Kudryavtsev, Pawlowski 2013 - Squamamoeba Japonica - Protist164,13-23

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Protist, Vol. 164, 13–23, January 2013http://www.elsevier.de/protisPublished online date 8 September 2012

ORIGINAL PAPER

Squamamoeba japonica n. g. n. sp. (Amoebozoa):A Deep-sea Amoeba from the Sea of Japan with aNovel Cell Coat Structure

Alexander Kudryavtseva,b,c,1, and Jan Pawlowskia

aDepartment of Genetics and Evolution, University of Geneva, Sciences III, 1211 Geneva, SwitzerlandbDepartment of Invertebrate Zoology, Faculty of Biology and Soil Science, St. Petersburg State University,Universitetskaja nab., 7/9 199034 Saint-Petersburg, Russia

cResearch Group Protozoology, Institute of Biology/Zoology, Free University of Berlin, Königin-Luise-Strasse,1-3, D-14195 Berlin, Germany

Submitted September 12, 2011; Accepted July 19, 2012Monitoring Editor: Sandra L. Baldauf

Squamamoeba japonica n. g. n. sp. was isolated and described from marine bottom sediments collectedat a depth of ca. 2700 m in the Sea of Japan. Trophic amoebae of this species are elongated and flattened,with a wide anterior hyaloplasm producing numerous ventral subpseudopodia for adhesion to thesubstratum. The cell coat consists of flat oval scales tightly packed together to form a continuous layerseparated from the plasma membrane. Amoebae can form cytoplasmic projections protruding throughthe scale layer and having tips covered only with the plasma membrane. Small subunit ribosomal RNAgene phylogeny shows that S. japonica forms a long branch in the amoebozoan tree, robustly groupingwith the marine strain ‘Pessonella’ sp. PRA-29. Morphological data available for the latter, althoughscarce, give additional support for the relatedness of both species. The resulting clade comprisingthe two taxa shows no close relationships to other Amoebozoa and seems to be a novel lineage thatdeveloped an ability to temporarily liberate local areas of the plasma membrane from the cell coatindependently from Himatismenida, Trichosida, Pellitida and Dermamoeba.© 2012 Elsevier GmbH. All rights reserved.

Key words: Amoebae; cell coat; deep-sea protists; phylogeny; SSU rRNA; taxonomy; ultrastructure.

Introduction

In spite of recent efforts (Arndt et al. 2003; Atkinset al. 2000; Hausmann et al. 2002a, b; Pawlowskiet al. 2011; Scheckenbach et al. 2005), very little isknown about the diversity of protozoa in deep-seacommunities, apart from Foraminifera and Komoki-acea which are slightly better characterised (e. g.Gooday 1994, 2002; Gooday and Bowser 2005;

1Corresponding author;e-mail [email protected] (A. Kudryavtsev).

Gooday et al. 2007). There are especially few dataon lobose amoebae (phylum Amoebozoa). Apartfrom three published records (Hausmann et al.2002a; Kudryavtsev et al. 2011b; Moran et al.2007), no information on amoebae inhabiting theocean bottom deeper than 200 m is available.During the last few years we have been investi-gating the diversity of amoebozoans isolated fromdeep-sea bottom sediments. Among other findingsan unusual amoeba with a novel set of morpho-logical characters was found in bottom sedimentscollected from the Sea of Japan (Pacific Ocean),

© 2012 Elsevier GmbH. All rights reserved.http://dx.doi.org/10.1016/j.protis.2012.07.003


14 A. Kudryavtsev and J. Pawlowski

representing a new genus and species of Amoebo-zoa. The purpose of this paper is to describe thisnew amoeba and analyse its phylogenetic positionusing microscopic and molecular tools.

Results

Morphology and Ultrastructure of theStudied Strain

The newly isolated amoeba could be culturedeasily, and readily adopted a locomotive form inculture dishes or when placed in plankton obser-vation chambers with a glass bottom. Locomotionoccurred less frequently in wet mounts on cover-slips, and many cells remained stationary duringmost of the time of observation. The rate oflocomotion at +18 ◦C was 3-8.7 �m/min (aver-age 5.3 �m/min) (n=21) or about 0.5-1 times thecell length per minute. The locomotive form wasflattened and elongated (Fig. 1A-C), with lengthgreater than breadth (all measurements are givenin the diagnosis). Most of the cells when viewedfrom above had an irregularly triangular shape,being broader anteriorly with a pointed posteriorend, while others were oval, with a rounded poste-rior end. A flat anterior hyaline area occupied aboutone-third to one-half of the cell length. The ante-rior and lateral edges of the hyaline area alwaysproduced short mammiliform subpseudopodia thatmoved after formation towards the ventral surfaceof the hyaloplasm (Fig. 1A-C). These subpseu-dopodia remained stationary with respect to thesubstratum as the cell advanced, and looked likerounded spots when the ventral surface of thecell was in focus. Non-directionally moving andstationary amoebae (Fig. 1B, D) were more flat-tened and occupied a larger area of the substratumthan in the locomotive forms. They produced dig-itiform subpseudopodia that were usually longerthan the subpseudopodia formed during locomo-tion. Sometimes the cell produced a single taperinghyaline projection terminating with a pointed tip,or several tiny projections. Cells rarely adopted adifferentiated floating form. When detached fromthe substratum, amoebae had the same shapeas when remaining stationary on the substratum,usually with several asymmetrically formed hyalinepseudopodia. Some of the cells after a long time onslides or in culture detached from the substratumand adopted a spherical shape with short hyalineprojections. These looked rather like dying cellsthan the normal floating forms; they were neverseen to settle down back to the substratum andresume normal locomotive activity. Amoebae were

