Department of Zoology and Animal Biology, University of Geneva, Switzerland
Department of Invertebrate Zoology, St. Petersburg State University, Russia
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Abstract |
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Introduction |
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Very little is known about the evolution of amoebae, and despite the rapid development of molecular studies only a few amoeba sequences are available. For over one decade, the naked amoebae were represented in phylogenetic trees only by three medically important genera, Acanthamoeba, Entamoeba (Gymnamoebae), and Naegleria (Heterolobosea). Although this situation is slowly changing, at least in what concerns the rRNA database (Sims, Rogerson, and Aitken 1999
; Amaral Zettler et al. 2000
), our knowledge of phylogenetic relationships among amoebae remains largely fragmentary.
One of the most enigmatic points concerns the phylogenetic origin of amoebae. The first molecular data confirmed the polyphyly of amoebae (Clark and Cross 1988
), in agreement with the morphology-based distinction between the classes Lobosea and Heterolobosea (Page and Blanton 1985
). Comparison of small subunit (SSU) rRNA gene sequences of Acanthamoeba castellanii (Lobosea) and Naegleria gruberi (Heterolobosea) shows that both species branch separately, the first one emerging within a radiation of eukaryotes (Gunderson and Sogin 1986
), the second one branching in the middle part of the SSU tree (Baverstock et al. 1989
). The independent origin of Naegleria was later confirmed by molecular studies on other Vahlkampfiidae (Hinkle and Sogin 1993
). The analysis of SSU rRNA also suggested a lack of any specific relationship between A. castellanii and Entamoeba histolytica (Sogin 1989
), leading to the exclusion of Entamoeba from the Lobosea and to the creation of a new parvkingdom of Entamoebia (Cavalier-Smith 1993
). Among all examined Gymnamoebia, only Hartmannella vermiformis shows a relationship to A. castellanii (Gunderson, Goss, and Sogin 1994
; Weekers et al. 1994
). The position of both species in the upper part (generally referred to as the crown) of the SSU tree was considered as representative for gymnamoebae. However, the SSU rRNA of Vannella anglica, another Gymnamoebia, did not associate with those of the two species (Sims, Rogerson, and Aitken 1999
).
The SSU rRNA sequence was also used to examine the phylogeny of other amoeboid protists. In particular, these data showed that the euglyphid filose amoebae (Bhattacharya, Helmchen, and Melkonian 1995
) and the anaerobic pelobiontid amoeboflagellate Phreatamoeba balamuthi (Hinkle et al. 1994
) have an independent origin. According to Simpson and others (Simpson et al. 1997
; Walker et al. 2001
), P. balamuthi should be considered to belong to the genus Mastigamoeba; nevertheless, despite their morphological similarity, the SSU sequences of P. balamuthi and Mastigamoeba invertens do not group together. Later, this discrepancy was interpreted as an artifact because of the heterogenous evolutionary rates (Stiller and Hall 1999
). On the other hand, M. invertens was shown to branch among the earliest eukaryotes in the tree based on the gene encoding the largest subunit of RNA polymerase II (RPB1) (Stiller, Duffield, and Hall 1998
).
In view of these data, based principally on ribosomal DNA sequences, the polyphyly of amoeboid protists seems to be well established. However, the results of some recent reanalyses of SSU rRNA sequences question the solidity of this hypothesis. Maximum likelihood reanalysis of SSU rRNA sequences (Cavalier-Smith and Chao 1996
) showed that E. histolytica, P. balamuthi, A. castellanii, and H. vermiformis branch together, in opposition to the previous results of the same data set. Moreover, in some maximum likelihood (ML) and maximum parsimony (MP) trees, Lobosea appears as a sister group to Archamoebae and Mycetozoa, supporting the inclusion of all the three groups in the phylum Amoebozoa (Cavalier-Smith 1998a
). The close relationships between gymnamoebae were later confirmed by the study of E. histolytica and Endolimax nana SSU rDNA sequences (Silberman et al. 1996
). The lobose amoebae grouped together also in the ML analysis of the SSU rRNA of leptomyxid amoebae (Amaral Zettler et al. 2000
), although the limited number of nonamoeboid species used in this study does not allow any general conclusion.
