Summary. A strain of marine amoeba has been isolated and studied from the bottom sediments of the Great Meteor Seamount (Atlantic Ocean, 29°36.29'N; 28°59.12'W; 267.4 m deep). This amoeba has a typical dactylopodiid morphotype, a coat of delicate, boat-shaped scales, and a Perkinsela-like organism (PLO), an obligatory, deeply-specialized kinetoplastid symbiont near the nucleus. These characters allow us to include this species into the genus Paramoeba. However, it differs from its only described species, P. eilhardi, in the structure of scales. P. atlantica n. sp. is established therefore to accommodate the studied strain. SSU rRNA gene sequence analysis suggests that P. atlantica belongs to the Dactylopodida, and is sister to a monophyletic clade of P. eilhardi and all Neoparamoeba spp., branching separately from P. eilhardi. Therefore, the genera Paramoeba and Neoparamoeba, currently defined based on the cell surface ultrastructure, might be paraphyletic and probably should be synonymized, as further evidence is accumulated. Based on the data available we emend the families Vexilliferidae and Paramoebidae to make them more consistent with the current phylogenetic schemes.
Key words: Amoebozoa, Dactylopodida, deep-sea protists, Paramoeba atlantica n. sp., phylogeny, SSU rDNA, taxonomy, ultrastructure.
Abbreviations: BS - bootstrap support; DAPI - 4',6-diamidino-2-phenylindole; DIC - differential interference contrast; PBS - phosphate buffered saline; PLO - Perkinsela-like organism; PP - Bayesian posterior probability; SEM - scanning electron microscopy; SSU rRNA - small-subunit ribosomal RNA; TEM - transmission electron microscopy
INTRODUCTION
Marine and aestuarine amphizoic amoebozoans of the genera Paramoeba Schaudinn, 1896, Janickina Chatton, 1953 and Neoparamoeba Page, 1987 (Discosea, Dactylopodida) possess a deeply-specialized kinetoplastid symbiont Perforisela amoebae (Hollande, 1980) Dyková et al., 2008 (or Perkins e la-like organism, PLO) located in the cytoplasm near the nucleus (Dyková et al. 2008, Hollande 1980). The dactylopodial locomotive morphotype (Smirnov and Brown 2004) is shared by Paramoeba and Neoparamoeba that may include both, free-living and parasitic species; entirely parasitic Janickina spp. live in chaetognaths and have a limax locomotive form with a villous-bulb uroid (Janicki 1912, Chatton 1953). Paramoeba has been a sole genus in the group until establishment of Janickina, where several species, formerly members of Paramoeba, were transferred (Chatton 1953). In the 1970's, description of several more marine and estuarine dactylopodial amoebae, then included in Paramoeba, revealed differences in cell surface structure. Amoebae of the type species Paramoeba eilhardi Schaudinn, 1 896 were covered with delicate, boat-shaped microscales (Grell and Benwitz 1966), while other species had an amorphous glycocalyx, sometimes containing hair-like structures but devoid of microscales (Page 1970, 1973; Cann and Page 1982). A thin stratified glycocalyx without scales was also demonstrated in Janickina (Hollande 1980). These observations allowed Page (1987) to create the genus Neoparamoeba to accommodate those members of Paramoeba without microscales. Neoparamoeba was included in the family Vexilliferidae, while Paramoeba was left in Paramoebidae, based on the differences in cell surface structure and presence of the microfilamentous core in subpseudopodia of the former genus. Molecular phylogenetic studies of the paramoebids and vexilliferids have mostly been focused on Neoparamoeba, as many members of this genus have been shown to cause mortal diseases in fish and invertebrates (e.g. Dyková et al. 2005, Fiala and Dyková 2003, Mullen et al. 2005, Young et al. 2007) and molecular diagnostics methods for these amoebae were sought (e.g. Wong et al. 2004). However, most of other available strains of Paramoebidae and Vexilliferidae were also sequenced (Dyková et al. 2011, Fahrni et al. 2003, Mullen et al. 2005, Peglar et al. 2003), including a strain of Paramoeba eilhardi on which the current description of this species and diagnosis of the genus Paramoeba is based (Grell 1961; Grell and Benwitz 1966, 1970; strain CCAP 1560/2). Small-subunit (SSU) rRNA gene sequence analysis (Mullen et al. 2005) has shown that this species branches as a sister to monophyletic Neoparamoeba spp., and the whole clade is classified in Dactylopodida (Smirnov et al. 2005). Later, Dyková et al. (2007, 2008) showed that P. eilhardi branched within a clade oí Neoparamoeba spp., mostly as a sister to Neoparamoeba perurans.
