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1 Department of Evolutionary Biochemistry, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia
2 Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland
3 Institute of Biology of Inland Waters, Russian Academy of Sciences, Yaroslavskaya obl., Borok, Russia
Correspondence
Sergey Nikolaev
Sergey.Nikolaev{at}medecine.unige.ch
Cédric Berney
Cedric.Berney{at}zoo.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the SSU rRNA gene sequences of Multicilia marina and the unidentified eukaryote clone Borok are AY268037 and AY626163, respectively.
A figure showing the predicted secondary structure of the small-subunit rRNA gene of Multicilia marina is available as supplementary material in IJSEM Online.
Present address: Department of Genetic Medicine and Development, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. 
Present address: Department of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK.
These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) at the Solovetskaya station (Onega Bay, White Sea). Its cells are usually spherical, about 30–40 µm in diameter, although some of them can have an oblong or irregular form. Typically, each cell has approximately 20–30 flagella. The basal apparatus of each flagellum is represented by a single kinetosome surrounded by a conical microtubular sheath. Submembrane interkinetosomal fibres connect each kinetosome to a neighbouring one and coordinate the movement of the flagella. No microtubular connectives between the basal apparatus and the nucleus have been observed (Mikrjukov & Mylnikov, 1996, 1998). The mitochondrial cristae are tubular. Multicilia marina normally feeds by phagocytosis, using lobopodia for the capture of large prey (gymnamoebae).
Cienkowski (1881) described Multicilia as an intermediate stage between flagellates and heliozoans. Kudo (1954) considered it to be a member of the order Rhizomastigina Butschli, together with the family Mastigamoebidae and two groups of heliozoans, the actinophryids and the dimorphids. Later ultrastructural studies revealed that the unusually weak and oscillating movements of the flagella of Multicilia marina (resulting in their superficial similarity to heliozoan axopodia), together with the poor coordination in their activity and a lack of anterior and posterior ends, make Multicilia marina distinct from all other known multiflagellated/ciliated protistan taxa, such as Caryoblastea, Pseudociliata, Hemimastigophora, Ciliophora, Opalinata and Parabasalida. A new phylum, Multiflagellata, of uncertain affinities was proposed to include the genus Multicilia (Mikrjukov & Mylnikov, 1996, 1998). In contrast, Cavalier-Smith (1998) proposed that Multicilia belongs in the phylum Amoebozoa, uniting lobose amoebae, the mycetozoan slime moulds and the amitochondriate entamoebids and pelobionts (Archamoebae), and that the genus is either related to Archamoebae (Cavalier-Smith, 2000) or to Vannellidae (Cavalier-Smith, 2003; Cavalier-Smith et al., 2004). In the second edition of the Illustrated Guide to the Protozoa, Multicilia was placed among ‘residual heterotrophic flagellates' (Patterson et al., 2000).
In spite of the rapid accumulation of sequences from amoeboid organisms during the past few years (Amaral Zettler et al., 2000; Bolivar et al., 2001; Fahrni et al., 2003; Peglar et al., 2003; Cavalier-Smith et al., 2004; Nikolaev et al., 2004, 2005), to date no molecular data have been reported for Multicilia. Previous molecular phylogenetic studies, based on actin and small-subunit (SSU) rRNA gene sequences, indicated the monophyly of Amoebozoa, although with very weak support (Bolivar et al., 2001; Fahrni et al., 2003; Cavalier-Smith et al., 2004). Furthermore, several higher-level taxonomic lineages have been distinguished within Amoebozoa (e.g. Smirnov et al., 2005). However, the relationships between and within these lineages remain unclear. It has been proposed that the ancestral amoebozoan may have been a flagellated organism similar to Phalansterium or Mastigamoeba and that lobose amoebae evolved by multiple flagella losses (Cavalier-Smith et al., 2004).
To ascertain the amoebozoan affiliation of Multicilia and to investigate the relationships between flagellated and non-flagellated members of the phylum, we obtained the complete SSU rRNA gene of Multicilia marina and performed phylogenetic analyses, including an exhaustive sampling of eukaryotes and all available sequences of Amoebozoa. We also constructed a predicted secondary structure of the SSU rRNA molecule for Multicilia marina and compared it with those of other eukaryotes. Our study confirmed that Multicilia is an amoebozoan and allowed us to propose a new hypothesis about the phylogenetic relationships within the phylum Amoebozoa.