A common origin for Amoebozoa was also proposed based on the analysis of combined protein data (Baldauf et al. 2000
). The grouping of Acanthamoeba with Mycetozoa (Dictyostelium and Physarum) is supported by protein sequences, like actin (Bhattacharya and Weber 1997
; Philippe and Adoutte 1998
) and actin-related proteins ARP2 and ARP3 (Kelleher, Atkinson, and Pollard 1995
; Schafer and Schroer 1999
), as well as by similarities in genome organization between Acanthamoeba and Dictyostelium (Iwamoto et al. 1998
). However, the position of gymnamoebae in other protein trees is extremely variable. For example, E. histolytica branches at the base of the EF-1
tree (Baldauf and Palmer 1993
) and as a sister group to Euglenozoa in the EF-2 tree (Moreira, Le Guyader, and Philippe 2000
), whereas A. castellanii branches with plants in the RPB1 tree (Stiller and Hall 1997
). Given that in most cases, the sequences of both amoebae are not available for the same protein, their mutual relationships cannot be inferred.
Here, we report the first SSU rRNA gene sequences of four typical large lobose amoebae of the family Amoebidae. Among them is the most commonly studied species, Amoeba proteus. These sequences were compared to those of other amoebae and various groups of eukaryotes. The phylogenetic relations within and between amoeboid protists were analyzed using different evolutionary models and methods to test the hypothesis of gymnamoebae monophyly.
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Materials and Methods |
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Molecular Genetics
For total RNA extraction the cells were lysed in Catrimox (Iowa Biotechnology Corp.), a surfactant that selectively precipitates nucleic acids (Dahle and Macfarlane 1993
). Total RNA was then purified from the Catrimox precipitate with Tri-Reagent (Molecular Research Center, Inc.) (Chomczynski and Sacchi 1987
). The whole SSU rRNA of A. proteus strain Ge was amplified in three overlapping fragments by RT-PCR using universal primers.
The sequences of A. leningradensis, C. nobile, C. carolinensis, and a partial sequence of another A. proteus, the Warsaw strain (data not shown), were obtained by amplifying DNA extractions of these cells with universal as well as A. proteusspecific primers. The sequence of A. proteus strain Ge was also obtained from DNA amplification and found to be identical to the cDNA sequence. DNA extractions, amplifications, cloning, and sequencing were performed as described previously (Pawlowski et al. 1999
). The new sequences reported in this paper have been deposited in the EMBL/GenBank database (accession numbers AJ314604, AJ314605, AJ314607, and AJ314606).
Sequence Analysis
Four sequences obtained in this study and seven sequences of other amoebae were added to the secondary structure-based alignment of the SSU rRNA (Van de Peer et al. 2000
). The sequences were aligned manually using SEAVIEW software (Galtier and Gouy 1996
). The 19 unambiguously aligned regions selected for phylogenetic analyses are composed of 1,110 sites (excluding gaps), which include 774 variable characters, among which 713 are parsimony informative. The sequences were analyzed using the following methods: the Neighbor-Joining (NJ) method (Saitou and Nei 1987
) using K2P, K3P, F84, HKY85, Tamura-Nei, and general time reversible model (GTR) substitution models; the MP method; and the ML method using HKY85 and GTR models, all as implemented in PAUP 4.0 (Swofford 1993
). The NJ and ML analyses were performed with or without gamma distribution (G) with estimated parameter
= 0.45 and six rate categories. The proportion of invariable sites (I) was estimated at 0.04. In the HKY85 model, the transition-transversion ratio was estimated at 1.3. In GTR models, the estimated substitution probabilities were 1.02 (AC), 2.17 (AG), 1.25 (AT), 0.84 (CG), 3.85 (CT), 1 (GT). All model parameters were estimated via ML. Additionally, the ML tree was constructed using fastDNAml program (Olsen et al. 1994
) as implemented in PHYLO_WIN (Galtier and Gouy 1996
). The reliability of internal branches in NJ, MP, and ML trees was assessed, respectively, by 1,000, 1,000, and 100 bootstrap replicates (Felsenstein 1988
). The relative rate test was carried out using RRTree program (Robinson et al. 1998
).
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Results |
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Evolutionary Rates
The relative rate test was used to examine the evolutionary rates within gymnamoebae, and between them and other eukaryotes. The test shows that most gymnamoebae and the amoeboflagellate M. invertens evolve at rates similar to those of species which branch in the crown of the SSU eukaryotic tree (table 2
). Significantly higher rates (at 1% level) are observed only in Entamoebidae (Entamoeba + Endolimax), as well as in Heterolobosea (Naegleria + Vahlkampfia) and Mycetozoa (Dictyostelium + Physarum). Among other gymnamoebae and amoeboflagellates, the most rapidly evolving are P. balamuthi, F. nolandi, Gephyramoeba sp., and Saccamoeba limax. Their evolutionary rates are significantly different from those of crown species at the 5% level (table 2
). Comparison of rates within gymnamoebae shows significant differences (at the 1% level) only in the case of Entamoebidae and S. limax.
