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Protistology 18 (2): 143-160 (2024) | doi:10.21685/1680-0826-2024-18-2-5 Pl'OtiStOlO&y
Original article
Polychaos insularis n. sp. (Amoebozoa, Tubulinea - one more amoeba species belonging to "Polychaos fasciculatum-like" species group
Oksana Kamyshatskaya12*, Elena Nassonova12, Yelisei Mesentsev1, Natalia Bondarenko1 and Alexey Smirnov1
1 Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia
2 Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, 194064 St. Petersburg, Russia
| Submitted November 2, 2024 | Accepted December 5, 2023 |
Summary
The diversity of amoebae of the genus Polychaos comprises a group of three hardly distinguishable "Polychaos fasciculatum-like" species: P. annulatum, P. fasciculatum and Metachaos gratum. Polychaos annulatum was twice re-isolated and thoroughly studied. There is little data on P. fasciculatum, and M. gratum has never been investigated since its description by A.A. Schaeffer in 1926. A strain of polytactic amoebae resembling in appearance Polychaos fasciculatum was isolated from a freshwater sample collected from the ditch in the surroundings of the Ostrov town, Pskov Region, Russia. This organism did not grow well in culture; we managed to get only 11 cells. Yet, with these cells, we performed light and electron-microscopic studies and obtained 18S rRNA gene sequence using single-nucleus DNA isolation technique followed by NGS-sequencing. To compare this strain with P. fasciculatum, we utilized light microscopic data and embeddings of the strain P. fasciculatum CCAP 1564/1 made by A. Smirnov in 1999, during his research at the Culture Collection of Algae and Protozoa (CCAP). The analysis of molecular data robustly placed our strain in the genus Polychaos as a sister clade of P. annulatum. Based on the morphological and molecular data, here we describe a new species, Polychaos insularis. Our study provides a comprehensive description of the nuclear structure of the "Polychaos fasciculatum-like" species, and demonstrates that re-investigations of available materials of the previously studied strains using modern technical facilities could be of significant benefit.
Key words: Amoebozoa, Tubulinea, phylogeny, systematics, diversity, Polychaos, nuclear structure
https://doi.org/10.21685/1680-0826-2024-18-2-5
Corresponding author: Oksana Kamyshatskaya. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; oksana.kamyshatskaya@gmail.com
© 2024 The Author(s)
Protistology © 2024 Protozoological Society Affiliated with RAS
Introduction
Amoebae of the order Euamoebida Lepsi 1960 sensu Smirnov et al. (2011) are among the most popular and recognizable amoebae (e.g., Amoeba proteus). However, the species diversity of this group remains poorly studied. Descriptions of many species of these amoebae rest on "classical observations" made during 1980-1990s that are confined to light microscopic characters, size data and, for some species, details of their ultrastructure (Page and Baldock, 1980; Page and Robson, 1983; Page and Kalinina, 1984; Page, 1986; Siemensma and Page, 1986; Smirnov and Goodkov, 1997, 1998; Goodkov et al., 1999). Molecular data are available for a limited number of species only (Bolivar et al., 2001; Fahrni et al., 2003; Kamyshatskaya et al., 2017, 2020, 2021; Patsyuk, 2023). Hence, every finding of these amoebae in the environment requires careful morphological study aimed at associating the observed organism with already known species or describing it as a new one.
The problem is further complicated because it is hard to obtain stable laboratory cultures of these amoebae. Many of them are large polyphagous organisms, feeding on a wide spectrum ofprokaryo-tic and eukaryotic food, for example, Polychaos annulatum (Smirnov and Goodkov, 1998) and Polychaos fasciculatum (Page and Baldock, 1980). Others are established primarily on a single food object, such as Deuteramoeba mycophaga (Old et al., 1985; Kamyshatskaya et al., 2022) and Metachaos gratum (Schaeffer 1926). Therefore, DNA extraction requires a preliminary procedure: prolongated starvation of cells (Kamyshatskaya et al., 2017). The majority of large polyphagous amoebae die during this process. Moreover, these amoebae contain numerous endocytobionts in the cytoplasm (Jeon and Lorch, 1967; Page, 1986; Willumsen et al., 1987; Smirnov et al., 1995; Ossipov et al., 1997; Park et al., 2004). As a result, preparations ofthe total DNA obtained from these organisms usually are heavily contaminated and cannot be used for the amplification of amoebae genes by conventional PCR methods. The 18S rRNA gene sequences for some species were obtained using RT-PCR (Bolivar et al., 2001). In the recent years, methods of single-cell genomics and modern light and electron-microscopic techniques allowed obtaining congruent morphological and molecular data using a low number of individual cells (Kamyshatskaya et al., 2020).