uninucleate; the nucleus was usually very difficultto see in the living cells due to its small size. It wasspherical, vesicular, with an inconspicuous centralnucleolus (Fig. 1E). During locomotion the nucleuswas usually located in the anterior part of the gran-uloplasm, which was filled with minute sphericalgranules and food vacuoles containing bacteria.There was no contractile vacuole. Encystment wasnever observed.

Scanning electron microscopy (Fig. 1F-G) usu-ally demonstrated non-directionally moving orstationary amoebae with long digitiform pseu-dopods, often with very fine tips. Short conicalsubpseudopodia used for adhesion to the substra-tum were clearly seen in some cells (Fig. 1G).The surface of every cell observed was unevenand carried minute papillate structures (Fig. 1G),but no other remarkable surface features wereseen. Transmission electron microscopy (Fig. 1H,2) revealed a pronounced flexible cell coat consist-ing of a single layer of flat, electron dense scaleshaving an oval shape in tangential sections, andwith the margins slightly bent distally (Fig. 2A, B,D). Thin filamentous structures were seen arisingvertically from the center of each scale (Fig. 2A).In favorable tangential sections the scales wereseen to possess denser points in their centersthat probably correspond to the bases of these fil-amentous structures (Fig. 2B). The scales wereconnected to each other with a material of mediumelectron density (Fig. 2B). The size of the scaleswas 95-145(125)x 60-90(75)x10 nm (n=17), andthe measured length of the distal filaments was160-420(258) nm (n=20). The plasma membraneunderlying the scale layer was folded (Fig. 2A),and an electron transparent space 25-85 nm widewas always present between the plasma mem-brane and the scale layer (Fig. 1H, 2A-D). All thecells observed in the sections carried eventual localexpansions of the scale layer where it was widelyseparated from the plasma membrane (Fig. 2C).These expansions always contained membranousmaterial of unclear origin. No structures that mightcorrespond to these expansions could be seen withlight microscopy. Several points of disruption of thescale layer were seen in some cells (Fig. 2B, D). Inthese points the neighboring scales were separatedfrom each other and small tapering cytoplasmicprojections were seen to expand beyond the scalelayer (Fig. 2D). These projections were filled withthe electron dense cytoplasm. The nucleus in sec-tions (Fig. 1H) was rounded or lobate, with aninconspicuous central nucleolus of irregular shape.The nuclear envelope was underlain with a layerof electron dense material (Fig. 2A). Most of the


Squamamoeba japonica n. g. n. sp. from the Deep Sea 15

Figure 1. Squamamoeba japonica n. g. n. sp. Light (A-E), scanning (F, G) and transmission electron micro-graphs (H). A-C. Amoebae on the glass substratum (A, B: DIC, C: Phaco); most of the depicted cells arelocomotive forms (direction of locomotion indicated by arrows). Arrowheads indicate ventral subpseudopodia.D. Non-directionally moving and stationary amoebae, DIC. E. Stationary amoeba demonstrating nucleus (N),DIC. F-G. Scanning electron micrographs of amoebae on the substratum. F. Two cells seen from above demon-strating long digitiform pseudopodia. G. Side view of an amoeba showing ventral subpseudopodia (arrowheads).H. TEM section through the whole cell at a smaller magnification showing nucleus (N), dictyosome (D) and foodvacuoles (FV). Scale bar = 10 �m in A-E, 2.5 �m in F-H.

cells observed had a large dictyosome consisting ofca. 6 flattened cisternae located near the nucleus(Fig. 2E). Rounded or oval mitochondria with tubu-lar cristae and cisternae of the rough endoplasmicreticulum were scattered throughout the cytoplasm(Fig. 2F).