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METHODS |
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, between the settlement Noviy Svet and the village Vesyolye (Sudak district of Crimea). DNA was extracted using a DNeasy Plant Minikit (Qiagen). The complete SSU rRNA gene sequence of Multicilia marina was amplified using the universal primers sA (5'-ACCTGGTTGATCCTGCCAGT-3') and sB (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). PCR was performed in a total volume of 50 µl with an amplification profile consisting of 40 cycles of 30 s at 94 °C, 30 s at 50 °C and 2 min at 72 °C, followed by 5 min at 72 °C for the final extension. The PCR products were purified using a High Pure PCR Purification kit (Roche), then ligated into the pGEM-T vector system (Promega), transformed in XL-2 ultracompetent cells (Stratagene), sequenced using an ABI PRISM BigDye Terminator cycle sequencing kit and analysed using an ABI-377 DNA sequencer (Perkin Elmer), all according to the manufacturers' instructions.
In addition, we obtained the SSU rRNA gene sequence (as described above) of an unidentified amoeba (unidentified eukaryote clone Borok), which was a contaminant in a culture of the centrohelid Clathrulina elegans isolated from waste treatment plants in Borok.
SSU rRNA gene phylogenetic analyses and secondary-structure modelling.
Complete SSU rRNA gene sequences of Multicilia marina and the unidentified eukaryote clone Borok were aligned manually with sequences from diverse eukaryotes using the Genetic Data Environment software (Larsen et al., 1993), following the secondary-structure model proposed by Wuyts et al. (2001). A first dataset, including the two sequences obtained in this study and sequences of 23 lobose amoebae, 25 opisthokonts and 50 bikonts, was used to infer the position of Multicilia marina among eukaryotes (1421 unambiguously aligned positions). More-detailed phylogenetic relationships within the Amoebozoa were then assessed using an alignment of 64 sequences, including the two sequences obtained in this study and 58 sequences of other amoebozoans (including the more divergent mycetozoans, pelobionts and entamoebids) and four opisthokonts as an outgroup (1290 unambiguously aligned positions). GenBank/EMBL/DDBJ accession numbers for all sequences used in our analyses are indicated in Figs 1 and 2.
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), using the general time-reversible (GTR) model of evolution (Lanave et al., 1984; Rodriguez et al., 1990), taking into account a proportion of invariable sites and a gamma distribution of the rates of substitution for the variable positions, with eight rate categories. All parameters were estimated from the dataset. Bayesian analyses were performed on the two datasets with MRBAYES (Huelsenbeck & Ronquist, 2001), using the same model. Two runs starting from different random trees were performed for 1 200 000 generations and sampled every 100 generations, with four simultaneous chains. The trees sampled before the chains reached stationarity were discarded as a burn-in (2000). In addition, a ML bootstrap analysis was performed for the second dataset with PHYML (Guindon & Gascuel, 2003). The predicted secondary structure of the SSU ribosomal molecule was modelled with MFOLD (Zuker et al., 1999) and visualized with RnaViz (De Rijk & De Wachter, 1997). |
RESULTS |
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; KCl, 0.38 ; MgCl2.6H2O, 3.15 ; MgSO4.7H2O, 3.95 ; CaCl2.H2O, 0.83 ; KNO3, 0.06 ; K2HPO4.3H2O, 0.006 ; pH 6.5–7.5) and was fed with the lobose amoeba Vannella sp. All attempts to cultivate Multicilia using as food the bacterium Pseudomonas fluorescens (which grew after addition of rice or wheat grains to the culture), the alga Chlorella, the bodonids Bodo designis, Bodo saltans or Procryptobia sorokini, or the chrysomonads Spumella or Paraphysomonas, failed. During light microscopic observation, it was established that, at the time of feeding, cells of Multicilia marina used their ventral side to cover the amoeba Vannella, and then stood motionless. The predator resumed its motion several seconds after the capture of its prey.