The purpose of this paper is to describe a new marine dactylopodial amoeba possessing surface microscales and a PLO. This amoeba has the characteristics of the genus Paramoeba. Light and electron microscopy together with SSU rRNA gene sequence analysis were used to directly compare the new strain with the previously available strain of P. eilhardi (CCAP 1560/2) and to justify the naming of P. atlantica ?. sp. together with the re-evaluation of the families in Dactylopodida.
MATERIALAND methods
Strain isolation, culturing and microscopy
Amoebae were isolated from the sandy bottom sediments collected using a Van Veen grab from the Great Meteor Seamount (eastern Atlantic Ocean; 29°36.29'N; 28°59.12^) at the depth of 267.4 m on August 17th, 2009 during the cruise M79/1 of the German research vessel METEOR. Sediment subsamples were removed using a sterilized cut syringe and transferred into sterile 650-ml tissue culture flasks (Saerstedt) filled with Millipore-filtered (0.2 µp?) seawater (ca. 35%o). Samples were kept at 100C after collection and during transport to the laboratory. Aliquots of sediment (1-2 ml) were inoculated into 130-mm Petri dishes with addition of filtered seawater and autoclaved wheat grains. Samples were kept at 18-200C and regularly observed using an inverted microscope. Amoebae were isolated and cloned by transferring into the Petri dishes with fresh seawater using glass capillary pipettes. Cultures were maintained in filtered seawater (ca. 35%o) with addition of wheat grains. Living amoebae were observed and measured either in culture or on coverslips using a Zeiss Axiovert 200 inverted microscope with phase contrast and DIC optics. In total, several hundred cells were observed and the dimensions of 1 17 cells were measured. For DAPI-staining, cells were fixed on coverslips with 4% paraformaldehyde prepared with 1 ? PBS (pH 7.4) for 10 min., washed with the same buffer (3*5 min.), followed by application of DAPI at a final concentration of 2.5 µg/ml in the same buffer for 15 min. After staining, cells were washed with buffer, enclosed in anti-bleaching medium and observed using a Zeiss Axiophot fluorescent microscope.
For transmission electron microscopy (TEM) the following fixation protocols were applied: (1) (AU steps at room temperature.) Addition of several drops of 1% osmium tetroxide in a culture medium (5 min.); 2.5% glutaraldehyde in filtered seawater (40 min.); 1% osmium tetroxide in filtered seawater (60 min.). Cells washed with seawater (3 ? 5 min.) between fixation steps and before dehydration. (2) The same as (1), but sodium cacodylate buffer (0.05 M, pH 7.4) used instead of seawater. (3) (AU steps on ice.) 2% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) mixed 1 : 1 with the culture medium (60 min.). Cells washed with buffer (3 ? 5 min.) before dehydration. (4) 1% osmium tetroxide in KOH-Cr buffer at pH 7.4 after Dalton (1955) (60 min. on ice). Cells washed with buffer (3*5 min.) before dehydration. In all cases the fixation started in culture dishes, later amoebae were scraped away from the substratum, concentrated by gentle centrifugation and embedded in 2% agar before dehydration. Small pieces of agar (ca. 1 mm3) containing amoebae were cut out and dehydrated in a graded ethanol series followed by epoxy propane and embedded in Araldite M epoxy resin (Serva). Silver to light gold sections were cut on Reichert ultramicrotome using a diamond knife and stained with 2% uranyl acetate in 70% ethanol and Reynolds' lead citrate. Negatively stained whole mounts of scales were prepared by placing the cells on formvar-coated grids, allowing them to settle and fixing with osmium tetroxide vapours for 15 min. Grids were then rinsed with distilled water, followed by negative staining with 1% aqueous phosphotungstic acidas described in Harris (1999: 20-21). Sections and negatively stained whole mounts were observed using a Philips EM208 electron microscope at 80 kV.