SSU rRNA Gene Sequence andPhylogenetic Position of the StudiedAmoebae

Three sequenced clones of SSU rDNA had a lengthof 2084-2085 base pairs and an average G+C



Figure 2. Squamamoeba japonica n. g. n. sp., ultrastructural details. A. Cross section of the cell coat, arrow-heads indicate vertical filamentous structures. Part of the nuclear envelope (NE) is seen. B. Portion of thecell coat in oblique section, arrowheads indicate scales. C. Part of the scale layer separated from the plasmamembrane. D. Cytoplasmic projection (arrowhead) protruding through the layer of scales. E. Dictyosome. F.Cisternae of rough endoplasmic reticulum (E) and mitochondrion (M). Scale bar = 1 �m in C, 0.25 �m in otherfigures.



content of 42.51%. Minor variation between theclones accounted for the 0.3% observed sequencedifference among them. Phylogenetic analysis ofthe obtained sequences showed a robust positionof the studied species within Amoebozoa, althoughthe sequence was very divergent due to numer-ous unique substitutions, and formed one of thelongest branches in the amoebozoan tree (Fig. 3).Regardless of the subset of positions used or thealgorithm of analysis, the new species robustlybranched as a sister clade to a marine amoeba‘Pessonella’ sp. ATCC PRA-29 (GenBank acces-sion No EU273458). Besides branching together,SSU rRNA gene sequences of the two speciesshared a number of unique nucleotide motifs ran-ging from 1 to 15 base pairs in lengths that werenot found in any other Amoebozoa (Table 1).

The position of the Squamamoeba+‘Pessonella’clade within the amoebozoan SSU rRNA treewas unstable. In the ML analysis it tended tobranch within the Dactylopodida, as a sister toVexillifera spp., albeit with negligible support,whereas in a slightly better resolved Bayesian treethis clade was sister to Cochliopodiidae, and thewhole branch of ‘Pessonella’+Cochliopodiidae wassister to Vannellida. While our analysis reliablyyielded the major clades of Tubulinea and Conosasensu Smirnov et al. 2011, the Discosea sensuSmirnov et al. 2011 was always paraphyletic, andStygamoebidae Smirnov et Cavalier-Smith, 2011represented by Vermistella antarctica and an envi-ronmental SSU rDNA sequence AB330051 alwaysbranched within Longamoebia, as sometimes didthe Cochliopodiidae.

Features of the Partial Mitochondrial COIGene SequencesThe amplified COI fragment had a standard lengthof 666 base pairs excluding primers. Five ofthe 9 sequenced clones were completely identi-cal; the other 4 had occasional substitutions in1, 2 or 3 nucleotide positions causing 0.2-0.6%sequence difference. Resequencing of the samemolecular clones showed that these substitut-ions were not due to sequencing errors. Thesequencing error rate determined as a fraction ofnon-repeated substitutions in a total number ofsequenced base pairs was 0.025%. The deducedpolypeptide sequences showed that in two clonesnucleotide sequence differences were at synony-mous sites only, while in the other two clones thenucleotide sequence differences resulted in aminoacid substitutions in one or two positions.

Discussion

Identification of the Studied Strain andJustification for a New Genus

Based on morphology and ultrastructure thestudied amoebae clearly can be included inAmoebozoa, although they demonstrate a combi-nation of several peculiar characters occurring inonly a very limited number of amoebozoan taxa.The cell coat consisting of scales is similar to thatof Korotnevella spp., and the scale morphology dis-tantly resembles that of K. discophora (Smirnov1999). This similarity is reinforced by the dactylopo-did pseudopodial pattern, sometimes occurring instationary cells. However, the facts that the scalesare not separated from each other but embedded ina kind of common matrix (Fig. 2B), the scale layeris clearly separated from the plasma membrane,and the cell is capable of forming cytoplasmic pro-jections with the tips not covered with scales, arenot typical of Korotnevella. A slightly similar scalepattern was previously found in a himatismenidCochliopodium gallicum (Kudryavtsev and Smirnov2006) and a protostelid Ceratiomyxella tahitiensis(Furtado and Olive 1971, 1972), however, no othercharacters are shared between these species andthe one studied here. The morphological featuresof the locomotive form, namely wide anterior hyalo-plasm with short ventral subpseudopodia usedfor adhesion to the substratum, together with theability to form cytoplasmic projections penetrat-ing the external cell coat resemble those of thegenus Pellita (Smirnov and Kudryavtsev 2005), andto a lesser extent Trichosphaerium. However, theamoeba studied here does not fit the diagnosisof any of these genera. The diagnostic featureof Pellita is an integration of the cell coat withthe plasma membrane (Smirnov and Kudryavtsev2005) that does not occur in the species describedhere, while the cell coats of both Pellita and Tri-chosphaerium are very different in structure fromthe new species (Angell 1975, 1976; Schuster1976; Sheehan and Banner 1973; Smirnov andKudryavtsev 2005). Another poorly known marineamoeba that is superficially similar to the speciesstudied here is Rhabdamoeba marina Dunkerly,1921, redescribed by Rogerson et al. (1998). Ourstrain is similar to it in size range, having slowlymoving cells, and forming subpseudopodia thatresemble the knob-like pseudopods described inR. marina. However, differences in the ultrastruc-ture of R. marina (in particular, absence of amorphologically differentiated glycocalyx) as wellas differences in the appearance of the knob-like