Unusual length of the SSU rRNA gene sequence of Multicilia marina
The length of the complete, amplified SSU rRNA gene sequence of Multicilia marina was 2746 bp, which is the second-longest amoebozoan SSU rRNA gene sequence after that of Pelomyxa palustris (3502 bp). The length of the SSU rRNA gene sequence of Multicilia marina is similar to those of pelobionts and Endolimax nana. In other Amoebozoa, the SSU rRNA gene is generally longer than that in most eukaryotes, but does not exceed 2200 bp, with the exception of Acanthamoeba spp. (Table 1).
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). One of the three insertions in region V2 corresponds to the additional helix E8_1, which is absent from most eukaryotes. The other two correspond to extensions in the terminal loops of helices 10 and 11. Two insertions are present in region V4 (extensions in the terminal loops of helices E23_12 and E23_14). The insertion in region V5 corresponds to an extension in the terminal loop of helix 29. The insertion in region V8 corresponds to the additional helix E45_1, which is absent from most eukaryotes, but present in all Archamoebae and some other amoebozoans. Remarkably, at 426 bp, variable region V7 (helices 43 and E43_1 to E43_4) of the SSU rRNA gene of Multicilia marina is among the longest of all eukaryotic sequences available to date, with longer insertions present to our knowledge only in the strepsipteran insects. The secondary structure proposed for this region in Supplementary Fig. S1 is tentative and other structures are possible.
Phylogenetic position of Multicilia marina
Fig. 1 shows the result of an ML analysis of 100 SSU rRNA gene sequences of eukaryotes, including Multicilia marina, the unidentified eukaryote clone Borok, 23 lobose amoebae and 75 other eukaryotes representing the most sequenced lineages of opisthokonts and bikonts. In the absence of a prokaryotic outgroup, the tree is presented in an unrooted format, with a basal trifurcation. The analysis clearly demonstrates that Multicilia marina belongs to the Amoebozoa, the monophyly of which was supported by a Bayesian posterior probability (PP) of 1.00. Opisthokonts also form a strongly supported clade (PP=1.00). In contrast, bikonts form two clusters: Ancyromonas and apusomonads branch together next to the opisthokonts, although without Bayesian support, whereas the remaining bikont lineages form a weakly supported group (PP=0.54) and resolution among them is typically poor.
In the tree presented in Fig. 1, Multicilia marina branches next to Gephyramoeba sp. (PP=0.84) and together they form a sister-group to a strongly supported clade (PP=0.97) comprising Filamoeba nolandi, the unidentified eukaryote clone Borok and an environmental clone (RT5iin44) that was described by Amaral Zettler et al. (2002). The Multicilia + Filamoeba + Gephyramoeba clade is supported by a PP of 0.80. Phalansterium solitarium branches at the base of this clade, but with lower support (PP=0.65).
Phylogenetic relationships among Amoebozoa
To further investigate the position of Multicilia marina among Amoebozoa, ML and Bayesian analyses were performed on a second dataset including the two sequences obtained in this study and those of 12 mycetozoans, 5 pelobionts, 6 entamoebids and 35 other amoebozoans, with 4 opisthokonts as an outgroup (Fig. 2). As in Fig. 1, Multicilia marina branches within a clade containing Phalansterium solitarium, Gephyramoeba sp., F. nolandi and the two unidentified clones Borok and RT5iin44. Analysis of the second dataset revealed that this clade, supported by a PP of 0.91 and a bootstrap value (BV) of 84 %, also includes Archamoebae and Mycetozoa.
Resolution within this clade was relatively good. As in Fig. 1, Multicilia marina branches as the sister taxon to Gephyramoeba sp. (PP=1.00; BV=91 %). F. nolandi and the two unidentified clones Borok and RT5iin44 form a strongly supported clade (PP=1.00; BV=93 %). Archamoebae (supported by a PP of 1.00 and a BV of 99 %) and Mycetozoa (PP=1.00; BV=99 %) branch together (PP=0.81; BV=68 %) as a sister group to the ‘Filamoeba clade’ (PP=0.93; BV=71). Finally, Phalansterium solitarium occupies the most basal position in the clade, with a PP of 0.99 and a BV of 80 %.
