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Protistology 11 (4), 231-237 (2017) Protistology
Phylogenetic position of the amoeba genus Deuteramoeba (Amoebozoa, Tubulinea)
Oksana Kamyshatskaya1, Elena Nassonova21 and Alexey Smirnov1
1 Department ofInvertebrate Zoology, Faculty of Biology, St. Petersburg State University, St. Petersburg, Russia
2 Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia | Submitted November 21, 2017 | Accepted December 6, 2017 |
Summary
Amoebae of the family Amoebidae are among the largest amoebozoans, most widely known to non-specialists as they are popular objects for teaching and various demonstrations. At the same time, they are among the groups, which are the least represented in the molecular phylogenetic trees. Of seven genera comprising the family, members of only two - Amoeba and Chaos - are present in SSU rRNA gene trees and only one species of Amoeba - in multigene trees. We have sequenced SSU rRNA gene of the CCAP 1586/1 strain representing the type strain of the species Deuteramoeba mycophaga, thus adding the third genus to the phylogeny of this family. Phylogenetic analysis robustly placed it as a sister group to Amoeba plus Chaos clade, thus confirming its position deduced earlier from the morphological characters. We also observed structures that may evidence for the presence of the intranuclear parasite in this strain of Deuteramoeba.
Key words: Amoebozoa, Amoebidae, systematics, phylogeny
Introduction
Amoebae of the family Amoebidae are among the largest and the most remarkable amoebozoans populating freshwater and soil habitats. However, they are not among the most widely distributed species. While amoebae ofthe genera Amoeba, Chaos and Polychaos were isolated several or even many times from a variety of natural habitats (Page and Baldock, 1980; Page and Kalinina, 1984; Page, 1986; Smirnov and Goodkov, 1997, 1999; Goodkov et al., 1999; Bolivar et al., 2001), members of other four genera - Deuteramoeba, Trichamoeba, Hydramoeba and Parachaos were seen few times or even once
only (Schaeffer, 1926; Page and Robson, 1983; Siemensma and Page, 1986; Pussard et al., 1980; Willumsen, 1982; Mrva, 2010/11). Four species belonging to the genera Amoeba and Chaos were sequenced by Bolivar et al. (2001) and grouped together in the phylogenetic tree; hence species of both genera were intermingled in the tree. Multigene data on Amoeba proteus were obtained by Kang et al. (2017), but in this tree no other species belonging to the family Amoebidae are present.
The genus Deuteramoeba was erected by Page (1986) to accommodate a species, first described as Amoeba algonquinensis by Baldock et al. (1983). The electron-microscopic study showed that this
doi:10.21685/1680-0826-2017-11-4-4 © 2017 The Author(s)
Protistology © 2017 Protozoological Society Affiliated with RAS
species, in contrast to those of the genus Amoeba, had amorphous but not filamentous glycocalix. At that time, this was a genus-level character; also later it was shown that filamentous and amorphous glycocalixes may be found in the same genus (Smirnov and Goodkov, 1997). The second species of this genus was first described as Trichamoeba mycophaga (Pussard et al., 1980) and transferred to the genus Deuteramoeba by Page (1988) because of its predominantly polypodial locomotive form. This species was isolated from soil (Pussard et al., 1980; Chakraborty and Old, 1986; Mrva, 2005, 2010/11; Page, 1988, 1991) and is cyst-forming; this maybe a specific adaptation to its soil lifestyle (Smirnov and Brown, 2004).
In the present study we have sequenced SSU rRNA gene ofthe CCAP 1586/1 strain representing the type strain of the species Deuteramoeba mycophaga. Phylogenetic analysis robustly placed it as a sister group to Amoeba plus Chaos clade, thus confirming its position deduced earlier from the morphological characters.
Material and methods
The strain CCAP 1586/1 was obtained from the Culture Collection of Algae and Protozoa (CCAP, nowadays it is a part of the UK National Culture Collection - UKNCC, Oban, Scotland, UK) in 2012 and maintained since that time on PJ medium (Prescott and James, 1955) and rice grains in 90 mm Petri dishes at + 18 °C and non-regulated light. Amoebae were transferred to fresh medium monthly.
