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Protistology 15 (4), 312-320 (2021)
Protistology
Description of Leptomyxa silvatica n. sp. (Amoebozoa, Tubulinea, Leptomyxida), a new soil amoeba species from Chernevaya taiga soil of West Siberia, Russia
Anna A. Glotova1, Sergey V. Loiko2, Georgy I. Istigichev2, Anastasia I. Kulemzina3, Evgeny V. Abakumov4, Alla L. Lapidus56 and Alexey V. Smirnov7
1 Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RRAS, 194064 St. Petersburg, Russia
2 National Research Tomsk State University, 634050 Tomsk, Russia
3 Institute of Molecular and Cellular Biology, 630090 Novosibirsk, Russia
4 Department of Applied Ecology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia
5 Center for Algorithmic Biotechnology, St. Petersburg State University, 199034 St. Petersburg, Russia
6 Department of Cytology and Histology, St. Petersburg State University, 199034 St. Petersburg, Russia
7 Department ofInvertebrate Zoology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia
| Submitted October 10, 2021 | Accepted November 12, 2021 |
Summary
During the studies of amoebae from the rare and highly productive soil of Chernevaya taiga (West Siberia, Russia) we have found an isolate of a leptomyxid amoeba showing 18s rDNA sequence significantly different from those of other known species of the order Leptomyxida. Here we describe this isolate as a new species, Leptomyxa silvatica n.sp. This species has both morphological and sequence differences from related ones. This finding confirms that reliable morphological differentiation and identification of leptomyxid amoebae is possible only for several remarkable species, while others require molecular data to be correctly labeled or described.
Key words: amoebae, Leptomyxa, morphology, phylogeny, Tubulinea
Introduction
The most remarkable character of amoebae belonging to the genus Leptomyxa (Amoebozoa, Tubulinea, Leptomyxida) is their ability to adopt two alternative body forms: monotactic (limax-like)
form with smooth non-anastamosing pseudopodia (lobopodia) in rapid locomotion and flattened, sometimes expanded branched (ramose or reticulate) form with loboreticulopodia when the cell moves slower (Page, 1987; Smirnov and Goodkov, 1999; Smirnov and Brown, 2004; Smirnov et al., 2005;
doi:10.21685/1680-0826-2021-15-4-7 © 2021 The Author(s)
Protistology © 2021 Protozoological Society Affiliated with RAS
2011). Trophozoites ofthese amoebae form adhesive uroidal filaments along the posterior edge of the cell (Smirnov et al., 2017). The alteration of the body shape may depend on various factors, not yet studied. It is shown that for the species Flabellula baltica the body shape depends on the amount of food resources available to the trophozoite (Fenchel, 2009).
Among amoebae of the order Leptomyxida, the most remarkably branched and ramose are species of the genus Leptomyxa Goodey, 1915 sensu Sminov et al. (2017). These organisms possess a simple life cycle, which includes subsequent stages of feeding, growth, multiplication and encystment (Goodey, 1915; Pussard and Pons, 1976a, 1976b, 1976c; Page, 1987, 1988; Smirnov et al., 2009; Del Valle et al., 2017). This genus nowadays includes eight species (see list in Smirnov et al., 2017; Del Valle et al., 2017; Smirnov, 2018). The type species of the genus is Leptomyxa reticulata Goodey, 1915. This is an irregularly triangular, expanded, large (up to several millimeters), often ramose multinucleate amoeba (usually recognized as a plasmodium) with numerous adhesive filaments along the posterior edge of the moving cell (Goodey, 1915; Page, 1988; Pussard and Pons, 1976a). Other members of the genus are significantly smaller and usually less ramose (Del Valle et al., 2017; Smirnov et al., 2017). Species that were previously classified as Rhizamoeba, like L. flabellata, L. australiensis, L. ambigua or L. neglecta almost do not demonstrate a ramose shape. They are monopodial, clavate or flattened, comet-shaped (Pussard and Pons, 1976c; Chakraborty and Pussard, 1985; Page, 1988; Smirnov et al., 2009; Smirnov et al., 2017).
Until recently, the species differentiation among the genus Leptomyxa was mostly based on light-microscopic morphology. However, due to the high level of morphological polymorphism in these amoebae, their recognition is difficult and demands continuous observation of numerous trophozoites in cultures, data on the number of nuclei and the structure of the nucleus, data on encystment and cyst wall structure (e.g., Pussard and Pons, 1976a, 1976b, 1976c). However, even careful study may not result in unambiguous differentiation of closely related species. Nowadays, molecular data are essential for reliable species distinction of leptomyxid amoebae, and often this is the molecular difference that forces investigators to seek for the morphological characters that can further distinguish newly found isolates (Smirnov et al., 2017). Moreover, the above-cited study shows that 18s rDNA gene sequences
among leptomyxid amoebae are rather conserved and even a small sequence difference may mark two species with clearly different morphology.