Among amoebae of the order Euamoebida, there is a remarkable group of the similar, hardly
distinguishable species. All of them are polypodial cells, often palmate with fasciculate uroid (Smirnov and Brown, 2004). They have the similar structure of nucleolus, resembling an interrupted ring or a system ofuneven concentric rings in the optical sections. This group initially included three amoebae species: Amoebafasciculata (Penard, 1902), Amoeba annulata (Penard, 1902), and Amoeba hyalobates (Penard, 1902). Further Schaeffer (1926) described one more similar species — Metachaos gratum, and moved A. fasciculata into the newly created genus Polychaos, as P. fasciculatum. Siemensma (1987) found a new isolate ofamoebae similar in morphology to these species. He described it as P. annulatum and proposed to synonymize it with A. annulata, P. fasciculatum, M. gratum, and A. hyalolobates. Later, a detailed revision of species belonging to this "Polychaos fasciculatum-Mke" group was published by Smirnov and Goodkov (1998). The authors suggested considering Amoeba hyalobates (Penard, 1902) as nomen dubium because of incomplete description. The species A. annulata, P. fasciculatum, and M. gratum were considered as closely similar though different species. A new isolate from this study was identified as A. annulata Penard, 1902 and transferred from the genus Amoeba to the genus Polychaos, based on the fine structure of the cell coat, nuclear structure, and morphology of the locomotive form. The species P. annulatum was thoroughly studied (Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019, 2020), while there is only one investigation on morphology and ultrastructure of P. fasciculatum (Page and Baldock, 1980). The species M. gratum has never been investigated since its first description by A.A. Shaeffer in 1926, and its status remains questionable. The same is true for Amoeba hyalobates Penard, 1902.
Thus, now the group of " Polychaosfasciculatum-like" species includes three species: P. annulatum, P. fasciculatum, and M. gratum. Morphological differences between these species are tiny. Smirnov and Goodkov (1998) mentioned the absence of mono-podial locomotive form and the presence of "well-pronounced leading pseudopodium" in M. gratum; more clavate monopodial form and length/breadth (L/B) ratio 3-4 in P. fasciculatum; and slightly clavate monopodial form and L/B ratio up to 9 in P. annulatum. Molecular data are available for P. annulatum only (Kamyshatskaya et al., 2020), and there is no way to compare these species at the molecular level.
Recently we isolated a strain labeled as Ostrov7II from the surroundings of the Ostrov town, Pskov
Region, Russia (57.335744N, 28.369557E). These amoebae had palmate locomotive form with fasciculate uroid and the organization ofnucleolar material, which is typical for the species of "Polychaos fasciculatum-like" group. During his work at the Culture Collection of Algae and Protozoa (CCAP, Windermere, UK) in 1999, Alexey Smirnov collected light microscopic data of P. fasciculatum CCAP 1564/1 and made the embeddings for electron-microscopic study of this strain. Using these materials, we were able to perform a detailed comparative analysis ofthe nuclear structure of amoebae from the "P. fasciculatum-like" species group.
Based on the results of LM and TEM studies of the strain Ostrov7II and on the results of18S rRNA gene sequencing we described it as a new species, Polychaos insularis n. sp.
Material and methods
Isolation and culturing
The samples of the top layer of the bottom sediment (upper 4—5 cm) were collected from the freshwater ditch at the depth of 20—25 cm in the surroundings of the Ostrov town, Pskov Region, Russia (57.335744N, 28.369557E). To obtain an enrichment culture, small amount of detritus and plant debris was inoculated in sterile 90 mm plastic Petri dishes filled with PJ medium (Prescott and James, 1955) and two rice grains per dish. Dishes were kept under +18 °C and non-controlled room light. Dishes were examined under inverted microscope; after two weeks of incubation few cells were found in one of the inoculated dishes. Attempts to clone amoebae or purify this mixed culture failed because the transferred cells never multiplied in fresh dishes. In total, we studied 11 isolated cells found in the initial culture; all ofthem were evidently co-specific.
Light microscopy
Live cells of the strain Ostrov7II were studied, measured and photographed in culture using inverted microscope Leica DMI3000 equipped with DIC optics and Leica DFC420 camera. Nuclei were photographed on the object slides (wet mounts in PJ medium) using an upright Leica DM2500 microscope equipped with DIC optics and Nikon DS Fi-3 camera powered by NIS-Elements software (Nikon Metrology, UK). Due to the small number
of available amoebae, after light microscopic documentation, cells were extracted from under the cover slides using tapered-tip glass Pasteur pipette and used for electron microscopy or DNA isolation.