Figure 3. Maximum likelihood tree of Amoebozoa rooted with Opisthokonta (151 sequences in total) based onsmall subunit (SSU) rRNA gene sequences. The tree shown was derived based on 1366 nucleotide positionsusing the program RaxML Version 7.2.6 (Stamatakis 2006) and has a LnL = -53423.28 and a gamma distribution� = 0.74. The sequence for the newly identified species is shown in bold and marked with an arrow. Numbers atnodes indicate Bayesian posterior probability/bootstrap values in percent if above 0.5/50. Solid circles=1.0/100.Dashes indicate values below 50/0.5, and an asterisk indicates that the branch does not exist in analyses usingthat method. Scale bar=0.2 substitutions/site.



Figure 3. (Continued).

pseudopodia, do not allow an assignment of thenewly described strain to the genus Rhabdamoeba.

In conclusion, based on morphology and ultra-structure the studied amoeba cannot be includedin any of the existing amoebozoan genera, and anew genus and species (Squamamoeba japonican. g. n. sp.) is established here to accommodateit. To facilitate an easier distinction of this speciesin the future, partial COI gene sequences wereobtained in this study and included with the speciesdiagnosis along with the SSU rRNA. COI appears

to be a promising DNA barcode for lobose amoe-bae (Nassonova et al. 2010), although it is currentlynot possible to evaluate its barcoding potential forSquamamoeba, as this genus is still monotypic.

Phylogenetic Relationships ofSquamamoeba japonica and theEvolution of Cell Coat in Amoebozoa

Among all sequenced amoebozoans, only ‘Pes-sonella’ sp. ATCC PRA-29 branches closely to



Table 1. Sequences and secondary structure location of the shared motifs in Squamamoeba japonica n. sp.and “Pessonella” sp. (EU273458) SSU rRNA gene sequences. Helix numbering according to Wuyts et al. (2000,2001).

Start position in ‘Pessonella’ sp. (EU273458) Helix number Length Sequence

262 11 CGTTTCGGCGC422 13 7 AGAGTTT483 5 1 T645 19 2 TG1133 E27-1 2 TG1269 22 3 CTA1319 32 1 C1446 38 1 C1750 45 15 TGAAAAAGAAAWAAG1968 Downstream of 48 1 A

Squamamoeba. Although this branching might looklike a potential long branch attraction artifact, asboth sequences are quite divergent form those ofthe rest of Amoebozoa, several unique sequencemotifs are also shared between these species(Table 1). In addition, several morphological char-acters known for the ATCC strain (Tekle et al. 2008)suggest that these species are truly related. Tekleet al. (op. cit.) depict the ATCC strain as discoid cellsless than 10 �m in diameter and note the presenceof “hyaloplasmic cone-like bosses” (p. 342) allowingthem to include the species in the genus Pes-sonella. However, no other evidence was presentedfor this, and the type species of Pessonella wasneither reinvestigated with electron microscopy, norsequenced since its initial description (Pussard1973). Based on the apparent close relationshipof these two taxa we suggest that the hyalinesubpseudopodia observed in Squamamoeba mayhave the same nature as the hyaloplasmic bossesof ‘Pessonella’ from ATCC and hence both speciesshare the same locomotive morphotype. This canbe a morphological justification of their true related-ness. Unfortunately, no other characters, that couldbe used for a more comprehensive comparisonhave been published for the ATCC strain.

As the clade of Squamamoeba+’Pessonella’does not find any close relatives among Amoebo-zoa, it is possible to suggest that Squamamoebajaponica demonstrates a novel set of structuralcharacters that evolved independently from theother major branches of Amoebozoa. An abilityto liberate part of the plasma membrane sur-face from the external cell coat for adhesionto the substratum and phagocytosis is consid-ered to be a synapomorphy of Himatismenida(Kudryavtsev 2012; Kudryavtsev et al. 2011b).However, the amoebae studied here are not specif-ically related to the himatismenids in the molecular

phylogenetic analysis, and do not demonstratea himatismenid morphotype during locomotion,i.e., one that involves formation of a ventral flat-tened disc of hyaloplasm (Kudryavtsev 2012).Thus the temporary removal of the cell coatfrom the local sites of plasma membrane surfacefor adhesion to the substratum and/or phago-cytosis has apparently evolved independently inSquamamoeba, Himatismenida, Pellita spp. andTrichosphaerium spp. (Angell 1975, 1976; Schuster1976; Sheehan and Banner 1973; Smirnov andKudryavtsev 2005).