During studies of amoebae from the soil of West Siberia (Russia) we have found an isolate of a leptomyxid amoeba showing a peculiar morphology; its 18s rDNA sequence was significantly different from other known ones. Here we describe this isolate as a new species, Leptomyxa silvatica n. sp.
Material and methods
The strain described in the present study was isolated from the sample of the top 10 cm of Cher-nevaya taiga soil (see Abakumov et al., 2020 for the description ofthe sampling site) from a location near Tomsk city (Russia, West Siberia, Tomskaya oblast; 56.30693° N, 85.47063° W). To establish enrichment cultures, 0.01 g of mixed soil was placed in a 60 mm sterile Petri dish filled with a 0.025% cerophyl infusion made on PJ medium (Page, 1988; Prescott and James, 1955).
Enrichment cultures were incubated at room temperature and light. After 7—14 days samples were examined using a Nikon TMF 100 inverted microscope. Detected cells were individually transferred into 60 mm Petri dishes filled with the same medium using a tapered-tip Pasteur pipette. One of the successful clones was used to establish the studied culture.
Light microscopic data on living trophozoites and cysts were obtained using Nikon TMF 100, Leica DM2500 and Leica DMI3000 microscopes equipped with phase contrast and DIC optics. Both living cells and cysts were measured using an inverted microscope on the plastic surface of the Petri dish. For detailed study ofthe nuclear structure trophozoites were transferred to the glass-made object slides, left to adhere for several hours and further fixed with Bouin's solution and stained with iron hematoxylin as described by Page (1988).
For the single-cell PCR, cells were washed in autoclaved PJ medium, collected with the minimal possible amount of medium and placed in 200 mkl PCR tubes using freshly made tapered-tip Pasteur pipette for each cell. Tubes were exposed to several freezing-defreezing cycles (4 cycles from -18 °C to room temperature). Ready PCR mixture was added to the tube content to the final volume of 50 mkl. Primer pairs RibA/S12.2r and S12.2/SB (Pawlowski, 2000) were used for amplification. Thermal cycle parameters were: initial denaturation (10 min at 95
°C) followed by 39 cycles of 30 seconds at 94 °C, 60 seconds at 50°C and 120 seconds at 72 °C, followed by 10 min at 72 °C for the final extension. Amplicons were sequenced directly using the ABI-PRISM Big Dye Terminator Cycle Sequencing Kit with the S12.2, S12.2r, S6f, S4fc, S6ra and S20r primers (Adl et al., 2014; Medlin et al., 1988; Pawlowski, 2000). The resulting sequences were edited and assembled using ChromasPro software (Technilesium).
Obtained sequences were added to the alignment of Leptomyxida 18s rDNA sequences. Sequences were aligned automatically using the Muscle algorithm (Edgar, 2004) as implemented in SeaView 4.0 (Gouy et al., 2010), then the alignment was polished manually. The phylogenetic analysis was performed using the maximum likelihood method implemented in the PhyML program (Guindon and Gascuel, 2003) with the GTR + y model. The number of invariant sites, alpha parameter and tree topology was optimized by PhyML; 1000 bootstrap pseudoreplicates were used; 1866 sites were selected for the analysis. The Bayesian analysis was performed on the same dataset using MrBayes 3.2.6 run at the CIPRES portal (Miller et al., 2010), the GTR model with gamma-correction for intersite rate variation (8 rate 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 1.7.1 (Rambaut et al., 2018); based on the estimates by Tracer, the first 35% of generations were discarded for burn-in.
The obtained consensus sequence was deposited in GenBank under the number MZ687121.
Results
1. Morphological description of Leptomyxa silvatica n. sp.
The general morphology of trophozoites was assessed during observation of cells on the plastic surface of the Petri dish, using an inverted microscope. Smaller and compact amoebae were comet-shaped or elongated, with several hyaloplasmic projections ending with a number of anastomosing lobes forming a small area of loboreticulopodia
(Fig. 1, A-D). Rapidly moving amoebae had trapezoid-shaped with a wider frontal edge, which was divided into several lobes. They might carry adhesive filaments on the posterior edge of the cell (Fig. 1, E-J). Stationary and relatively small cells might produce several flattened pseudopodia of irregular shape extending in different directions. When a cell started locomotion, it usually formed one leading pseudopodium extending along the direction of movement, but also retained several lateral lobes (Fig. 1, A; Fig. 2, H). Sometimes the cell extended a pseudopodium or a hyaloplasmic eruption opposite to the direction of locomotion. Furthermore, this pseudopodium retracted and joined the main cytoplasmic body of the cell.