Amoebae ofthe strain P. fasciculatum CCAP 1564/1 were maintained on the Chapman-Andresen's modified Pringsheim's solution with rice grains (Chapman-Andresen, 1962; Page, 1986). Amoebae in the culture fed on Chilomonasparamecium, other eukaryotes and accompanying bacteria (Page, 1986). Light microscopic observations, imaging and video records were performed on the glass object slides under room conditions using Olympus BH2 microscope equipped with the phase contrast and DIC optics. Images were made by A. Smirnov in the year 1999.
Electron microscopy
For transmission electron microscopy, three cells of the strain Ostrov7II were utilized. They were collected and transferred with tapered-tips glass Pasteur pipettes, then fixed individually using a mixture of 2.5% glutaraldehyde and 1.6% formaldehyde prepared in 0.1M phosphate buffer (pH 7.4) for 90 min at room temperature (RT). After fixation, the cells were washed 3 * 5 min in the same buffer at RT, postfixed with 1% osmium tetroxide (final concentration) for 1 h at +4 °C. Subsequently, the cells were washed in the same buffer 3 * 10 min before dehydration at RT. Further, they were dehydrated in graded ethanol series followed by two changes of 100% acetone. Embedding was performed in SPI-PON 812 resin (SPI, an analog of Epon 812 resin) according to the manufacturer's instructions.
Amoebae ofthe strain P. fasciculatum CCAP 1564/1 were fixed in the year 1999, individually in glass embryo dishes with 2.5% glutaraldehyde prepared in 0.05M phosphate buffer (pH 7.4) for 40 min. Afterwards, they were washed in the same buffer 3 * 5 min, postfixed for 1 h with osmium tetroxide prepared in the same buffer at the final concentration of ca. 2%. Further, amoebae were washed again, prior to dehydration, 3x5 min in the same buffer. All fixation procedures were carried out under RT. Cells were dehydrated in a graded ethanol series followed by propylene oxide and embedded in Spurr's resin according to the manufacturer's instructions.
The embeddings of both strains were sectioned in 2023 using a Leica Ultracut 7 ultramicrotome and double-stained using 2% aqueous solution of uranyl acetate and Reynolds' lead citrate. Samples
were examined with a JEOL JEM-1400 (JEOL, Ltd., Tokyo, Japan) electron microscope at 80 kV.
DNA EXTRACTION, AMPLIFICATION, AND SEQUENCING
For DNA isolation, two individual amoeba cells were transferred into 40 mm plastic Petri dishes filled with Millipore-filtered (0.22 ^m) PJ medium, and left to starve for three days. Every day the medium was replaced with the fresh one. The cells were examined using an inverted Leica DMI 3000 microscope equipped with phase contrast optics to check for the absence ofvisible food vacuoles. After that, one cell was collected, washed in three subsequent changes of PJ medium, and placed with 1—2 ^l of the medium in 200 ^l PCR tubes. Another cell was transferred to the fresh dish filled with the sterile medium and destroyed by intensive pipetting. The nucleus of the amoeba was picked out using tapered-tip glass Pasteur pipette, washed in three subsequent changes of the same medium and placed with 1 — 2 ^l of the medium in a 200 ^l PCR tube. DNA from the single cell and from the isolated nucleus was extracted using Arcturus PicoPure DNA Extraction Kit (Thermo Fischer Scientific, Waltham, MA, USA). The extraction mixture was prepared according to the manufacturer's instructions. Then, 10 ^l of the mixture was added to the tube containing the single cell/nucleus. Further, we performed the whole genome amplification of the nuclear DNA using REPLI-g Single Cell DNA Amplification Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The resulting MDA products were sequenced using Illumina HiSeq 2500 system at the core facility center "Development of Molecular and Cell Technologies" of the SPSU Research Park. Two independent DNA samples (originating from the single nucleus and from the single cell, respectively) were sequenced; ca. 25 mln paired reads with length 150 bp were obtained for each sample. Quality control check of raw sequence data was performed using FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/ fastqc/). SPAdes assembler was used for de novo mitochondrial genome assembly (Bankevich et al., 2012). The contigs from both samples contained the identical fragments of 18S rRNA gene; they were identified using BLAST (Altschul et al., 1997). Obtained sequence was deposited with GenBank under the number OR947210 (Polychaos insularis strain PO7II, length 2002 bp).