Light-microscopic observations and photographing were performed in cultures, using Leica DMI3000 inverted microscope equipped with Phase contrast optics and on the glass object slides using upright Leica DM2500 microscope equipped with Phase contrast, DIC optics and Nikon DSFi-1 camera linked to the computer with NIS-Elements software. The size of the cells was measured in culture using inverted microscope, the size of the nucleus — on the object slides; special attention was paid to not pressing the amoebae with the coverslip to avoid deformation of the nucleus.
To obtain the SSU rRNA gene sequence, individual amoeba cells floating in the water were collected with tapered-tips glass Pasteur pipettes to the 60 mm Petri dishes filled with fresh Millipore-filtered (0.22 ^m) medium. Amoebae were left in these
dishes overnight, next day they were transferred to the dishes with fresh medium in the similar manner. After one more day, cells in 1-2 ^l of the medium were placed in 200 ^l PCR tubes and frozen under -20 °C. Right before the PCR, the tubes containing cells were defrozen under room temperature and frozen back for approximately 3 min; this cycle was repeated three times. After that, 48 ^l ofready-made PCR mixture were added to the tubes.
The SSU rRNA gene was amplified by PCR using universal eukaryote primers RibA (5'> ac ctg gtt gat cct dcc agt <3') and RibB (5'> tga tcc atc tgc agg ttc acc tac <3') complementary to the 5' and 3' end of the gene (Medlin et al., 1988). Thermal cycle parameters were: initial denaturation (10 min at 95 °C) followed by 39 cycles of 30 s at 94 °C, 60 s at 50 °C and 120 s at 72 °C, followed by 10 min at 72 °C for final extension. Amplicons were purified using Cleanup mini Purification Kit (Eurogene) and sequenced using ABI-PRISM Big Dye Terminator Cycle Sequencing Kit. Besides amplification primers, additional primer F6 (5'> cng cgg taa ttc cag ctc <3') was used for sequencing.
Obtained sequences were added to the alignment containing all culture-derived sequences of Amoebida, a sample of other tubulinean sequences and some discosean sequences used as the outgroup. Sequences were aligned manually using SeaView 4.0 (Gouy et al., 2010). The phylogenetic analysis was performed using maximum likelihood method as implemented in PhyML program (Guindon and Gascuel, 2003) with GTR + y + I model suggested by the program Modeltest (Posada and Crandall, 1998); 1471 sites were selected for the analysis. The number of invariant sites, alpha parameter and tree topology were optimized by PhyML, 100 bootstrap pseudoreplicates were performed. Bayesian analysis was performed on the same dataset using MrBayes 3.1.2, GTR model with y - correction for intersite rate variation (8 categories) and the covarion model (Ronquist and Huelsenbeck, 2003). Trees were run as two separate chains (default heating parameters) for 10 million generations, by which time they had ceased converging (final average standard deviation of the split frequencies was less than 0.01). The quality of chains was estimated using built-in MrBayes tools and additionally - using the software Tracer 1.6 (Rambaut et al., 2014); based on the estimates by Tracer, the first 25 % of generations were discarded for burn-in.
Results and discussion
Light-microscopic observations show cells, fitting the previously published descriptions of this species (Old et al., 1985; Chakraborty and Old, 1986; Page, 1988, 1991; Mrva, 2010/11). Amoebae were polytactic, with relatively short and non-numerous pseudopodia (Fig. 1 A-B). Actively moving cells were nearly orthotactic (Fig. 1 C-E). Cells often formed a morulate uroid (Fig. 1 C, E-F); some cells had two or even three uroidal structures simultaneously, of those only one was functional while the others were at different stages of degradation. Sometimes cells in locomotion became rather flattened and even formed a sort of longitudinal dorsal ridges in the anterior part of the hyaloplasm (Fig. 1 D). The cytoplasm of the cell contained a lot of crystals. Some of them were truncate bipyramidal (Fig. 1 G), but the most of crystals should rather be recognized as truncate spindle-shaped, because they were rather smooth and had no pronounced edges. The abundance of crystals makes a lot of problems during observations as they obscure the internal structures and are highly refractive. Other inclusions were opaque granules of different size and vacuoles containing food remnants. Few cells contained large crystalloids of irregular form, sometimes they were comparable in size with the cell nucleus; such cells did not look healthy and were not actively moving.