Another interesting case of temporary removalof glycocalyx from part of the plasma mem-brane surface during feeding is demonstrated byDermamoeba algensis (Smirnov et al. 2011a).Seemingly different mechanisms are involvedin this process within these groups: while, forexample, Pellita and Trichosphaerium seem to lib-erate their pseudopodial tips “mechanically”, bypushing the glycocalyx elements apart, in D. algen-sis chemical digestion might be involved in thisprocess which occurs only during food uptake(Smirnov et al. 2011a). All of the above-mentionedgroups of amoebae, with the exception of himatis-menid Parvamoeba (Kudryavtsev 2012), are unifiedby their relatively thick and hardly permeable cellcoat. In Squamamoeba the situation is different: thescale layer of this amoeba is thin as compared tothe above mentioned taxa. However, tightly packedscales in this taxon form an almost continuous layerthat may also be hardly permeable to different sub-stances as well as complicating adhesion to thesubstratum and locomotion. Therefore, it is possi-ble that in this case as well, the mechanism allowingthe cell to liberate part of the plasma membranesurface, probably by formation of a cytoplasmic pro-jection pushing the neighboring scales aside, hasevolved independently.



The phylogenetic relationships of Squa-mamoeba+’Pessonella’ to other Amoebozoacannot be unambiguously reconstructed herebased on SSU rRNA gene sequence analysisprobably due to very heterogeneous evolutionaryrates. Nonetheless, the set of morphological andultrastructural characters described here allowsa tentative inclusion of this clade into Discoseasensu Smirnov et al. 2011. Some of the MLtrees presented here and published earlier for‘Pessonella’ (Kudryavtsev et al. 2011a, b; Lahret al. 2011; Smirnov et al. 2011b; Tekle et al.2008) also suggest that this clade belongs toDactylopodida. This is also strongly suggested bythe features of dactylopodial morphotype seen inthe locomotive forms and non-directionally movingSquamamoeba. If this is confirmed by furtherstudies of other related species, a separate familywithin this order will need to be established.

Culturing Conditions and PossibleEcological Preferences of the NewSpecies

The described amoeba was found after introduc-tion of material collected from deep-sea bottominto enrichment culture and incubation under ambi-ent conditions (normal atmospheric pressure andambient temperature) in contrast to those expectedat the original depth. This may raise a concernas to whether the studied organism is a genuineinhabitant of the deep-sea bottom, or its isolationresults from contamination of the collected materialfrom the surface water. However, previous experi-ments have shown that the sampling method usedis resistant to contamination with water from otherdepths (Scheckenbach et al. 2005), and during thisstudy all possible measures were taken to avoidcontamination of the samples during processing onboard. Indirect evidence for the absence of contam-ination from the surface comes from the fact thatthis species was only isolated from one core outof 16 used during the cruise and never occurredin surface water samples. This suggests that thestudied species indeed inhabits the deep-sea bot-tom and implies either a wide tolerance range of thetrophic amoebae to pressure and temperature, oran ability to form resistant cysts, that however werenot observed in the cultures. Such broad toleranceranges were previously demonstrated for amoe-bae and flagellates isolated from deep-sea samples(Arndt et al. 2003; Atkins et al. 1998, 2002; Moranet al. 2007). Whether this is also typical for thespecies studied here, remains to be determined.

This can be done in future, as the culture hasbeen deposited in the CCAP culture collection(accession number CCAP 1593/1) and is open forfurther studies.

Taxonomic appendix

Phylum Amoebozoa Lühe, 1913Subphylum Lobosa Carpenter, 1861Order Dactylopodida Smirnov et al. 2005

Squamamoeba n. g. Diagnosis: amoebae with flattened ante-rior hyaloplasm producing short conical subpseudopodia fromthe margin, and on the ventral side, for adhesion to the sub-stratum. Cell coat consisting of scales encloses the entire cell;cytoplasmic projections with the distal parts covered solely bythe plasma membrane, may be formed protruding through thescale layer. Type species: Squamamoeba japonica n. sp. (bymonotypy).