Phylogeny reconstruction
The obtained sequence was mounted in the alignment containing all culture-derived sequences of amoebae species of the family Amoebidae, a representative set of Hartmannellidae, other Tubu-linea, and a number of discosean sequences used as an outgroup. Sequences were automatically aligned using the Muscle algorithm as implemented in SeaView 4.0 (Gouy et al., 2010); the alignment was further refined manually. Initial selection of nucleotide sites for tree inference was done using GBlocks (Castresana, 2000). The phylogenetic analysis was performed using maximum likelihood method as implemented in RAxML program (Stamatakis, 2014) with GTR + y model; 1563 sites were selected for the analysis, 1000 bootstrap pseudoreplicates were used. Bayesian analysis ofthe same dataset was performed using MrBayes 3.2.6, GTR model with gamma 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 % ofgenerations were discarded as burn-in. RaxML and MrBayes programs were run at Cipres V.3.3 website (Miller et al., 2010).
Results
Light microscopic observations
The cells of our isolate were predominantly polytactic. Rapidly moving cells gradually elongated and became oblong and orthotactic (Fig. 1, A), but never were monotactic. Polytactic locomotive form varied from palmate with fasciculate uroidal structures (Fig. 1, B, D) to oblong, with fewer pseudopodia and morulate uroid (Fig. 1, C, E). Large food vacuoles and a single contractile vacuole usually were located at the posterior end of the locomotive cell (Fig. 1, A—E), while the nucleus was always localized in the central part of the cell (Fig. 1, E—F). When the cell changed the direction
Fig. 1. Light microscopic images of Polychaos insularis n. sp., DIC. A—D — Amoeba rapidly moving at the bottom of the Petri dish. A — Orthotactic locomotive form; B—D — polytactic forms; E — subsequent stages of the locomotion of the individual cell on the slide surface under the pressure of coverslip — from expanded slowly moving cell, to the one with pronounced leading pseudopodium (the background of the image is natural and was not edited, this is just a mosaic of original photographs); F — palmate cell with fasciculate uroid; G — an area of cytoplasm with truncate bipyramidal crystals and more rare flattened plate-like crystals (marked with white arrow); H — an area of cytoplasm with truncate bipyramidal crystals, each in its own vacuole (black arrow) and spherical refractive bodies, also localized in the vacuoles; I — an area of cytoplasm showing a nucleus and large food vacuole, containing a ciliate. Abbreviations: u — uroid, h — frontal hyaline area of pseudopodium, cv — contractile vacuole, n — nucleus, cr — truncate bypiramidal crystals, rb — refractive body, fv — food vacuole, fo — food object. Scale bars: A-F — 50 ^m, G, H — 10 ^m, I — 25 ^m.
of movement, two large frontal pseudopodia could be observed. One of them soon became a leading pseudopodium (Fig. 1, E). The hyaline cup at the anterior part of the pseudopodia usually was quite extensive and well visible (Fig. 1, D—E). The length of the locomotive form was 146—223 ^m (n=11), breadth 48—120 ^m (n=11). The L/B ratio varied from 0.8 in palmate cells up to 3 in elongated cells.
During non-directed movement, cells spread over the substrate and extended one or several hyaline lobes (Fig. 1, E). At the beginning of the movement, the cell most often moved slowly and produced 8—12 pseudopodia of various size and shape. The remnants of these pseudopodia moved laterally while the cell was progressing forward and formed a fasciculate uroid when reached the posterior end of the cell (Fig. 1, F).
Amoebae of this strain had a single nucleus with a complex structure of nucleolus (Fig. 2). Generally, the nucleus had a spherical shape (Fig.
1, E-F, I; Fig. 2, A-H, I). When it moved with the rapid flow of the cytoplasm, it could adopt flattened and elongated shape, especially squeezing near large food vacuoles. The size of the nucleus in maximal dimension was 20-31 ^m (n=11). The form ofnucleolus (Fig. 2, A-H) resembled a hollow perforated sphere (Smirnov and Goodkov, 1998). In optical sections, it looked like an open or complete ring of the nucleolar material (Fig. 2, G-K). The thickness of this ring in different areas varied from wide lobes (Fig. 2, D, G-I, K) to a thin, hardly recognizable "line" connecting these lobes (Fig.
2, H-I). Some lobes harbored a single large (Fig. 2, K) or several small lacunae (Fig. 2, E). In some focal planes, the ring of the nucleolar material was approximately equal in the thickness along its entire perimeter (Fig. 2, J). In optical sections through the periphery of the nucleolus, islands of the nucleolar material were connected to each other (Fig. 2, A-F). The body ofthe granulated loose material was located inside the sphere of the nucleolar material (Fig. 2, D, K). Usually, one central or several (up to 7) spherical bodies (Fig. 2, B, F, G, I-K) were located in the central part of the nucleus. The size of nucleoli measured along the outer side of the sphere was 17-25 ^m (n=11).