For scanning electron microscopy amoebae were placed on coverslips (18 ? 18 mm), allowed to attach and then fixed and dehydrated. Fixation was for 30 min. in a mixture of 2.5% glutaraldehyde and 1% osmium tetroxide in seawater. After a brief wash in seawater of decreasing concentration amoebae were dehydrated in a graded ethanol series (30 to 100%) and critical-point dried with liquid CO2, sputter-coated with gold and observed using a FEI Quanta scanning electron microscope.
For comparative purposes, a strain of Paramoeba eilhardi was obtained from Culture Collection of Algae and Protozoa, Oban, UK (accession number CCAP 1560/2). Culture maintenance and observations were conducted as described above.
DNA isolation, sequencing and phylogenetic analysis
For molecular phylogenetic study the genomic DNA was isolated from the cell cultures using the guanidine isothiocyanate (Maniatis et al. 1982) method. Small subunit ribosomal RNA gene was amplified and sequenced as a single piece essentially as described previously (Kudryavtsev et al. 2009, 2011) using the universal primers sAF (5'-CTGGTTGATYCTGCCAG-3') in combination with RibB (Medlin et al. 1988). In total, one molecular clone of full-length SSU rDNA was sequenced for Paramoeba atlantica in both directions (GenBank accession No JN202436). Partial sequences of the three molecular clones of the symbiont's SSU rDNA co-amplified in the same reaction were obtained (the final sequence used is a consensus of them; GenBank accession No JN202437). For P. eilhardi four molecular clones of full-length SSU rRNA were sequenced in both directions (GenBank accession No JN202438JN202441). Phylogenetic analysis was done as described in Kudryavtsev et al. (2011). Seaview (Galtier et al. 1996) was used for manual alignment; RaxML Version 7.2.6 (Stamatakis 2006) and MrBayes Version 3. 1 .2 (Altekar et al. 2004, Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003) run at the Bioportal computer service (http://www.bioportal.uio.no) were used for tree reconstruction. Alternative tree topologies were produced and tested using Treefinder (Jobb 2008, http://www.treefinder.de).
RESULTS
Culture growth, morphology and ultrastructure of Paramoeba atlantica ?. sp.
The cells first appeared in the inoculated samples after 3 weeks of incubation, and multiplied to considerable densities in around 5-6 weeks of incubation in one of the 10 dishes inoculated. Following purification and cloning, amoebae multiplied in culture and formed dense aggregations of the cells, with very low densities of bacteria, after three weeks of incubation. Cultures could remain stable in this condition for more than three months (repeatedly observed since January 2010) if the Petri dishes were sealed with Parafimi. If dishes were not sealed, amoebae did not demonstrate fast growth and the culture degraded quickly.
Typical locomotive forms are shown in Figs 1-7. During rapid locomotion amoebae were generally oval, with length greater than breadth (all measurement data are given in the diagnosis); in slower moving cells breadth was sometimes the greatest dimension (Fig. 4). The cytoplasm was clearly separated into anterior hyaloplasm, occupying 1/4-1/3 of the cell length, and posterior granuloplasm. The anterior edge of the cell produced numerous hyaline subpseudopodia (Figs 1-4) usually up to 10 µ?t? in length. These subpseudopodia could be withdrawn shortly after formation or moved laterally towards the uroid as the cell advanced. Most of the locomotive cells had 2-4 dorsal longitudinal ridges (Figs 1, 6, 7) bearing subpseudopodia. These extended forward, sometimes their tips reached beyond the anterior edge of the cell. Some of these subpseudopodia moved forward over the dorsal surface of the cell and ventrally towards the substratum. The posterior end of the locomotive form was usually blunt (Figs 1, 2, 6) with several small folds over the surface. Less frequently it was pointed, and the cell adopted an elongated triangular shape (Figs 3, 5). Rate of locomotion over the glass substrate at 18°C was 10-32 µm/min. (average 17.4 µm/min.) (n = 17) equaling about half of a cell length per minute.