As in Fig. 1, other groupings within the Amoebozoa are congruent with a recent molecular phylogeny and classification of the phylum (Smirnov et al., 2005), and were named accordingly in Fig. 2. Three well-supported clades are recognized, the classes Tubulinea and Flabellinea and the order Acanthopodida. As shown previously (Fahrni et al., 2003; Cavalier-Smith et al., 2004; Smirnov et al., 2005), the relationships between these clades and the three independent lineages formed by Dermamoeba algensis, Mayorella sp. and ‘Platyamoeba’ stenopodia were very poorly resolved (the generic status of the latter species needs revision; see, for example, Smirnov et al., 2005). In the tree shown in Fig. 2, the Tubulinea occupy a basal position within Amoebozoa, with a PP of 0.56 and a BV of 81 %.
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DISCUSSION |
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), in spite of the inclusion of several bikont lineages of unknown affinities, such as apusomonads, centrohelids, Ancyromonas, Diphylleia, Trimastix and Breviata anathema [after this paper had been accepted, ‘Mastigamoeba’ invertens was redescribed as Breviata anathema (Walker et al., 2006)], some of which have been shown to group near or with lobose amoebae in previous large-scale phylogenies of eukaryotes (e.g. Brugerolle et al., 2002; Cavalier-Smith & Chao, 2003; Cavalier-Smith, 2004). This result contradicts the initial idea that Multicilia is an intermediate stage between flagellates and heliozoans, as proposed by Cienkowski (1881) on the basis of the similarity of its body form, mode of locomotion and the presence of multiple flagella. It is also in disagreement with the more recent placement of Multicilia in the phylum Multiflagellata of uncertain affinities (Mikrjukov & Mylnikov, 1996, 1998) or among heterotrophic flagellates of uncertain affinities (Patterson et al., 2000).
The results of our study confirm the hypothesis that Multicilia belongs in the phylum Amoebozoa, as first suggested by Cavalier-Smith (1998). However, the position of Multicilia among Amoebozoa in our SSU rRNA gene trees differs from the classification of this genus in the class Discosea, together with Vannellidae, Vexilliferidae and Paramoebidae (Cavalier-Smith, 2003; Cavalier-Smith et al., 2004). This classification was based on observations of cell surface glycostyles in Multicilia (Mikrjukov & Mylnikov, 1996), possibly similar to the glycostyles present in vannellids (Bovee, 1965). However, it cannot be excluded that the glycostyles observed in Multicilia originate from its prey, Vannella. According to our analyses, the closest sequenced relative of Multicilia is Gephyramoeba sp. ATCC 50654, and both organisms branch far from Vannellidae and Vexilliferidae. Although well-supported in our analyses, the relationship between Multicilia and Gephyramoeba is difficult to assess morphologically, because the ATCC strain of Gephyramoeba sequenced by Amaral Zettler et al. (2000) was not described in detail and was possibly misidentified (A. Smirnov, personal communication).
Monophyly of all flagellated amoebozoans
Our SSU rRNA gene analyses, including the first sequence of Multicilia to be obtained and an exhaustive sampling of available complete sequences of Archamoebae and Mycetozoa, allow us to recover for the first time a monophyly of all flagellated amoebozoans (pelobionts, myxogastrids, Multicilia and Phalansterium), which branch together with a few non-flagellated lineages (entamoebids, dictyostelids, Filamoeba and Gephyramoeba) within a strongly supported clade (PP of 0.91 and BV of 84 %). As this clade contains all flagellated amoebozoans examined to date, we propose to name it Conosea (after Cavalier-Smith, 1998), in agreement with the new system of Amoebozoa published recently by Smirnov et al. (2005).