The space between the nucleolus and the nuclear envelope, filled with the karyoplasm, was small and in normal cell, not squeezed with the coverslip, measured 0.5-1 ^m. When the nucleus was deformed, the shape and size of the nucleolus changed insignificantly, while the space between the
nucleolus and the nuclear envelope could increase twice in size (Fig. 2, J). In the cells pressed with the coverslip, the nucleolus was flattened, and the space reached 11 ^m (Fig. 2, K).
The granuloplasm of the cells was densely filled with a large number of small opaque granules, spherical refractive bodies enclosed in vacuoles, and numerous crystals (Fig. 1, G, H). The majority of crystals had a truncate bipyramidal shape (Fig. 1, G, H). Plate-like crystals were occasionally observed (Fig. 1, G). In the cell pressed by the coverslip, it was visible that every crystal was enclosed in an individual vacuole (Fig. 1, H). Many food vacuoles containing food objects digested to a different degree were seen in the cytoplasm (Fig. 1, A-D, I). In mixed culture, amoebae fed on accompanying bacteria and eukaryotic organisms (algae, ciliates). We never observed cysts formation by amoebae of this strain.
An observation on the nuclear organization of the type strain of Polychaos fasciculatum, strain CCAP 1564/1, was performed by A. Smirnov in 2000 and in 2002. Numerous microphotographs made at that time show various configurations of the nucleolar material. Many of them differ from the earlier descriptions of this strain (Baldock and Backer, 1980; Page and Baldock, 1980). Available microphotographs show kidney-shape lobes (Fig. 5, A); the nucleolus with a single wide lobe (Fig. 5, C); nuclei containing several compact nucleoli (Fig. 5, D), sometimes with large lacunae (Fig. 5, E), localized on the periphery ofthe sphere.The nucleus with two unequal lobes of the nucleolar material, connected by a poorly visible septum (Fig. 5, B), presumably corresponds to the Fig. 15 in Page and Baldock (1980) and Fig.16G by Page (1991).
Transmission electron microscopy of the strain Ostrov7II
The cell surface was covered with a glycocalyx comprising two layers (Fig. 3, A, B). The basal layer consisted of the amorphous electron-dense material, approximately 15 nm in thickness. The second layer was composed of fine filaments extending from the basal layer. The length of these filaments varied from 180 to 300 nm (Fig. 3, A). The filaments were not very abundant and not tightly packed. In some areas of our sections, there were electron-dense dots on the surface of the basal layer of the glycocalyx (Fig. 3, B). They resembled the collapsed and coagulated filaments described in Polychaos
Fig. 2. Light microscopic images of the nucleus of Polychaos insularis n. sp., DIC. A—H — 8 optical sections through the single nucleus. From its apical part to the center (A—D), and from the opposite apical part to the center (E—H). In the central optical sections, (D, G—H) the nucleolus looked like an enclosed ring of variable thickness. In the apical — like distinct islands of nucleolar material with numerous lacunae (black arrows) — A—C, E—F; I — overall view of the nucleus, showing its typical appearance, observed in the majority of cells. The space (marked with asterisk) between nuclear envelope and nucleolus (white arrows) is quite narrow. The central part of karyoplasm contains several little spherical bodies; J — nucleus under the pressure of coverslip. The granular material in the center of the nucleolus becoming clearly visible, and the space between nuclear envelope and the nucleolus becoming wider; K — nucleus, squeezing through large organelles by the fast flows of the cytoplasm. Note the wide space between nucleolus and nuclear envelope (asterisk). Abbreviations: nu — nucleolus, ne — nuclear envelope, sb — spherical body, g — granular material. Scale bars: A-I — 25 ^m, J-K — 10 ^m.
annulatum earlier (Smirnov and Goodkov, 1998). The peripheral area ofthe cytoplasm contained bundles of fine filaments resembling actin (Fig. 3, B).
Many mitochondria were observed in the cytoplasm. All of them had elongate or ovoid shape in the sections, electron-dense matrix, and curved cristae of the tubular type (Fig. 3, C); mitochondrial heteromorphism was not found. Mitochondria were
especially abundant around the contractile vacuole. The latter, together with numerous small vesicles surrounding it, formed a spongiome (Fig. 3, C).