During slower and non-directed movement amoebae were rounded and flattened, with strongly wrinkled dorsal surface and numerous hyaline subpseudopodia produced from the narrow peripheral hyaloplasm in all directions. Floating forms were adopted for a long time by some cells in dense mature cultures, and, for several minutes, when amoebae were artificially detached from the substratum (Figs 8, 9). At the initial stages of formation they were spherical with short papillate projections (Fig. 8); mature floating forms had rounded cell body with narrow radiating hyaline pseudopodia (Fig. 9). Some of the floating forms were slightly asymmetrical, especially in cultures.
Amoebae possessed a single, spherical nucleus of vesicular type with the central nucleolus (Figs 5, 10, 12). The nucleus was located centrally in the granuloplasm. The symbiont (PLO) was clearly seen always closely associated with the nucleus (Fig. 5). It was ovoid, and had a usual structure, with the large central "Mittelstück" and two "Seitenteile" (terminology according to Grell 1961). 1-2% of all cells observed had two symbionts (Fig. 10). In D API-stained preparations (Figs 11, 12) the "Mittelstück" was strongly positive, while "Seitenteile" demonstrated less intensive fluorescence. The nucleus showed a ring-shaped fluorescence pattern around DAPI-negative nucleolus (Fig. 11). The granuloplasm contained numerous transparent vesicles and food vacuoles with bacteria. Some of the cells contained numerous spherical, yellowish bright granules of different sizes, not exceeding 3 µp? in diameter. There were neither contractile vacuole nor crystals. In our cultures amoebae never formed cysts.
Figures 13-18 show some ultrastructural features of the studied strain. In spite of various fixation protocols applied for TEM, the fixation quality of the nucleus, PLO and cytoplasm was never adequate, being slightly better when protocols (1) and (4) were used. Cell surface structure was preserved more or less identically with every protocol and corresponded well to the SEM observations. The plasma membrane surface was completely covered with a layer of delicate, boat-shaped microscales of medium electron density. The microscales could be easily detected even in the lower magnification SEM micrographs (Fig. 6). Higher magnification SEM (Fig. 13) and the ultrathin sections (Figs 14-17) have shown that there was only one type of microscales; they all were distally open boat-shaped structures consisting of the flat bases (Figs 16, 17) and slightly curved walls arising from the periphery of the bases (Figs 14-16). Bases and walls of the microscales did not contain any holes and appeared to have homogeneous structure. Diagrammatic reconstruction of the microscale is shown in Fig. 19. The nucleus in sections was rounded and showed an electron-dense central nucleolus; one or two structures resembling parasomes were often seen close to the nuclear membrane. However, the fixation quality of nucleus and parasomes was never sufficient for a detailed description. Lipid droplets and food vacuoles were regularly seen in the cytoplasm; mitochondria were probably destroyed, as a structure resembling a poorly preserved mitochondrion was seen only once in the sections. Dictyosomes were also not found, although numerous vesicles probably deriving from the dictyosomes, many of which contained scales (Fig. 18) were seen in the cytoplasm close to the nuclear envelope.