A common origin for Archamoebae and Mycetozoa was proposed previously by Cavalier-Smith (1998). In a multigene analysis of expressed sequence tag data from Mastigamoeba, Entamoeba and Dictyostelium, Bapteste et al. (2002) later claimed to have confirmed the monophyly of an Archamoebae + Mycetozoa clade, but the lack of any lobose amoebae in their study made this conclusion irrelevant. On the other hand, analyses of actin and SSU rRNA genes from a broad taxonomic sampling of Amoebozoa provided opposite results, showing Archamoebae and Mycetozoa as separate lineages (Fahrni et al., 2003). Finally, these two taxa did not group together, even with the inclusion of Phalansterium (Cavalier-Smith et al., 2004). It appears that the addition of the sequences of Multicilia and one unidentified amoeba was important to recover the monophyly of all flagellated amoebae. The monophyly of Conosea is indeed sensitive to taxon sampling, as Phalansterium does not usually branch with Filamoeba, Gephyramoeba, Archamoebae or Mycetozoa in the absence of Multicilia and the two unidentified clones Borok and RT5iin44 (data not shown). Comparison of the present results with those of previous analyses also suggests that the use of a larger sampling of Archamoebae and Mycetozoa was important to stabilize the relationships within Conosea (data not shown). In particular, the addition of two recently published sequences of Trichiales (Fiore-Donno et al., 2005) allows the breaking of the very long stem branch leading to myxogastrids. This apparently increases the bootstrap support for the close relationship between Multicilia and Gephyramoeba, as well as for the monophyly of Mycetozoa and their sister-group relationship with Archamoebae.
Apart from Multicilia, the flagellated amoebae include pelobionts, myxogastrids and Phalansterium, a genus that was previously considered as a zooflagellate (Ekelund, 2002) and was only recently transferred to the Amoebozoa, based on an analysis of its SSU rRNA gene sequence (Cavalier-Smith et al., 2004). These flagellated Amoebozoa are all characterized by a unikont flagellar apparatus, with a kinetid composed of one centriole and one flagellum, with a microtubular cone (Cavalier-Smith, 1998). Ultrastructural studies of Multicilia marina (Mikrjukov & Mylnikov, 1996, 1998) revealed that the basal apparatus of each flagellum is also represented by a single centriole and possesses a microtubular cone. However, in contrast with other flagellated amoebozoans, which possess a kinetid with the broad end of the conus of microtubules orientated towards the nucleus (Cavalier-Smith, 1998), in Multicilia the kinetid broad end is orientated towards the cell surface (Mikrjukov & Mylnikov, 1996). This might be an ancestral state or a consequence of the multiciliarity, where the flagellar apparatus does not lean on the nucleus but on the surface skeletal elements.
Some of the features observed in the primary and secondary structures of the SSU rRNA gene of Multicilia suggest its possible close relationship to Archamoebae (Table 1). The extraordinary length of the SSU rRNA gene in Multicilia is shared with the majority of Archamoebae (Hinkle et al., 1994; Milyutina et al., 2001), although this is not strong evidence for a common origin, because even closely related amoebae species may differ widely in their SSU rRNA gene length (in the Acanthopodida, for example, it varies from 1972 bp in Balamuthia mandrillaris to greater than 2500 bp in some Acanthamoeba spp.). Multicilia and all Archamoebae, except Entamoeba, possess an insertion in variable region V4, in helix E23_14 (data not shown). This insertion corresponds in Multicilia to an additional hairpin in the loop of helix E23_14. Unfortunately, because the secondary structure of region V4 has not yet been fully determined in Archamoebae, we do not know whether this region also corresponds to an additional hairpin in the loop of helix E23_14 in this taxon. Multicilia also shares with all Archamoebae an insertion in region V8 (additional helix E45_1). The presence of helix E45_1 is a characteristic feature of Archamoebae, although it is present in some other Amoebozoa (Acanthamoeba spp., Clydonella sp. and Vexillifera minutissima).