There were many food vacuoles in the cytoplasm. Most of them contained large eukaryotic food objects and/or bacteria (Fig. 3, D). Dictyoso-mes of the Golgi complex were not numerous: 1—3 per section. They were represented by stacks
Fig. 3. General ultrastructure of Polychaos insularis n. sp., TEM. A—B — Filamentous cell coat. Filaments (black arrowheads) are based on the tiny amorphous layer adjacent to the surface of the cell membrane. Note the filaments (black arrows), resembling actin microfilaments in the peripheral cytoplasm; C — mitochondria with dense matrix and dictyosomes of the Golgi complex arranged near the spongiome area; D — an area of cytoplasm containing huge food vacuole with eukaryotic food object; E — dictyosome of the Golgi complex; F — the rounded body, which is localized in the cytoplasm and bounded with the electron-dense multilayered envelope (white arrows); G — a vacuole (white arrowheads) enclosed several bacteria, which are looked not digested; H — endocytobiotic bacteria located freely in the cytoplasm. Abbreviations: pm — plasma membrane, s — spongiome, mt — mitochondria, cyt — cytoplasm, fo — food object, fv — food vacuole, d — dictyosome of the Golgi complex, rb — bounded body, b — bacteria. Scale bars: A, B, E — 200 nm, C—D — 500 nm, F—H — 1 ^m.
consisting of 5-6 cisternae (Fig. 3, C, E) and never grouped together. No distinct microtubules or any evidence of a MTOC were found in the cytoplasm surrounding the dictyosome.
We observed multiple rounded or nearly rounded bodies ofunknown nature in the cytoplasm (Fig.
3, F). They reached several micrometers in diameter and were bounded by a multilayered envelope. Every layer ofthis envelope was 7-10 nm in thickness and resembled the cell membrane. The inner content was represented by the finely granulated electron-dense material, which was diffused in a more electron-transparent matrix (Fig. 3, F). Earlier, we described similar bodies in Polychaos annulatum (Kamyshatskaya et al., 2019).
Two types of presumably endocytobiotic bacteria were found in the cytoplasm. Bacteria of the first type were electron-dense in sections. Aggregation of them was enclosed in the vacuole (Fig. 3, G). Bacteria ofthe second type were elongate, more electron-transparent, and apparently localized freely in the cytoplasm (Fig. 3, H). They were surrounded by an electron-transparent halo, but we have seen nothing that resembled a cell membrane around them. The size of both types of bacteria was almost the same; in our sections, it was 800-1500 nm in length and 400-700 nm in breadth. We have seen the division of the second type of bacteria in the cytoplasm of the cell (Fig. 3, H). Both types of bacteria were similar in appearance to bacteria described in previous studies for the other representatives of the order Euamoebida (Page, 1986; Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019, 2022; Kamyshatskaya and Smirnov, 2023).
Nuclei in our sections had an irregularly ovoid shape. The karyoplasm was distinctly subdivided into two parts. The "external karyoplasm" - ka-ryoplasm outside the nucleolus, according to Smirnov and Goodkov (1998), was more finely granulated. It appeared to be denser than the "internal karyoplasm" and contained a layer of curved filaments (Fig. 4, A-F). In tangential sections, this layer looked like rounded alveoli. The walls of these structures consisted of curved, separate bundles of filaments. We have never seen a direct contact of these bundles with the nuclear envelope.
Nucleolar material was organized in a hollow ellipsoid body. Its external walls consisted of electron-dense material packed in a bent cord (Fig.
4, D). Thicker areas of walls contained additional patches of the nucleolar material of heterogeneous density (Fig. 4, E-F). Some areas of the nucleolar
material were highly condensed (Fig. 4, E-G). Thus, in tangential sections through the apical part of the nucleus (Fig. 4, D), the nucleolar material could be distinguished as bent electron-dense cords located at the border of external and internal karyoplasm. The nearly longitudinal sections of the nucleus demonstrated the nucleolar material, which looked concentrated as a closed ring, with walls varying in thickness from a hardly distinguishable layer to broad lobes (Fig. 4, E-F).
In the central part of the nucleus, the body of loose, finely granulated material, resembling in appearance the condensed chromatin, was recognizable (Fig. 4, F). It probably corresponded to the central body observed in this area by using LM (Fig. 2, A, J, K).
Outside the nucleus, in the surrounding cytoplasm, separate bundles of microfilaments resembling actin were observed (Fig. 4, G). Nuclear pore complexes were especially well visible in the peripheral part of the nucleus, where the nuclear envelope was sectioned tangentially (Fig. 4, A-C). They had a typical structure, showing the peripheral spoke ring assembly and the central plug.