Gene sequence data analysis
Small-subunit ribosomal RNA gene of Paramoeba atlantica was 2100 b.p. long and had a G + C content of 37.9%. All typical eukaryotic secondary structure elements could be identified in this sequence. There were no long introns. Preliminary phylogenetic analysis of the sequence has shown that P. atlantica belongs to Amoebozoa and constantly groups within the Dactylopodida. For detailed analysis of the phylogenetic relationships of this species we have selected the datasets of Dactylopodida and Vannellidae (selected (79) sequences, 1426 alignment positions; Fig. 20) and Dactylopodida only (all available (66) sequences, 1556 alignment positions; Fig. 21). In both datasets branching oîParamoeba atlantica was the same regardless of the algorithm of tree reconstruction. This species was always sister to a monophyletic clade of Neoparamoeba spp. with moderate to high support (PP = 0.95-1; BS = 69-93%). The sequence of P. eilhardi CCAP 1560/2 never formed a clade with P. atlantica, branching instead in a poorly resolved position (PP = 0.6; BS = 53%, Fig. 21) at the base of the Neoparamoeba spp. clade, or as sister to Neoparamoeba perurans strains with a restricted dataset (Fig. 20). Different strains of other Neoparamoeba spp.: N. aestuarina, N pemaquidensis and N. branchiphila, formed clades mainly corresponding to species (Fig. 21). Korotnevella spp. were basal to a clade of Neoparamoeba + Paramoeba spp. Relationships at the base of the dactylopodid tree were not stable and the tree topology depended on the dataset used. With a restricted dataset (Fig. 20) Korotnevella spp. were always sister to a clade oîParamoeba + Neoparamoeba, while a robust clade of "Vexillifera armata" and Pseudoparamoeba pagei was always sister to Korotnevella + ParamoebalNeoparamoeba. With the more expanded dataset positions of "Vexillifera" + Pseudoparamoeba and Korotnevella were swapped (Fig. 21). Korotnevella was either paraphyletic (Figs 20, 21), or sometimes monophyletic (not shown) depending on the algorithm of analysis and the choice of nucleotide positions. The clade of Vexillifera spp. and "Pessonella sp." PRA-29 always demonstrated the same topology regardless of the dataset and the algorithm of analysis and was sister to the whole clade of Paramoebal Neoparamoeba/ Korotnevella (Fig. 20). Several molecular signatures shared by P. atlantica with Korotnevella, Pseudoparamoeba and Vexillifera, but absent in P. eilhardi and Neoparamoeba spp. were found in the sequence at positions 226, 319, 547, 1022, 1044 and 1081.
Small-subunit rRNA gene attributed to a PLO was co-amplified, cloned and sequenced together with the nuclear SSU rRNA gene of an amoeba. In phylogenetic trees this sequence was robustly a sister branch to a monophyletic clade of PLOs from different species of Neoparamoeba spp. (Fig. 22). The clade of PLOs from Neoparamoeba spp. and P. atlantica was sister to Ichthyobodo spp.
Morphology, cell coat and SSU rDNA of P. eilhardi CCAP 1560/2
A CCAP culture of P. eilhardi showed a good growth under the culturing conditions used, and no traces of the contamination with other eukaryotes were ever seen. During locomotion (Figs 23-27) amoebae were mostly longer than broad, and had a dactyl opodial morphotype. Numerous blunt dactylopodia were formed from both, edge of the cell (Figs 23, 25) and its dorsal surface (Figs 24, 29). They could be as long as the entire locomotive form (Fig. 27). Many cells formed dorsal longitudinal ridges that continued anteriorly into dactylopodia (Fig. 24). An uroid was mostly plicate (Fig. 26). Length of the locomotive form was 28-63 µp? (average 45 µp?), breadth 11^40 µp? (average 26 µp?), length : breadth ratio was 0.93-3.10 (average 1.80) (n = 48). Rate of locomotion at 180C was 14-35 µ??/min (average 24 µm/min.) (n = 6) comprising approximately 0.5-1 cell length per minute. Locomotion was very unstable; amoebae often changed the direction of movement, and the locomotive rate of the same cell could change in up to ca. 2 times within minutes. Floating form formed numerous tapering, slender, hyaline pseudopods radiating from the central mass of the cytoplasm. Amoebae had a single vesicular nucleus (Fig. 28) 5-10 µm in diameter (average 8 µm) with a central nucleolus, 3-6 µm in diameter (average 4 µm) (? = 14). About 75% of the cells contained two parasomes adjacent to the nucleus (Fig. 28), the rest of the cells had three or, rather exceptionally, one parasome. Length of the parasome was 6-8 µm (average 7 µm), breadth 3-5 µm (average 4 µm) (? = 18). Scanning electron microscopy and transmission electron microscopy of the negatively-stained whole mounts (Figs 29-31) shows that the cell surface was entirely covered with the boat-shaped scales consisting of a base plate and an upper rim connected to the periphery of the base plate with eight upright bars (Figs 30, 31). Length of the scale was 357- 490 nm (average 415 nm), width 179-238 nm (average 212 nm), height 107-200 nm (average 154 nm) (n = 20).