Among larger trophozoites comet-like forms were also documented. These large fan-shaped amoebae formed a kind of palisade of numerous short subpseudopodia directed frontally and laterally as well as a number of wide anterior lobes. Moving cells usually had a number of adhesive filaments along the posterior edge (Fig. 1, E-J; Fig. 2, F, H). Such comet-shaped amoebae were more inclined to fast, direct movement than reticulate forms and rarely formed pseudopodia extending opposite to the direction of movement.
The largest trophozoites were remarkably ramose and reticulate (Fig. 2, A-E). Short subpseudopodia were noticed along the outer contour of moderately branched cells. Amoebae also formed bunches of subpseudopodia or distinct flattened areas carrying short subpseudopodia as the end of major branches (e.g. Fig. 1, K, L; Fig. 2, I). When these amoebae moved, the leading part of the cell had a comet-shaped form, while in the central and posterior parts of the cell one or several elongated branches could be observed.
The length of actively moving trophozoites varied from 66 to 160 ^m (mean 146 ^m). The maximum width varied from 38 to 188 ^m (mean 85 ^m) and the minimum width varied from 5 to 94 ^m (mean 30 ^m).
According to our observations, cells contained numerous elongate vesicular nuclei from 5 to 11 ^m in maximal dimension (mean 7 ^m) (Fig. 2, F-L). Up to 30 nuclei were seen in large, comet-shaped cells. The nucleolus was rounded or, rarely, oblong, sometimes it had the shape of a thick spindle or of a long drop. This was especially well-visible in stained preparations (Fig. 2, H-I), while in DIC optics it was hard to distinguish nuclei among food vacuoles and other inclusions (Fig. 2, J-L). The refractile index
Fig. 1. Light microscopy of Leptomyxa silvatica n. sp. (A-D) — Small comet-like trophozoites, (E-J) — large comet-like trophozoites, (K) — an example of intermediate variant between comet-like and ramose forms, (L) — fragmentation in trophozoite with intermediate shape. Phase contrast. Scale bar is 50 ^m throughout.
of the nucleolus was probably close to that of the karyoplasm and thus the nucleolus was very lowly contrasted in live cells. Numerous synchronously working contractile vacuoles were visible in the cytoplasm, their number apparently depended on cell volume (Fig. 2, J). The cytoplasm contained a high number of rounded small granules. Neither crystals, nor other opalescent inclusions were documented (Fig. 2, J-L). Sometimes noticeable food vacuoles were observed in the central or posterior part of the granuloplasm of moving cells. In ramose cells vacuoles tended to concentrate in the most massive area of the cytoplasm.
Single-walled cysts were rounded or ovoid, from 24 to 45 ^m (mean 34 ^m) in the largest dimension. Observed cysts always contained a single nucleus
located in the central part of the cytoplasm (Fig. 2, M-Q).
2. Molecular phylogeny
The 18s rDNA gene sequence of our isolate was always grouped within a clade, containing several environmental sequences, hence showing a certain distance from them. The sister group to this clade, and the closest named relative of our strain was the species Leptomyxa arborea (Fig. 3). The support for this position was always from average to low, but the entire clade containing this species and some more environmental sequences was properly supported (94/0.9 BS/PP supports). The entire genus Leptomyxa, and all the other genera
Fig. 2. Light microscopy of Leptomyxa silvatica n. sp. (A-E) — Rreticulate plasmodia-like trophozoites. Phase contrast. Scale bar is 50 цт. (F-H) — Stained preparations of trophozoites, iron hematoxylin. Several (not all visible) nuclei are indicated with arrows. Phase contrast, scale bar is 50 цт. (I) — Trophozoite in non-directed movement; (J) — higher magnification ofthe cytoplasm, numerous nuclei are indicated with arrows. DIC, scale bar is 25 (M-Q) — Large and small living cysts in aggregations. DIC, scale bar is 25
of Leptomyxida, were fully supported, as well as the entire Leptomyxida clade. The arrangement of species within the genus Leptomyxa generally corresponded to that obtained by Smirnov et al. (2017).