Finally, there are also some ultrastructural features of the flagellar root system that may suggest a relationship between Multicilia and Archamoebae (Cavalier-Smith, 2000). These include the presence of the microtubular cone and of a short flagellar band around the flagellum, as well as a single microtubular band (Brugerolle, 1991; Simpson et al., 1997; Mikrjukov & Mylnikov, 1998; Brugerolle & Müller, 2000; Walker et al., 2001). The loss of some components of the axoneme of the flagellum (Brugerolle & Patterson, 2000), such as the outer dynein arms, and the weak motion of the flagella in some Archamoebae (Mastigina, Pelomyxa) are also shared by Multicilia. Perhaps the ancestor of Archamoebae lived, as Multicilia, in an aerobic environment, possessed tubulocristate mitochondria and was multiflagellated. If this hypothesis is correct, then Pelomyxa, with its multiple flagella (Griffin, 1988; Goodkov & Seravin, 1991), may have maintained the ancestral state of the group and, because of a sedentary lifestyle, its flagella have been preserved in a reduced form only.
Where is the root of Amoebozoa?
The monophyly of flagellated amoebae depends not only on the sequences included in the analyses, but also on the position of the root of Amoebozoa. Because the radiation of Amoebozoa is not well resolved, its rooting varies depending on the gene, type of analysis, taxonomic sampling and selected outgroup. In the present study, the root of Amoebozoa is either placed between Tubulinea + Mayorella + ‘Platyamoeba’ stenopodia + Acanthopodida and Flabellinea + Dermamoeba algensis + Conosea (Fig. 1) or between Tubulinea and all other amoebozoans (Fig. 2). The latter result was also recovered in actin phylogenies published by Fahrni et al. (2003) and Nikolaev et al. (2005) and in the SSU rRNA gene phylogeny published by Smirnov et al. (2005). In other SSU rRNA gene trees, the root was placed between Tubulinea + Archamoebae and Mycetozoa + other lobose amoebae (Fahrni et al., 2003) or between Acanthopodida and other Amoebozoa (Cavalier-Smith, 2002). Cavalier-Smith et al. (2004) used Breviata anathema as an outgroup, but this species is not related to Amoebozoa (Fig. 1; see also Edgcomb et al., 2002) and did not increase the stability of the relationships within Amoebozoa. Finally, in other protein trees, the taxonomic sampling is too small to make any assumption about the position of the root.
Alternatively, the position of the root might be deduced from such features as the presence of flagella. Assuming that the ancestor of Ameobozoa was flagellated, as proposed by Cavalier-Smith (1998, 2000), the flagellum was lost several times during the evolution of amoebae. For example, a flagella loss occurred at least once within Archamoebae (in the Entamoebidae), or twice if Entamoeba and Endolimax are not monophyletic, as suggested by SSU rRNA gene analyses (see Fig. 2). In this particular case, the loss of flagella may be related to the parasitic mode of life of members of these genera. In the whole Amoebozoa, up to ten flagella losses have been proposed to match the observed tree topology (Cavalier-Smith et al., 2004). However, the number of flagella losses depends on the phylogenetic position of the flagellated species within Amoebozoa and the position of the root in the group. According to the topologies shown in Figs 1 and 2, which place Multicilia and Phalansterium in the same clade, the number of flagella losses can be reduced to eight. If the rooting possibilities suggested by our analyses are not correct and the root actually lies between Conosea and all other amoebozoans, as observed in some ML trees (data not shown), this number would be reduced to six (Fig. 3). In this most-parsimonious case, the flagellum would have been lost once in the common ancestor of Tubulinea and Flabellinea, once in Gephyramoeba sp., once in the common ancestor of the Filamoeba clade, once in the common ancestor of dictyostelids and once or twice in the parasitic Archamoebae.
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; Richards & Cavalier-Smith, 2005), the Amoebozoa are close to the eukaryotic root. This root has been placed between the ancestrally uniflagellate opisthokonts and Amoebozoa on the one hand and the ancestrally biflagellate bikonts on the other. However, it cannot be excluded that the Amoebozoa might be paraphyletic. If this was the case, it would be obvious to search for early eukaryotes among flagellated amoebae. One of them, Phalansterium, has already been proposed as a candidate for the closest relative to the ancestral eukaryotes (Cavalier-Smith et al., 2004). Further multigene studies of this and other flagellated amoebozoans, including Multicilia, may possibly bring answers to some of the questions concerning early eukaryote evolution.
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ACKNOWLEDGEMENTS |
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