Sequenced molecular clones of SSU rDNA were 2137-2142 base pairs long and had a G + C content of 41.46^1.97%. A slight sequence variation between clones was seen. This variation was comparable to that occurring between the newly obtained sequences and the previously published SSU rDNA sequence attributed to P. eilhardi (Mullen et al. 2005; GenBank accession No AY686575). Identity percentage between the newly obtained sequences and a previously published one was 96.9-97.5 (average 97.2) while that within the newly obtained sequences was 97-97.8 (average 97.5). Regular substitutions (i.e. those present in all molecular clones compared to a sequence AY686575) occurred in 0.5% of all nucleotide positions.
DISCUSSION
Species identification
Morphology and ultrastructure of the newly isolated amoeba fully correspond to the diagnosis of the genus Par amoeba as emended by Page (1987). We made a direct comparison of this strain with the only described species of this genus, P. eilhardi, using CCAP strain 1560/2 on which all current knowledge on P. eilhardi is based. A re-investigation of this strain has shown that its light microscopic and cell surface characteristics are in accordance with the published descriptions of P. eilhardi (Cann and Page 1982; Grell and Benwitz 1966, 1970; Page 1983), except that the size of the amoebae studied here was somewhat smaller than reported in the literature. Re-sequencing of the SSU rDNA of this strain demonstrates that it is identical with the previously obtained sequence (AY686575; Mullen et al. 2005), that was hence correctly attributed to P. eilhardi.
The new isolate differs from P. eilhardi in having a broader frontal hyaloplasm, not producing long subpseudopodia during locomotion (like shown in Fig. 27), smooth uroid, structure of the microscales and a very small fraction of amoebae cells hosting two symbionts as well as absence of the cells with more than two symbionts. Though the latter character may be more strain- than species-specific (e.g. Page 1983), it is still mentioned here, as the nature of host-symbiont relationships in these amoebae, and of the observed variability is not yet clear. This does not allow to exclude the species-specificity of this character completely. It is impossible to compare the details of the cytoplasmic ultrastructure, as the problems with fixation quality of a new isolate were not overcome. Interestingly, similar problems occurred during the ultrastructural study of P. eilhardi (Grell and Benwitz 1970), that partly could be overcome by the application of the Dalton's (1955) protocol. Yet, the application of this protocol in our study did not significantly improve the results. Smallsubunit rDNA sequence analysis demonstrates remarkable differences between the strains and shows that in the phylogenetic trees (Figs 20, 21) P. eilhardi always branches distantly from our isolate. Therefore, we establish Paramoeba atlantica ?. sp. to accommodate the studied amoeba. Interestingly, the strain studied here is morphologically similar to an unnamed amoeba isolated by Smirnov (1999) from the anaerobic sediments of the Nivâ Bay (The Sound, Baltic Sea) and identified as Neoparamoeba sp. Both strains are similar in size and shape of the locomotive form, nucleus and subpseudopodia. However, the ultrastructure of the Nivâ Bay strain is unknown, therefore it is not possible to conclude whether both amoebae really belong to the same morphospecies and the same genus.
Phylogenetic position of P. atlantica and taxonomy ofDactylopodida
In the molecular phylogenetic analysis presented here two microscale-bearing species P. eilhardi and P. atlantica branch separately. The former species is within Neoparamoeba spp. and forms a clade with an uncultured amoebozoan that is sister to N. perurans (Fig. 21), while P. atlantica is sister to the whole clade of Neoparamoeba spp./P. eilhardi. Only part of the tree is well-resolved: while position of P. atlantica as well as the clade of Neoparamoeba + P. eilhardi are supported well, the position of P. eilhardi is never highly supported, but at the same time it never alters with the algorithm of tree reconstruction. An alternative branching for P. eilhardi shown by Dyková et al. (2008) in maximum parsimony trees was never reproduced in our analysis. Topology tests also reliably reject all hypotheses that imply a monophyletic clade of two Paramoeba spp.