The length of the majority of environmental sequences of leptomyxids available in GenBank rarely exceeds 600 bp. This makes the pairwise comparison of sequences difficult, because the difference in short fragments may not reflect the entire sequence divergence. The level of sequence identity between our isolate and the closest described species L. arborea measured in the longest possible fragment (1755 bp shared by both species) is 96.66%. The differences between the sequences of the studied isolate and L. arborea are the following (all positions are given in the sequence of L. silvatica): 241-314bp — one binucleotide and eight single nucleotide insertions; 756-757 bp CG vs AA in L. arborea; 1098-1129 bp — single nucleotide replacement and 26 nucleotides fragment that L. arborea lacks. More single and binucleotide replacements are distributed across the sequence. Other neighboring sequences are much shorter. When we perform the comparison in 569 bp fragment shared by the most of nearest neighbors, the level of identity of our isolate with L. arborea rises to 97.53%, with the highest level of identity with the sequence FO181391, originating from the soil ofthe province of Limburg, North-East Belgium (GenBank record data). It counts 99.64% (almost full identity). Further sequences FN394923 (98.38%) and FN394941 (95.77%) follow. This correlates with the position of sequences in the phylogenetic tree.
Discussion
The appearance of flattened extended reticulate trophozoites with posterior adhesive filaments convincingly indicates that the observed strain of naked lobose amoebae belongs to the genus Leptomyxa Goodey, 1915 sensu Smirnov et al. (2017).
Both compact and large comet-shaped forms of the studied isolate can be compared with typical trophozoites of L. variabilis Geisen et Burberg, 2017, but they produce numerous frontal spine-like subspeudopodia (Smirnov et al., 2017). Ramose forms of the studied isolate may resemble reticulate plasmodia-like trophozoites of L. arborea Berney, Geisen et Burger, 2017 and L. valladaresiDel Valle, Lorenzo-Morales et Maciver, 2017, but expanded
cells of the studied strain are less branched and often produce short subpseudopodia along the outer contour. Intermediate forms of trophozoites are rather compact and produce frontal lobes but still contain slightly branching parts were never clearly shown in L. arborea or L. valladaresi (Smirnov et al., 2017; Del Valle et al., 2017). The species L. valladaresi was described as primary uninucleate or possessing a few rounded vesicular nuclei (oval only when compressed), well-visible in stained preparations (Del Valle et al., 2017). They are very different in morphology from our pictures. The data on the nuclear morphology of L. arborea are not detailed enough, and available images do not clearly show the nucleus.
We have never documented a true limax mono-podial form in the studied isolate, which is not a unique case. For example, L. arborea and L. variabilis are mostly described as expanded and reticulate (Smirnov et al., 2017), while in L. flabellata, L. valladaresi, L. australiensis and L. ambigua limax-like forms are frequently seen (Chakraborty and Pussard, 1985; Page, 1988; Smirnov et al., 2017; Smirnov, 2018). It seems that in many strains the detection of the limax-like form is a matter of observation duration and specific culture conditions, so it may be difficult to see them in fresh or mixed cultures.
The 18s rDNA sequence identity level of96.66% is a rather high similarity for an eukaryotic organism. However, among leptomyxids 18s rDNA sequences are very stable, in contrast with many other amoebae lineages. This is the only group ofamoebae where 18s rDNA sequences of distant isolates show complete identity and the sequence divergence between morphologically different species may be very low (Smirnov et al., 2009, 2017). For leptomyxid amoebae the difference found in the present study is significant, and if we consider it together with the morphological peculiarities, this all clearly votes for the species status of the studied isolate. Based on enlisted characteristics we consider the studied isolate as a new species and suggest naming it Leptomyxa silvatica n. sp. The origin ofthis species from Chernevaya taiga soil is interesting, because this soil represents a kind of rare, highly productive ecotope, known for its pronounced plant gigantism (see Abakumov et al., 2020). Hence, the closest similarity of its 18s rDNA sequence with several 18s rDNA fragments of unnamed isolates originating from Belgian soil may be evidence of the wider distribution of this species.
ÎAF293901 Rhizamoeba sp. ATCC50743 AY121847 Rhizamoeba sp. cf. saxonica ATCC50742 EU852655 Flabellula sp. strain SEDF/I
ÎEU852656 Flabellula sp. strain N ETC3/1 EU852658 Flabellula sp. strain STAR2 LC340972 Flabellula sp. CSP3 AF293899 Paraflabellula cf. hoguae ATCC30733 AY277797 Paraflabellula cf. hoguae strain UP16 -EU273463 Rhizamoeba sp. ATCC50933
- AF293900 Paraflabellula cf. reniformis ATCC50741 -FN598467 Flabellulidae strain M02 El
96/1.0 90/1.0
54/0.791/1.0-
75/1.0 64/0.7.