Squamamoeba japonica n. sp. Diagnosis: measured lengthof the locomotive form 5.6-11.3 �m (average 8.1 �m), breadth1.9-5.5 �m (average 3.5 �m), length:breadth ratio 1.25-4.5(average 2.39) (n=112). Elongated flattened amoebae, ovalor triangular during locomotion with anterior hyaloplasm occu-pying one-third to a half of the cell; hyaline subpseudopodiaproduced from the ventral surface of the cell have a shapeof rounded granules when viewed from above. Single vesic-ular nucleus 1.5-2.5 �m in diameter (average 1.9 �m) (n=8)with inconspicuous central nucleolus 0.7-1.4 �m in diame-ter (average 1 �m) (n=7). Cell coat a single layer of tightlypacked scales separated from the plasma membrane, form-ing a number of papillate expansiones along the cell surface.Scales flat, oval with the tiny filament arising from the center;scale size 95-145(125)x60-90(75)x10 nm (n=17). Type habi-tat: bottom sediments of the Sea of Japan, Pacific Ocean(42◦26.4337′N 133◦08.7581′E, depth 2709 m); type mate-rial: type culture deposited with CCAP (Oban, UK), accessionnumber CCAP 1593/1; type sequences: GenBank acces-sion numbers JN638030- JN638032 (SSU rRNA), JN638033-JN638037 (COI); etymology: Squamamoeba, squama (l.)“scale”, refers to the scale-bearing cell surface, japonica refersto isolation site of the species (Sea of Japan); differential diag-nosis: may resemble a small Pellita or a small Korotnevellabut differs from the members of the former genus in the cellcoat structure and uneven margin during locomotion, produc-ing short subpseudopodia; differs from the latter genus in thepresence of ventral subpseudopodia, tight connection of sur-face scales to each other and their separation from the plasmamembrane, and formation of cytoplasmic projections protrudingthrough the cell coat.

Methods

Sample collection, culturing and microscopy: Amoebaewere isolated from a bottom sediment sample collected onAugust 26, 2010 from the Sea of Japan (42◦26.4337′N133◦08.7581′E, depth 2709 m) during the joint Russian-German expedition SoJaBio on board of the Russian researchvessel “Akademik MA Lavrentyev”. Samples of the soft aerobicbottom sediment were collected using multicorer gear. Subsam-ples of the sediment surface mixed with a several centimeter



thick layer of overlaying water were picked aseptically, trans-ferred into sterile 650-ml tissue culture vessels, and furtherdistributed into 90-mm petri dishes and 50-ml tissue culturevessels with addition of sterile (Millipore-filtered, 0.22 �m) sea-water and autoclaved wheat grains (2-3 per culture vessel) asa source of nutrients. Samples were incubated at room temper-ature. Amoebae observed in the samples were cloned, studiedwith light microscopy (LM), and scanning electron microscopy(SEM) essentially as described in Kudryavtsev et al. (2011b).For transmission electron microscopy (TEM) amoebae werefixed on ice in 2.5% glutaraldehyde prepared with seawa-ter for 40 minutes followed by 1% osmium tetroxide preparedwith seawater for 1 hour. Cells were washed with seawater(3x5 minutes) between fixation steps. Before dehydration theseawater concentration was gradually decreased, and the cellswere embedded in agar prepared with distilled water. Piecesof agar (ca. 1 mm3) containing amoebae were cut out, dehy-drated in a graded ethanol series followed by epoxy propaneand embedded in Araldite M epoxy resin (Serva). Silver to lightgold sections were cut on a Reichert ultramicrotome using a dia-mond knife and stained with 2% uranyl acetate in 70% ethanoland Reynolds’ lead citrate. Sections were observed using aPhilips EM208 electron microscope at 80 kV.

DNA isolation, sequencing and phylogenetic analysis:Total DNA was isolated from the cell culture, and full-lengthnuclear small-subunit (SSU) ribosomal RNA gene was ampli-fied as a single piece, cloned and sequenced as described inKudryavtsev et al. (2011b). Partial sequence of mitochondrialcytochrome C oxidase subunit 1 gene (COI) was obtained asdescribed in Nassonova et al. (2010). Three SSU rRNA geneclones and 9 COI gene clones were sequenced in both direc-tions (GenBank accession numbers JN638030- JN638032 forSSU rRNA and JN638033- JN638037 for COI). The obtainedSSU rRNA gene sequences were manually aligned with ourdatabase of amoebozoan and opisthokont sequences usingSeaview (Gouy et al. 2010). The final alignment used forphylogenetic analysis comprised 151 sequences, includingrepresentatives of all major lineages of Amoebozoa and 13opisthokonts as an outgroup. All of the 1366 unambiguouslyaligned nucleotide positions were included in the analysis.The phylogenetic analysis was done using RaxML Version7.2.6 (Stamatakis 2006) and MrBayes Version 3.1.2 (Altekaret al. 2004; Huelsenbeck and Ronquist 2001; Ronquist andHuelsenbeck 2003) run at the Bioportal computer service(http://www.bioportal.uio.no). The GTR model of nucleotidesubstitution (Lanave et al. 1984) was used in both analy-ses using a gamma model of among-site rate heterogeneitywith a proportion of invariable sites (as suggested by MrMod-eltest; Nylander 2004, 2006; Posada and Crandall 2001) ina maximum likelihood analysis; covarion and autocorrelationmodels for among-site rates were used in the Bayesian anal-ysis. All model parameters were estimated from the data.The stability of the clades in the ML analysis was assessedusing non-parametric bootstrap with 1000 replicates in RaxML(Stamatakis 2006).

