstmann. 1993. Entamoeba histolytica as a model for the primitive eukaryotic cell. Parasitol. Today 9:27-31.[CrossRef][ISI]

    Baldauf, S. L., and J. D. Palmer. 1993. Animals and fungi are each other's closest relatives: congruent evidence from multiple proteins. Proc. Natl. Acad. Sci. USA 90:11558-11562.[Abstract]

    Baldauf, S. L., A. J. Roger, I. Wenk-Siefert, and W. F. Doolittle. 2000. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290:972-977.[Abstract/Free Full Text]

    Bapteste, E., H. Brinkmann, and J. A. Lee, et al. (11 co-authors). 2002. The analysis of 100 genes supports the grouping of three highly divergent amoebae: Dictyostelium, Entamoeba, and Mastigamoeba. Proc. Natl. Acad. Sci. USA 99:1414-1419.[Abstract/Free Full Text]

    Bhattacharya, D., and K. Weber. 1997. The actin gene of the glaucocystophyte Cyanophora paradoxa: analysis of the coding region and introns, and an actin phylogeny of eukaryotes. Curr. Genet. 31:439-446.[CrossRef][ISI][Medline]

    Bolivar, I., J. F. Fahrni, A. Smirnov, and J. Pawlowski. 2001. SSU rRNA-based phylogenetic position of the genera Amoeba and Chaos (Lobosea, Gymnamoebia): the origin of gymnamoebae revisited. Mol. Biol. Evol. 18:2306-2314.[Abstract/Free Full Text]

    Bovee, E. 1985. Class Lobosea Carpenter 1861. Pp. 158–211 in J. Lee, S. Hutner, and E. Bovee, eds. An illustrated guide to the Protozoa. Society of Protozoologists, Lawrence, Kansas.

    Butler, H., and A. Rogerson. 2000. Naked amoebae from benthic sediments in the Clyde Sea area, Scotland. Ophelia 53:37-54.[ISI]

    Cavalier-Smith, T. 1987. Eukaryotes with no mitochondria. Nature 326:332-333.[CrossRef][ISI][Medline]

    1993. Kingdom Protozoa and its 18 phyla. Microbiol. Rev. 57:953-994.[ISI][Medline]

    1998. A revised six-kingdom system of life. Biol. Rev. Camb. Philos. Soc. 73:203-266.[CrossRef][ISI][Medline]

    2000. Flagellate megaevolution: the basis for eukaryote diverification. Pp. 361–390 in J. R. Green and B. S. C. Leadbeater, eds. The flagellates: unity, diversity and evolution. Taylor and Francis, London.

    Cavalier-Smith, T., and E. E. Chao. 1996. Molecular phylogeny of the free-living archezoan Trepomonas agilis and the nature of the first Eukaryote. J. Mol. Evol. 43:551-562.[ISI][Medline]

    Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[CrossRef][ISI][Medline]

    Clark, C. G., and G. A. Cross. 1988. Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas. Mol. Biol. Evol. 5:512-518.[Abstract]

    Clark, C. G., and A. Roger. 1995. Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 92:6518-6521.[Abstract]

    Corliss, J. O. 1994. An interim utilitarian (‘user friendly’) hierarchical classification and characterization of the protists. Acta Protozool. 33:1-51.[ISI]

    Edgcomb, V. P., A. G. B. Simpson, L. Amaral Zettler, T. A. Nerad, D. J. Patterson, M. E. Holder, and M. L. Sogin. 2002. Pelobionts are degenerate protists: insights from molecules and morphology. Mol. Biol. Evol. 19:978-982.[Abstract/Free Full Text]

    Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:368-376.[ISI][Medline]

    1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791.[ISI]

    2002. PHYLIP (phylogeny inference package). Version 3.6a3. Distributed by the author, Department of Genetics, University of Washington, Seattle.

    Finlay, B. J., H. I. J. Black, S. Brown, K. J. Clarke, G. F. Esteban, R. M. Hindle, J. L. Olmo, A. Rollett, and K. Vickerman. 2000. Estimating the growth potential of the soil protozoan community. Protist 151:69-80.[ISI][Medline]

    Forget, L., J. Ustinova, Z. Wang, V. A. R. Huss, and B. F. Lang. 2002. Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi. Mol. Biol. Evol. 19:310-319.[Abstract/Free Full Text]

    Galtier, N., M. Gouy, and C. Gautier. 1996. SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 12:543-548.[Abstract]

    Hasegawa, M., and H. Kishino. 1994. Accuracies of the simple methods for estimating the bootstrap probability of a maximum likelihood tree. Mol. Biol. Evol. 11:142-145.[Free Full Text]

    Hausmann, K., and N. Hülsmann. 1996. Protozoology. Georg Thieme Verlag, Stuttgart, Germany.