The nucleus had the single central homogeneous nucleolus, often with a central lacuna (Fig. 1 H). The size of the nucleolus varied from 12 to 16 ^m. Interestingly, some of the observed nuclei (Fig. 1 I-K) contained structures that could be interpreted as presence of an intra-nuclear parasite of amoebae, like Paramicrosporidium (Scheid, 2007; Michel et al., 2000, 2009a) or Nucleophaga (Michel et al., 2009b, 2012). We observed nuclei with a number of rounded bodies, 2-4 ^m across, surrounding the nucleolus (Fig. 1 I-J). Each body had one or (rarely) two depressions or lacunas in the middle area when viewed with DIC optics. In the most of presumably infected cells these bodies surrounded the nucleolus forming a monolayer. These patterns are comparable to those of the developmental stages of Paramicrosporidium as illustrated by Schneid (2007) and Michel et al. (2009a). To the lesser extent they resemble Nucleophaga (Michel et al., 2009b, 2012). We also observed nuclei, where the nucleolus appeared to be hypertrophied and filled with the granular material (Fig. 1 K), which is comparable to the images of the sporogony in
Paramicrosporodium (Schneid, 2007; Michel et al., 2000, 2009a). No other stages have been observed yet; should this observation be confirmed by the electron microscopy or molecular methods, this will expand the known range of hosts of these parasites.
It is noticeable that Page (1988 p. 60 Fig. 18, J) and Page (1991, p. 65, Fig. 20, h-i) showed the nucleus of D. mycophaga containing exactly the same structures as shown here (which is not surprising, given that those are the images of the same CCAP strain 1586/1). The depositor of this strain, according to CCAP record, was Old in the year 1984. In the formal description of this species, Chakraborty and Old (1986) noted that the studied culture was deposited with CCAP (without indication of the exact time). Neither Old et al. (1985) nor Chakraborty and Old (1986) who studied the ultrastructure of this amoeba species, found anything resembling these structures, neither in light microscopy nor in the electron-microscopic images of the nucleus. This suggests that either the culture probably got infected during its maintenance in CCAP but the putative infection was not recognized since this group of organisms was not known at that time, or a different strain rather than the one studied by Chakraborty and Old (1986) was deposited. Chakraborty and Old (1986) wrote that "amoebae were isolated from a sandy loam wheat field soil from Eyre Peninsular region of South Australia and from a red basaltic loam soil supporting a mixed eucalypt rainforest community, south of Burnie, Tasmania, Australia" (op. cit., p. 564); so, this presumes that more than one strain might have been available initially. Mrva (2010/11) reported isolates identified as D. mycophaga from several different habitats in Slovakia; however, he did not note any suspicious structures in the vesicular nucleus ofthese amoebae.
In the SSU phylogenetic tree, Deuteramoeba mycophaga robustly grouped with the other members of the family Amoebidae as a basal group for this family. This position is fully supported and evidences that Deuteramoeba is an independent genus within the family Amoebidae, as it was suggested from the morphological data (Page 1988). As in all other trees, members of the genera Amoeba and Chaos are intermingled in the tree, and their grouping has moderate support. Arrangement of the other clades in the tree corresponds to the generally recognized topology (Smirnov et al., 2005, 2011, 2017; Kang et al., 2017; Cavalier-Smith et al., 2016) and all these clades are highly supported. These data evidence once more that morphological
Fig. 1. Light microscopy of Deuteramoeba mycophaga CCAP 1586/1 strain. DIC. A-B — Polytactic locomotive form; C-D — orthotactic locomotive form, note tiny dorsal longitudinal ridges arrowed in D; E — elongate cell that just started to change the direction of movement; F — uroidal structures; G — crystals in the cytoplasm, of them the only one that can be considered as a truncate bipyramidal is arrowed; H — intact nucleus; I-K — nuclei, presumably infected with the parasite (several parasite cells are arrowed in I-J and the aggregation of granular material replacing the nucleolus in K). Abbreviations: u — uroid; n — nucleus; nu — nucleolus. Scale bars: 20 ^m in A-E, 10 ^m in F-K.