Acknowledgements

We are greatful to the captain, crew, and allmembers of the research team in the SoJaBiocruise on board the RV “Akademik MA Lavrentyev”(August-September 2010). Cruise organization andsampling were financially supported by the grant

12-I-P30-07 from the Far Eastern Branch of theRussian Academy of Sciences to the A. V. Zhir-munsky Institute of Marine Biology. Further studywas supported by DFG grant HA 818/22-1 and bythe research grant IZLR Z3_128338 from Scienceand Technology Cooperation Program Switzerland– Russia.

References

Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004)Parallel metropolis-coupled Markov chain Monte Carlo forBayesian phylogenetic inference. Bioinformatics 20:407–415

Angell RW (1975) Structure of Trichosphaerium micrum sp. n.J Eukaryot Microbiol 22:18–22

Angell RW (1976) Observations on Trichosphaeriumplatyxyrum sp. n. J Eukaryot Microbiol 23:357–364

Arndt H, Hausmann K, Wolf M (2003) Deep-sea heterotrophicnanoflagellates of the Eastern Mediterranean Sea: qualitativeand quantitative aspects of their pelagic and benthic occur-rence. Mar Ecol Prog Ser 256:45–56

Atkins MS, Anderson OR, Wirsen CO (1998) Effect ofhydrostatic pressure on the growth rates and encystment offlagellated protozoa isolated from a deep-sea hydrothermalvent and a deep shelf region. Mar Ecol Prog Ser 171:85–95

Atkins MS, Teske AP, Anderson OR (2000) A survey of flagel-late diversity at four deep-sea hydrothermal vents in the easternPacific Ocean using structural and molecular approaches. JEukaryot Microbiol 47:400–411

Atkins MS, Hanna MA, Kupetsky EA, Saito MA, Taylor CD,Wirsen CO (2002) Tolerance of flagellated protists to highsulfide and metal concentrations potentially encountered atdeep-sea hydrothermal vents. Mar Ecol Prog Ser 226:63–75

Furtado JS, Olive LS (1971) Ultrastructure of the protostelidCeratiomyxella tahitiensis including scale formation. Nova Hed-wigia 21:537–576

Furtado JS, Olive LS (1972) Scale formation in a primitivemycetozoan. Trans Am Microsc Soc 91:594–596

Gouy M, Guindon S, Gascuel O, Galtier N, Gouy M, Gau-tier C (2010) SeaView version 4: a multiplatform graphical userinterface for sequence alignment and phylogenetic tree build-ing. Mol Biol Evol 27:221–224

Gooday AJ (1994) The biology of deep-sea foraminifera: areview of some advances and their applications in paleoceanog-raphy. Palaios 9:14–31

Gooday AJ (2002) Organic-walled allogromiids: aspects oftheir occurrence, diversity and ecology in marine habitats. JForam Res 32:384–399

Gooday AJ, Bowser SS (2005) The second species of Gromia(Protista) from the deep sea: its natural history and associa-tion with the Pakistan margin oxygen minimum zone. Protist156:113–126

Gooday AJ, Kamenskaya OE, Cedhagen T (2007) New andlittle-known Komokiacea (Foraminifera) from the bathyal andabyssal Weddell Sea and adjacent areas. Zool J Linn Soc151:219–251



Hausmann K, Weitere M, Wolf M, Arndt H (2002b) Meteorasporadica gen. nov. et sp. nov. (Protista incertae sedis) – anextraordinary free-living protist from the Mediterranean deepsea. Eur J Protistol 38:171–177

Hausmann K, Hülsmann N, Polianski I, Schade S, Weitere M(2002a) Composition of benthic protozoan communities alonga depth transect in the eastern Mediterranean Sea. Deep-SeaRes I 49:1959–1970

Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesianinference of phylogeny. Bioinformatics 17:754–755

Kudryavtsev A (2012) Microscopic evidence for inclusion ofParvamoeba Rogerson, 1993 into the order Himatismenida(Amoebozoa). Eur J Protistol 47:85–88

Kudryavtsev A, Smirnov A (2006) Cochliopodium gallicum n.sp. (Himatismenida), an amoeba bearing unique scales, fromcyanobacterial mats in the Camargue (France). Eur J Protistol42:3–7

Kudryavtsev A, Pawlowski J, Hausmann K (2011a) Descrip-tion of Paramoeba atlantica n. sp. (Amoebozoa, Dactylopodida)– a marine amoeba from the Eastern Atlantic, with emendationof the dactylopodid families. Acta Protozool 50:239–253

Kudryavtsev A, Wylezich C, Pawlowski J (2011b) Oval-opodium desertum n. sp. and the phylogenetic relationshipsof Cochliopodiidae (Amoebozoa). Protist 162:571–589