Based on our re-investigation of P. eilhardi, we can exclude the possibility that its sequence has been misattributed to P. eilhardi, being instead a sequence of a Neoparamoeba sp. (e.g. contaminant in the culture) as suggested earlier (Dyková et al. 2007). Therefore, two explanations remain possible for the revealed branching of two Paramoeba spp. First, the poorly supported position of P. eilhardi in the phylogenetic tree may indicate an insufficient taxon sampling for Paramoeba, and the tree configuration may change substantially, once more scale-bearing species are added. In this case P. eilhardi may finally form a clade with P. atlantica. However, SSU rRNA sequence signatures shared by P. atlantica with Korotnevella and P seudopar amoeba but not with P. eilhardi and Neoparamoeba spp. weaken this suggestion. Second, if the position of P. eilhardi in the tree is correct, the results obtained here suggest that Paramoeba and Neoparamoeba as defined by Page (1987) are paraphyletic, and the latter name should be abandoned as a junior synonym of Paramoeba. This explanation implies that presence of boat-shaped surface microscales is ancestral to the clade oí Paramoeba/ Neoparamoeba + Korotnevella; one or several losses of microscales must have then occurred in the clades of Neoparamoeba spp., probably correlated with the development of an amphizoic way of life. This is in accordance with the hypothesis of the cell coat evolution proposed by Smirnov et al. (2007), in the sense that the microscales of Paramoeba/ 'Neoparamoeba might have evolved in a similar way to the glycostyles of Vannellal Platyamoeba, having been lost several times independently in different evolutionary lineages of amoebae. In this case additional differences used by Page (1987) to separate Neoparamoeba from Paramoeba (like dorsal longitudinal ridges during locomotion) also seem to be non-valid at the generic level, as both, P. atlantica and P. eilhardi have microscales and longitudinal ridges. Therefore a future re-definition of the genus Paramoeba could be possible based on the dactylopodial morphotype and a PLO shared by Paramoeba and Neoparamoeba, regardless of the cell surface organisation. Currently this formal change seems to be premature, as the position of P. eilhardi is poorly supported and its alternative explanation is possible, but it should be kept in mind for future taxonomic work. A re-unification of Paramoeba and Neoparamoeba would not affect the validity and position oîJanickina. It should remain incerate sedis until gene sequence data are available, as it has a limax locomotive form and a villous-bulb uroid (Chatton 1953, Hollande 1980) never observed in other parasome-bearing species.
Based on the present phylogenetic analysis and recent data of Dyková et al. (201 1), the families Paramoebidae Paramoeba and Korotneve Ila) and Vexilliferidae (Vexillifera, Neoparamoeba and Pseudoparamoeba) re-defined by Page (1987) based on the cell surface structure, the shape of subpseudopodia and presence or absence of dorsal folds during locomotion, and remaining unchanged since then (Adi et al. 2005, CavalierSmith et al. 2004, Smirnov et al. 2005, Smirnov et al. 201 1), need a reassessment. Our data and all previously published phylogenetic trees show that if Paramoebidae includes Korotnevella and Paramoeba, it should also include Neoparamoeba and P seudopar amoeba, otherwise both Vexilliferidae and Paramoebidae are paraphyletic. Vexilliferidae in this case should comprise Vexillifera spp. (but not "K armata' ATCC 50883 branching with Pseudoparamoeba pagei, that was most probably misidentified and requires a reinvestigation; Dyková et al. 2011) and an amoeba PRA-29 identified as "Pessonella sp." (Tekle et al. 2008). Both families are then monophyletic, and we provide new diagnoses for them to accommodate the proposed changes in the taxonomic composition.
Being isolated from the distant and poorly accessible locality, P. atlantica is a good example of how the survey of amoebae from poorly studied habitats may lead to expansion of our knowledge on the diversity of this group and influence the established classification schemes. The sediment sampling method used (Van Veen grab, Van Veen 1933) did not allow a precise determination of whether amoebae were really isolated from the bottom or water column (hence whether they can be considered deep-sea), but this is further evidence highlighting the extent to which the protozoan diversity in the oceans is understudied (e.g. Atkins et al. 2000; Hausmann et al. 2002a, b; Moran et al. 2007), especially in the bottom sediments. For Amoebozoa, for example, only two papers are available with in total 1 1 morphospecies recorded (Hausmann et al. 2002a, Moran et al. 2007), and only the latter one, where 3 morphospecies were found, contains complete descriptions and illustrations of the species observed.
Diagnoses of new and emended taxa
Position in the system according to Smirnov et al. 2011.