0.05
Chaos nobile CCAP1511/2! AJ31460'6 —Amoeba leningradensis CCAP 1503/6 AJ314605
Chaos carolinense strain WW13-1324 AJ314607
Amoeba proteus strain Geneva AJ314604 -Deuteramoeba mycophaga CCAP 1586/1
r—Copromyxa sp. strain 4/3Da/10 AY680841
1-Copromyxa protea strain BF09-2-2-B GU938872
Copromyxa cantabrigiensis CCAP1534/8 AY294147 -Copromyxa sp. strain ZEB4 JQ271679
Saccamoeba sp. strain MSED6 JQ271718 Saccamoeba Umax strain NTSHR EU869301 Saccamoeba lacustris CCAP1572/4 GQ221845 -Glaeseria mira CCAP1531/1 AY294146
r-Sa
r-Cs
I—Sacc
0/99 ^Nolandella sp. strain AFSM9 JQ271706 Nolandella sp. ATCCPRA27 EU273456
Nolandella abertawensis strain JKS1 JQ271707 Nolandella abertawensis CCAP1534/9 DQ190241 Nolandella sp. ATCC50913 EU273451
Hyalosphenia papilio JF694282
-Nebela flabellulum EU392152
-Heleopera sphagni AY848964
-c"
-Paraflabellula reniformis ATCC50741 AF293900 tParaflabellula hoguae AY277797 -Flabellula citata CCAP1529/2 EU852654 Leptomyxa variabilis (former 'Ripidomyxa' sp.) AY549563 ^ ^Leptomyxa reticulata ATCC50242 AF293898 —Rhizamoeba saxonica CCAP1570/2 EU719197 -Echinamoeba thermarum AJ489261
-Echinamoeba exundans ATCC50171 AF293895
-Vermamoeba vermiformis KU519742/1
|-Stenamoeba.stenopodia CCAP1565/3 AY294144
-Stenamoeba sp. JQ271721 -Stenamoeba limacina GU810183 -Mayorelia sp. strain JJP2003 AY294143
^ r-Stenai I-Stena
-Paradermamoeba levis CCAP1555/2 JN247435
-Dermamoeba algensis AY294148
-Acanthamoeba lenticulata ATCC50703 U94730 -Acanthamoeba tubiashi ATCC30867 AF019065
-Protacanthamoeba bohemica AY960120
-Balamuthia mandrillaris AF477019
Euamoebida
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Arcellinida
Leptomyxida
Echinamoebida Thecamoebida Dermamoebida
Acanthopodida
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Fig. 2. Phylogenetic tree based on SSU rRNA gene, showing the position of Deuteramoeba mycophaga. 1477 sites; MrBayes (GTR+ у model, 25 rate categories) and PhyML (GTR + у + I model). Supports indicated as PP/BS; black dotes mean full (1.0/100) support of branches.
systematics of amoebae, when it concerns species with remarkable morphological characters, remains in many cases valid and that the "proteus-like" organization of the cell (large cells forming discrete tubular pseudopodia) may represent a valid taxono-mic character that probably unifies the family Amoebidae (Bovee, 1985; Page, 1986, 1987).
Acknowledgments
Supported by the Russian Science Foundation grant 17-14-01391 (concept, systematics and molecular studies), RFBR grant 16-04-01454 (cultivation, light microscopy) and RFBR grant 15-04-08870 (identification and light-microscopy of a putative intranuclear parasite). The present study utilized equipment of the core facility centres "Development of molecular and cell technologies" and "Culture Collection of microorganisms" of Saint Petersburg State University. Infrastructure was supported by the SPSU grants 1.40.539.2017
and 1.42.1101.2016.
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Address for correspondence: Oksana Kamyshatskaya. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia; e-mail: oksana.kamyshatskaya@gmail.com