Transmission electron microscopy of the strain P. fasciculatum CCAP 1564/1
In total, three nuclei from cells of acceptable fixation quality were available for our study. Nuclei in our sections had nearly rounded shapes with uneven edges. The karyoplasm contained a layer of filaments. Bundles offilaments were localized along the nuclear envelope (Fig. 5, G). The peripheral part ofthe karyoplasm, bounded by this layer, seemed to be slightly denser and more finely granulated than the rest of the karyoplasm (Fig. 5, F). Nucleolar material in each observed nucleus was organized as an individual lobe, located eccentrically in the internal part of the karyoplasm. Probably, this pattern corresponded to the LM images in the Fig. 5 (A or C). It was not homogenous: the patches or distinct granules of more electron-dense material were distinguishable (Fig. 5, F). The central part of the karyoplasm contained a "cloud" of coarse granular material, resembling in appearance the condensed chromatin (Fig. 5, F, H). This was congruent with the granular parts of the nucleus visible in light-microscopic images (Fig. 5, A, C, D). Distinct electron dense bodies were associated with this material (Fig. 5, H).
Fig. 4. Ultrastructure of the nucleus of Polychaos insularis n. sp., TEM. A—C — Tangential sections through the single nucleus, showing the presence of layer of curved filaments (white arrows) in the peripheral karyoplasm; D — tangential section, showing the subdivision of karyoplasm into external and internal parts, curved filaments on their boundary, and highly condensed bent cords of nucleolar material (black arrows) representing the thin walls of the ring of nucleolar material; E-F — nearly longitudinal sections through the nucleus, showing the typical appearance of the nucleolar material, forming, on a plane, partly closed ring with additional sealing lobes of different thickness, note the granular material in the central area of the internal karyoplasm, resembling in appearance densely condensed chromatin (F); G — an enlarged region of E, showing fine details of nuclear organization: the placement of filaments, the heterogeneous nucleolar material, lacunae inside it (black asterisk) and the cytoplasmic filaments distinguishable near the nuclear envelope (black arrowheads). Abbreviations: np — nuclear pores, ek — external karyoplasm containing microfilaments, ik — internal karyoplasm, lob — lobe of nucleolar material, g — granulated material resembling in appearance a condensed chromatin, ne — nuclear envelope, cyt — cytoplasm. Scale bars: A—D, G — 1 ^m, E—F — 2 ^m.
Fig. 5. Structure of the nucleus of P. fasciculatum CCAP 1564/1, DIC, TEM. A—E — Different variations of configuration of nucleolar material, visible in the optical sections. A — Two kidney-shaped lobes with several little lacunae; B — two lobes (one of them with huge lacuna) connected by the thin string (black arrowhead); C — a single lobe with numerous lacunae and acentric placement of granular material; D — numerous lobes of the nuclear material concentrated on the periphery of the nucleolus; E — the same nucleus with D, squeezing through large organelles by the fast flows of the cytoplasm. F — Nearly cross section through the nucleus, showing the slightly visible differentiation of karyoplasm on external and internal parts. The type of organization of nucleolar material of this nucleus is congruent with that, demonstrated in light microscopic image C. Nucleolar material is not homogenous — it arranged in a single lobe, includes electron-dense patches and tiny granules (white arrows) and more electron-transparent areas; G — peripheral region of the nucleus, showing the bundles of filaments (black arrows) localized along with the nuclear envelope and properly preserved nuclear pore complexes with a typical structure; H — an area of nucleus, showing granulated material in the internal karyoplasm and a little dense body, associated with it. Abbreviations: ne — nuclear envelope, g — granulated material resembling in appearance a condensed chromatin, lac — lacunae, lob — lobe of nucleolar material, ek — external karyoplasm containing filaments, ik — internal karyoplasm, np — nuclear pores, db — dense body. Scale bars: A—E — 10 ^m, F — 2 ^m, G — 500 nm, H — 1 ^m.
Table 1. Levels of sequence identity (%) between the 18S rRNA gene sequences of Polychaos species.
Species P. annulatum MN633272 P. centronucleolus MT874850
P. insularis strain Ostrov7II n.sp. 91.02 (1748bp) 90.61 (1150bp)
P. centronucleolus MT874850 89.81 (1148bp)
Molecular phylogenetic analysis
The length of the obtained 18S rRNA gene sequence of the strain Ostrov7II was 2002 bp. Phylogenetic analysis based on the alignment of the 18s rRNA gene sequences showed that the genus Polychaos is a robust and fully supported clade in the family Hartmannellidae (Fig. 6). The strain Ostrov7II occupied a sister position to P. annulatum, while the species P. centronucleolus formed a basal branch in the Polychaos clade. The rest of the tree topology was almost the same as in the earlier study (Kamyshatskaya et al., 2020). The comparison of the strain Ostrov7II sequence with other Polychaos species showed sequence identity at the level of 90.6-91.0%, based on 1150 bp and 1748 bp pairwise aliments, respectively (Table 1).