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Two other well-supported groups of gymnamoebae are Acanthamoeba + Balamuthia and Entamoeba + Endolimax. The last one also includes the pelobiont P. balamuthi. Both clades appear in all types of analyses supported, respectively, by 97%100% and 71%87% bootstrap values (fig. 1 ). The positions of both clades change depending on the method of analysis (table 3 ). The clade Acanthamoeba + Balamuthia branches with Gymnamoebia sensu stricto in MP and ML (GTR or F84) analyses, but clusters with Plantae in NJ trees without gamma correction. The grouping of Entamoeba + Endolimax + Phreatamoeba with Gymnamoebia sensu stricto (fig. 1 ) appears only in ML (F84) trees. In other analyses, the clade Entamoeba + Endolimax + Phreatamoeba branches separately.
Three gymnamoebae species V. anglica, Gephyramoeba sp., and F. nolandi, which appear as independent lineages in the ML tree (fig. 1 ), change their position in some other analyses. Gephyramoeba sp. and F. nolandi branch together in MP and some ML and NJ trees (table 3 ). Both species branch with Heterolobosea and Mycetozoa (or both) in some analyses (data not shown). Some affinities are also observed between V. anglica and the clade Entamoeba + Endolimax + Phreatamoeba. None of these relationships, however, is supported by a bootstrap value higher than 50%.
In the NJ tree with gamma and invariable sites corrections, all gymnamoebae group together (fig. 2 ). This grouping is not statistically supported, yet it remains stable whatever the model of nucleotide substitution. Depending on the model, the topologies differ in the position of the two mycetozoan species (Dictyostelium discoideum and Physarum polycephalum). Both species branch within the gymnamoebae when GTR or Tamura-Nei models are used (fig. 2 ), whereas they appear in the lower part of the tree, close to the Euglenozoa and the Heterolobosea, when K2P or other models are used.
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Discussion |
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The position of Amoebidae and related taxa in the middle part of the eukaryote tree (fig. 1
) contrasts with some previous studies that showed gymnamoebae (A. castellanii and H. vermiformis) branching in the crown of the SSU tree, as a sister group to Plantae (Schlegel 1991
) or Metazoa and Fungi (Bhattacharya, Helmchen, and Melkonian 1995
; Sogin and Silberman 1998
; Sims, Rogerson, and Aitken 1999
). Our data show that by adding new taxa, the position of amoebae in the SSU tree changes. It could be argued that this change results from attraction by the most rapidly evolving amoeboid lineages. Several studies demonstrate that the topology of SSU trees, in particular the position of early diverging lineages, is biased by the among-lineages rate variations (Philippe, Germot, and Moreira 2000
; Van de Peer et al. 2000
). It has also been argued that the position of some amoebae is biased by their rapid rates of evolution (Stiller and Hall 1999
). However, when the rapidly evolving species (E. histolytica, E. nana, P. balamuthi, Gephyramoeba sp., and Filamoeba sp.) are omitted from the analysis, the other amoeba species remain indeed below the crown. Moreover, the real place for the root of the SSU tree is not known, and therefore one cannot say which taxa diverged early and which late; other possible roots are discussed in Cavalier-Smith (2000a)
.
The System of Gymnamoebia sensu stricto
Traditionally, all naked amoebae possessing lobose pseudopodia are included in the subclass Gymnamoebia that comprises four orders and three incertae sedis families (Page 1987
). In our analysis, six independent lineages of gymnamoebae appear. The majority of the species (11) branch in a clade grouping mostly species from the orders Euamoebida and Leptomyxida. As these species represent the most typical gymnamoebae, we propose considering this clade as a representative for the subclass Gymnamoebia (fig. 1
). Among other amoeba lineages, two are composed of more than one species (A. castellanii + B. mandrillaris and E. histolytica + E. nana + P balamuthi), whereas three others are single species lineages (Gephyramoeba sp., F. nolandi, and V. anglica).
The clade Acanthamoeba + Balamuthia corresponds most probably to the order Acanthopodida, which traditionally includes a single family Acanthamoebidae (Sawyer and Griffin 1975
; Page 1987
). The close relationship between both genera has been demonstrated using rRNA data (Stothard et al. 1998
; Amaral Zettler et al. 2000
).