Phylum Amoebozoa Luhe, 1913
Subphylum Lobosa Carpenter, 1861
Class Discosea Cavalier-Smith, 2004
Subclass Flabellinia Smirnov et al., 2005
Order Dactylopodida Smirnov et al., 2005
Family Paramoebidae Poche, 1913, emend.
Flattened dactylopodial amoebae with blunt, hyaline subpseudopodia, conical or finger-shaped in outline. Cell coat consists of microscales, dense amorphous glycocalyx that may include hair-like structures, or dome-shaped glycostyles with hexagonal bases.
Genera: Paramoeba Schaudinn, 1896 (type genus), Korotnevella Goodkov, 1988, Neoparamoeba Page, 1987, Pseudoparamoeba Page, 1979.
Paramoeba atlantica n. sp.
Diagnosis: Length of the locomotive form 23-65 urn (average 36.5 µm), breadth 12-31 µm (average 21 µm), length : breadth ratio 0.92-3.42 (average 1.78) (n = 117). During locomotion flattened, with wide anterior hyaloplasm and dorsal longitudinal ridges; conical or finger-shaped hyaline subpseudopodia produced from anterior margin and dorsal surface of the cell. Single vesicular nucleus 4-9 µm in diameter (average 6 µm), spherical central nucleolus 1.5-5 µm in diameter (average 3 µm) (n = 37). Single PLO adjacent to nucleus, ovoid, 5-8 µm long (average 12 µm) and 1-2 µp? broad (average 1.6 µm) (n = 25); rarely two parasomes. Cell coat consists of boat-shaped microscales with delicate, homogeneous walls; length of the scale base 0.21-0.37 µm (average 0.32 µm), breadth 0.120.24 µm (average 0. 18 µm) (n = 74); height of the scale 0.08-0.17 µm (average 0.13 µm) (n = 54).
Observed habitat: marine, bottom sediments of the Great Meteor Seamount, eastern Atlantic Ocean (29°36.29'N; 28°59.12'W; depth 267.4 m).
Type material: type strain is deposited with CCAP (Oban, UK), accession number 1560/9.
Etymology: atlantica, refers to the Atlantic Ocean where the strain was collected.
Differential diagnosis: differs from P. eilhardi in the structure of microscales, shape during locomotion and predominating number of parasomes per cell; from P. perniciosa in size of the cell, nucleus and PLO.
Family Vexilliferidae Page, 1987, emend.
Elongated flattened amoebae of acanthopodial morphotype, with one or more long, slender, hyaline subpseudopodia, rarely formed in some species. Cell coat consisting of delicate glycostyles that can be prismatic with hexagonal cross-section, or t-shaped in vertical section.
Genera: Vexillifera Schaeffer, 1926 (type genus); a discosean amoeba PRA-29 identified as "Pessonella" sp. may also belong to this family, but currently available data (Tekle et al. 2008) do not provide a morphological evidence for this suggestion; therefore, the presented diagnosis is based entirely on features of Vexillifera spp.
Acknowledgements. We are grateful to the captain, crew and all scientists on board German RV METEOR during the cruise M79/1 (DIVA3 expedition), Dr. Nils Brenke (Senckenberg Research Institute, Wilhelmshaven, Germany) for help with the literature and Dr. Iva Dyková (Institute of Parasitology Academy of Sciences of the Czech Republic, Ceské Budejovice, Czech Republic) for sharing her sequences of Vexillifera spp. prior to publication. This study was supported by the DFG grant HA 818/22-1 to KH and the research grant IZLR Z3128338 from Science and Technology Cooperation Programme Switzerland - Russia to JP.
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Received on 15th May, 201 1; revised on 13th July, 201 1; accepted on 14th July, 2011
Alexander KUDRYAVTSEV1 2^, Jan PAWLOWSKI2, Klaus HAUSMANN1
1 Research Group Protozoology, Institute of Biology/Zoology, Free University of Berlin, Berlin, Germany; 2Molecular Systematics Group, Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland; department of Invertebrate Zoology, Faculty of Biology and Soil Science, St-Petersburg State University, St-Petersburg, Russia
Address for correspondence: Alexander Kudryavtsev, Molecular Systematics Group, Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland; E-mail: [email protected]; [email protected]
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