    Hinkle, G., D. D. Leipe, T. A. Nerad, and M. L. Sogin. 1994. The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi. Nucleic Acids Res. 22:465-469.[Abstract]

    Hinkle, G., and M. L. Sogin. 1993. The evolution of the Vahlkampfiidae as deduced from 16S-like ribosomal RNA analysis. J. Eukaryot. Microbiol. 40:599-603.[ISI][Medline]

    Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8:275-282.[Abstract]

    Kelleher, J. F., S. J. Atkinson, and T. D. Pollard. 1995. Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. J. Cell. Biol. 131:385-397.[Abstract]

    Kishino, H., T. Miyata, and M. Hasegawa. 1990. Maximum likelihood inference of protein phylogeny, and the origin of chloroplasts. J. Mol. Evol. 31:151-160.[ISI]

    Lanave, C., G. Preparata, C. Saccone, and G. Serio. 1984. A new method for calculating evolutionary substitution rates. J. Mol. Evol. 20:86-93.[ISI][Medline]

    Larsen, N., G. J. Olsen, B. L. Maidak, M. J. McCaughey, R. Overbeek, T. J. Macke, T. L. Marsh, and C. R. Woese. 1993. The ribosomal database project. Nucleic Acids Res. 21:3021-3023.[Abstract]

    Lee, J. J., G. F. Leedale, and P. Bradbury. 2000. An illustrated guide to the Protozoa. 2nd edition. Society of Protozoologists, Lawrence, Kansas.

    Levine, N. D., J. O. Corliss, and F. E. Cox, et al. (16 co-authors). 1980. A newly revised classification of the Protozoa. J. Protozool. 27:37-58.[ISI][Medline]

    Margulis, L. 1974. Five-kingdom classification and the origin and evolution of cells. Evol. Biol. 7:45-78.

    Margulis, L., J. Corliss, M. Melkonian, and D. Chapman. 1990. Handbook of Protoctista. Jones and Bartlett, Boston.

    Milyutina, I. A., V. V. Aleshin, K. A. Mikrjukov, O. S. Kedrova, and N. B. Petrov. 2001. The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication. Gene 272:131-139.[CrossRef][ISI][Medline]

    Neefs, J. M., Y. Van de Peer, P. De Rijk, S. Chapelle, and R. De Wachter. 1993. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res. 21:3025-3049.[Abstract]

    Page, F. 1987. The classification of ‘naked’ amoebae (phylum Rhizopoda). Arch. Protistenk. 133:199-217.[ISI]

    Page, F., and L. Blanton. 1985. The Heterolobosea (Sarcodina: Rhizopoda), a new class uniting the Schizopyrenida and the Acrasidae (Acrasida). Protistologica 21:121-132.[ISI]

    Pawlowski, J., I. Bolivar, J. F. Fahrni, C. De Vargas, and S. S. Bowser. 1999. Molecular evidence that Reticulomyxa filosa is a freshwater naked foraminifer. J. Eukaryot. Microbiol. 46:612-617.[ISI][Medline]

    Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817-818.[Abstract]

    Rodriguez, F., J. L. Oliver, A. Marin, and J. R. Medina. 1990. The general stochastic model of nucleotide substitution. J. Theor. Biol. 142:485-501.[ISI][Medline]

    Saitou, N., and M. Nei. 1987. The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]

    Schafer, D. A., and T. A. Schroer. 1999. Actin-related proteins. Annu. Rev. Cell. Dev. Biol. 15:341-363.[CrossRef][ISI][Medline]

    Silberman, J. D., C. G. Clark, L. S. Diamond, and M. L. Sogin. 1999. Phylogeny of the genera Entamoeba and Endolimax as deduced from small-subunit ribosomal RNA sequences. Mol. Biol. Evol. 16:1740-1751.[Abstract/Free Full Text]

    Sims, G. P., A. Rogerson, and R. Aitken. 1999. Primary and secondary structure of the small-subunit ribosomal RNA of the naked, marine amoeba Vannella anglica: phylogenetic implications. J. Mol. Evol. 48:740-749.[ISI][Medline]

    Smirnov, A. 2001. Vannella ebro n. sp. (Lobosea, Gymnamoebia), isolated from cyanobacterial mats in Spain. Europ. J. Protistol. 37:147-153.[ISI]

    Sogin, M. L. 1991. Early evolution and the origin of eukaryotes. Curr. Opin. Genet. Dev. 1:457-63.[Medline]

    Stechmann, A., and T. Cavalier-Smith. 2002. Rooting the eukaryote tree by using a derived gene fusion. Science 297:89-91.[Abstract/Free Full Text]

    Strimmer, K., and A. Von Haeseler. 1996. Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964-969.[Free Full Text]

    Swofford, D. L. 1998. PAUP*: phylogenetic analyses using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.

    Szenasi, Z., T. Endo, K. Yagita, and E. Nagy. 1998. Isolation, identification and increasing importance of ‘free-living’ amoebae causing human disease. J. Med. Microbiol. 47:5-16.[Abstract]

    Wang, H., M. Qi, and A. J. Cutler. 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res. 21:4153-4154.[ISI][Medline]

    Wuyts, J., P. De Rijk, Y. Van de Peer, G. Pison, P. Rousseeuw, and R. De Wachter. 2000. Comparative analysis of more than 3000 sequences reveals the existence of two pseudoknots in area V4 of eukaryotic small subunit ribosomal RNA. Nucleic Acids Res. 28:4698-4708.[Abstract/Free Full Text]

Accepted for publication June 16, 2003.