From a morphological point of view their grouping is quite unexpected. Balamuthia mandrillaris was initially described as a leptomyxid (Visvesvara et al. 1990
). However, more detailed study of B. mandrillaris showed that the species differs fundamentally from other leptomyxids (Gephyramoeba and Leptomyxa) in the morphological, physiological, and antigenic characteristics (Visvesvara, Schuster, and Martinez 1993
). At the same time, it has been observed that B. mandrillaris possesses a MTOC similar to those seen in A. castellanii (Visvesvara, Schuster, and Martinez 1993
). The presence of this basic cellular feature in both species reconciles somehow the molecular and morphological data. It will be interesting to compare our data with the sequence of Stereomyxa, which also possesses similar MTOC's (Von Benwitz and Grell 1971a, 1971b
).
Another well-supported clade of amoeboid protists is composed of E. histolytica + E. nana + P. balamuthi. This clade was shown previously in rRNA trees (Cavalier-Smith and Chao 1997
; Cavalier-Smith 1998b, 2000b;
Silberman et al. 1999
). The clade exists only if the sequence of E. nana is included. If E. nana is omitted, E. histolytica and P. balamuthi branch separately. This explains why the relationship between P. balamuthi and Entamoebidae has not been suggested by Hinkle et al. (1994)
. In fact, P. balamuthi is a pelobiont, i.e., a free-living amoeboflagellate that lacks mitochondria and Golgi bodies. It shares with Entamoebidae general features, such as an anaerobic life mode and a remarkably simplified intracellular organization (Silberman et al. 1999
), yet no shared derived morphological features have been identified so far.
Among the other three lineages composed of single species (Gephyramoeba sp., F. nolandi, and V. anglica), the first two are quite unusual gymnamoebae. Based on the morphological characteristics, they are classified, respectively, in the suborder Leptoramosina (order Leptomyxida) and in incertae sedis family (Page 1987
). In our analyses, both species tend to group together (table 2 ), but their grouping is weakly supported. The case of V. anglica is much more puzzling. Similar to a previous study (Sims, Rogerson, and Aitken 1999
), our analysis of the molecular data shows this species as an independent lineage branching in the middle part of the rRNA tree. Yet, the vannellids are quite typical gymnamoebae (Page 1987
) and no obvious morphological or ultrastructural feature could distinguish Vannella from the other Gymnamoebia. As the unexpected position of Vannella in the trees cannot be attributed to a significantly fast rate of evolution (table 2
), other explanations will have to be found.
Monophyly of Gymnamoebae?
The presence of several independent lineages of gymnamoebae in the SSU tree is in agreement with the hypothesis of a polyphyletic origin of the amoebae. This hypothesis is generally accepted; however, Heterolobosea alone shows an independent origin, based on solid morphological and molecular evidences (Page and Blanton 1985
; Page 1987
; Clark and Cross 1988
; Roger et al. 1996
). The polyphyly of other amoebae, based solely on SSU rDNA data, remains disputable. Possible close relationships of gymnamoebae have been discussed already based on some previous analyses of SSU (Cavalier-Smith and Chao 1996
; Cavalier-Smith 1998a;
Silberman et al. 1999
). The present study shows that these relationships appear stronger as the number of examined species increased. In all our analyses, the gymnamoebae branch close to each other; however, they group together only in NJ analysis using gamma correction for among-site rate variations (fig. 2 ). Although the statistical support for this grouping is very weak, it is possible that by adding more amoebae SSU sequences their relationships will be better resolved.
Most striking is the grouping of Lobosea together with Mycetozoa (represented by Dictyostelium and Physarum) in the phylum Amoebozoa (Cavalier-Smith 1998a
). The same situation is evident in our SSU-tree (fig. 2
), in the actin-tree of Bhattacharya and Weber (1997)
and in and a recently published eukaryote tree based on combined protein data (Baldauf et al. 2000
). Interestingly, the two mycetozoan species are closely related to Gephyramoeba; the latter was initially seen (Goodey 1914
) as a possible bridge between Amoeba and Mycetozoa. Indeed, the plasmodial stages of Gephyramoeba clearly evoke those of Mycetozoa (see discussion in Visvesvara, Schuster and Martinez 1993
).
Both SSU (this work) and protein trees (Baldauf et al. 2000
) also agree in grouping together the Euglenozoa and Heterolobosea in a unit corresponding to the infrakingdom Discicristata (Cavalier-Smith 1993
). The major handicap of the protein tree, however, is the very limited number of amoebae protein sequences. In fact, all analyses are based on the sequences of the single species A. castellanii. It is therefore difficult to make any conclusion before the number of available protein data on amoebae increases.
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Acknowledgements |
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Footnotes |
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Keywords: amoebae
rRNA
phylogeny
Address for correspondence and reprints: Jan Pawlowski, Station de Zoologie, 154, rte de Malagnou, CH-1224 Chêne-Bougeries, Switzerland. jan.pawlowski{at}zoo.unige.ch
.
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