Lahr DJG, Grant J, Nguyen T, Lin JH, Katz LA (2011) Com-prehensive phylogenetic reconstruction of Amoebozoa basedon concatenated analyses of SSUrDNA and actin Genes. PLoSONE 6:e22780

Lanave C, Preparata G, Saccone C, Serio G (1984) A newmethod for calculating evolutionary substitution rates. J MolEvol 20:86–93

Moran DM, Anderson OR, Dennett MR, Caron DA, Gast RJ(2007) A description of seven Antarctic marine gymnamoebaeincluding a new subspecies, two new species and a new genus:Neoparamoeba aestuarina antarctica n. subsp., Platyamoebaoblongata n. sp., Platyamoeba contorta n. sp. and Vermistellaantarctica n. gen. n. sp. J Eukaryot Microbiol 54:169–183

Nassonova E, Smirnov A, Fahrni J, Pawlowski J (2010) Bar-coding amoebae: comparison of SSU, ITS and COI genes astools for molecular identification of naked lobose amoebae. Pro-tist 161:102–115

Nylander JAA (2004) MrModeltest v2. Program Dstributed bythe Author. Evolutionary Biology Centre, Uppsala University

Nylander JAA (2006) Mailfit script, Distributed by the Author.Evolutionary Biology Centre, Uppsala University

Pawlowski J, Christen R, Lecroq B, Bachar D, ShahbazkiaHR, Amaral-Zettler L, Guillou L (2011) Eukaryotic richnessin the abyss: insights from pyrotag sequencing. PLoS ONE6:e18169

Posada D, Crandall KA (2001) Selecting the best-fit model ofnucleotide substitution. Syst Biol 50:580–601

Pussard M (1973) Description d’une amibe de type flabellulien:Pessonella marginata n. g. n. sp. (Mayorellidae, Amoebaea).Protistologica 9:175–185

Rogerson A, Hannah FJ, Anderson OR (1998) A rded-scription of Rhabdamoeba marina, an inconspicuous marineamoeba from benthic sediments. Invertebr Biol 117:261–270

Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesianphylogenetic inference under mixed models. Bioinformatics19:1572–1574

Scheckenbach F, Wylezich C, Weitere M, Hausmann K,Arndt H (2005) Molecular identity of strains of heterotrophicflagellates isolated from surface waters and deep-sea sedi-ments of the South Atlantic based on SSU rDNA. Aquat MicrobEcol 38:239–247

Schuster FL (1976) Fine Structure of the schizont stage of thetestate marine ameba, Trichosphaerium sp. J Eukaryot Micro-biol 23:86–93

Sheehan R, Banner FT (1973) Trichosphaerium—an extraor-dinary testate Rhizopod from coastal waters. Estuar Coast MarSci 1:245–250

Smirnov AV (1999) Korotnevella diskophora n.sp. (Gym-namoebia, Paramoebidae) - small freshwater amoeba withpeculiar scales. Protistology 1:30–33

Smirnov AV, Kudryavtsev AA (2005) Pellitidae n. fam.(Lobosea, Gymnamoebia) – a new family, accommodating twoamoebae with an unusual cell coat and an original mode oflocomotion, Pellita catalonica n. g., n. sp. and Pellita digitatacomb. nov. Eur J Protistol 41:257–267

Smirnov AV, Bedjagina OM, Goodkov AV (2011a) Der-mamoeba algensis n. sp. (Amoebozoa, Dermamoebidae) – analgivorous lobose amoeba with complex cell coat and unusualfeeding mode. Eur J Protistol 47:67–78

Smirnov AV, Chao E, Nassonova ES, Cavalier-Smith T(2011b) A revised classification of naked lobose amoebae(Amoebozoa: Lobosa). Protist 162:545–570

Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixedmodels. Bioinformatics 22:2688–2690

Tekle Y, Grant J, Anderson OR, Nerad TA, Cole JC, Pat-terson DJ, Katz LA (2008) Phylogenetic placement of diverseamoebae inferred from multigene analyses and assessment ofclade stability within ‘Amoebozoa’ upon removal of varying rateclasses of SSU-rDNA. Mol Phylogenet Evol 47:339–352

Wuyts J, Van de Peer Y, De Wachter R (2001) Distribu-tion of substitution rates and location of insertion sites on thetertiary structure of ribosomal RNA. Nucleic Acids Res 29:5017–5028

Wuyts J, De Rijk P, Van de Peer Y, Pison G, Rousseeuw P,De Wachter R (2000) Comparative analysis of more than 3000sequences reveals the existence of two pseudoknots in areaV4 of eukaryotic small subunit ribosomal RNA. Nucleic AcidsRes 28:4698–4708

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Kudryavtsev, Pawlowski 2013 - Squamamoeba Japonica - Protist164,13-23

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