EU - I EU
94/0.9
100/0.8, EU852652 Flabellula baltica strain SMA17/I 77/0.9 JAM087465 environmental clone DGGE band 77 L- KT945248 Flabellula baltica strain TF
LC340976 Flabellula sp. PH21 EU852657 Flabellula sp. strain M4M/I EU852653 Flabellula sp. strain M9M/I [r EU852654 Flabellula citata CCAP1529/2 GU320577 Flabellula sp. strain COHH/IO KT986071 Flabellula sp. AK2016a KT945249 Flabellula baltica strain AS KT986070 Flabellula sp. 5004 KT986072 Flabellula sp. 5001 KT986068 Flabellula sp. AK2016a.5002 KT986069 Flabellula sp. AK2016a.5003 EF024771 environmental clone Eimer3 EF024925 environmental clone Eimer2 EF024454 environmental clone Eimer4 EF024279 environmental clone Oxytrl L EF024969 environmental clone Eimerl
Flabellula
IKT945254 Leptomyxa arborea strain 23Netherlands 78/o 8j \ KT945253 Leptomyxa arborea strain LA2wmllK 58/o.6^s_ r F0181391 environmental clone RPZ5YH12 80/0.7 r j FN394941environmental clone 01RPZ110600071
64/0.6---V\
85/0.7—H FN394923 environmental clone 01RPZ110600053 64/0.6rt L_ Leptomyxa silvatica n. sp.
MT531200 environmental clone OTU265 FN396498 environmental clone 01RLS110600079 ■ MK946187 environmental clone ¡652
58/0.6 i-EF023245 environmental clone Amb_18S_547
84/0.91 KT945251 Rhizamoeba neglecta type strain complete AF293898 Leptomyxa cf. reticulata ATCC50242 KT945252 Rhizamoeba cf. australiensis CCAP KX792145 Leptomyxa valladaresi LLT AY549563 Ripidomyxa sp. strain RP009 KT945250 Leptomyxa variabilis AY549564 Ripidomyxa sp. strain RP010 Fj844435 Rhizamoeba neglecta type strain partial LC222981 clone HRT6hrs.A5.pssu9 AB238161 clone 18S.KM.B.2
AB505542 environmental clone RM2SGM34 EU719197 Rhizamoeba saxonica CCAP 1570/2 EF023499 environmental clone Amb_18S_1090
AY605217 environmental clone Sey088 AF293895 Echinamoeba exundans AJ489264 Echinamoeba thermarum
Leptomyxa
Rhizamoeba
OUTGROUP
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Fig. 3. Phylogenetic tree based on the SSU gene and showing the relationships between available SSU sequences of the order Leptomyxida. Bootstrap support (PhyML) / Posterior probability (MrBayes) are indicated; 1866 nucleotide positions used. Black dots indicate 1.0/100 supports (BS/PP).
Diagnosis: Leptomyxa silvatica n. sp.
Flattened, expanded or ramose amoebae. Moving cells form an area of loboreticulopodia on the frontal edge. Moving cells are 60-160 ^ across in length and up to 190 ^ in width. Trophozoites contain up to 20-30 ovoid vesicular nuclei up to 7 ^ in largest dimension. Single-walled rounded or ovoid uninucleate cysts are 24-45 ^ across.
Type material: Holotype, permanent preparation No 1049, stained with iron hemotoxylin, deposited in the collection of preparations of the Laboratory of Cytology of Unicellular Organisms, Institute
of Cytology, St. Petersburg, Russian Academy of Sciences.
Type location: Chernevaya taiga soil from a location nearby Tomsk city (Russia, West Siberia, Tomskaya oblast; 56.30693° N, 85.47063° W)
Etymology: from latin word silva, forest. The name means "originating from the forest".
Acknowledgements
Supported by the RSF No 19-16-00049 project. The present study utilized equipment of the Core
facility centers "Development of molecular and cell technologies, "Biobank", "Computing Center SPSU" and "Culture Collection ofMicroorganisms" of the research park of Saint Petersburg State University.
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Address for correspondence: Anna Glotova. Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky ave. 4, 194064 St. Petersburg, Russia; e-mail: glotova.anna@gmail.com