Discussion
Based on the predominantly polytactic, often palmate locomotive form with broad fasciculate uroidal lobes and complex organization ofnucleolus (which in optical sections looked like a ring), we
identified amoebae ofthe studied strain as members ofthe group of " Polychaosfasciculatum-Uke" species (Smirnov and Goodkov, 1998).
Comparison with closely related species
In the group of "Polychaos fasciculatum-Mke" species, amoebae of the studied strain are among the largest. Their size is exceeded only by the sizes of Polychaos annulatum strains, isolated by Penard (1902) and Smirnov and Goodkov (1998): 146-223 ^m in length versus 250/220-325 ^m in length, respectively (Table 2). In general, this character varies greatly in P. annulatum (Penard, 1902; Schaeffer, 1926; Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019). This may be explained by the fact that elongated, mono- and orthotactic forms dominate in some isolates, while in others, polytactic ones are more numerous. However, comparing different types of locomotive forms separately, the difference in length remains within the range of the size variability of amoebae species (Page, 1988; Smirnov et al., 2002, 2007).
The extremum values of the sizes are typical for elongated, fast-moving cells, the length of which
Characteristics P. annulatum P. fasciculatum M. gratum Present isolate
Penard 1902 Schaeffer 1926 Smirnov and Goodkov 1998 Kamyshatskaya et al. 2019 Penard 1902 Page and Baldock 1980 strain CCAP 1564/1 Schaeffer 1926
length (pm) 250 70-100 220-325 60-188 140 65-180 200 146-223
L/B ratio - - up to 9 0.85-3.4 - 3-4 - 0.8-3
nucleus (pm) 15 16-25 11-22 18-19 11-23 25 20-31
glycocalyx basal layer filaments (nm) * - distinct filaments - - 13 330 7-10 250 - 15-17 80 (280-300*) - 15 180-300
Table 2. Comparison of the dimensional characteristics of "Polychaos fasciculatum-like" species
and strain Ostrov7II.
Fig. 6. Molecular phylogenetic tree based on 18S rRNA gene sequences of representative set of tubulinean amoebae and a number of discosean sequences used as out-group. 1563 sites used in the analysis. Labeling of nodes: ML/PP support. Black circles are used to label fully supported nodes (100/1.0 support). Other supports are labeled only if either of them exceeds 75% (ML) or 0.75(PP).
exceeds the breadth several times. The maximum L/B ratio, 9, which was noted in P. annulatum (Smirnov and Goodkov, 1998), strongly differs from other species. The maximum L/B ratio for them does not exceed 3—4 (Table 2). The locomotive form of such oblong cells moving on the substrate tends to the monotactic, as observed for P. annulatum and P. fasciculatum (Page and Baldock, 1980, Fig. 17; Page, 1988, Fig. 16, H; Smirnov and Goodkov, 1998, Figs 3, 4; Kamyshatskaya et al., 2019, Fig. 1, H). However, amoebae of the present isolate never adopted a monotactic form (which is also typical for Metachaos gratum).
During rapid directed movement, amoebae of the strain Ostrov7II elongate, but retained polytactic or, less often, orthotactic locomotive form (Fig. 1, A-B, E). They have one or two large leading pseudopodia, which simultaneously determine the direction of locomotion, and short pseudopodia along the side of them (Fig. 1, A-E). In contrast to that, M. gratum has "numerous pseudopods of all sizes", and the leading pseudopodium "may be
detected at all times" (Schaeffer, 1926, p. 39; Fig. 11, Plate 3: Fig. 3). The flowing of the cell through the single main pseudopodium is generally a distinctive feature of the genus Metachaos, according to Schaeffer (1926, p. 34). In addition, the presence of a palmate locomotive form was not shown for M. gratum, contrary to the present isolate and amoebae of the genus Polychaos.
The most typical shape of crystals in the cytoplasm of "Polychaos fasciculatum-like" species is the truncate bipyramidal shape (Page and Baldock, 1980; Page, 1986, 1988; Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019). The plate-like crystals are less common in this group. They are more characteristic for P. dubium, as well as for amoebae of the genera Chaos and Amoeba (Griffin, 1960, 1961; Page, 1986, 1988). Among the numerous inclusions in the cytoplasm of the studied strain, bipyramidal crystals predominated (Fig. 1, G, H). However, these crystals appeared to be more elongated and less truncate than in any of the mentioned species. In addition, occasionally, we
have seen parallelepiped plate-like crystals in the cytoplasm of the observed cells (Fig. 1, G). For M. gratum, Schaeffer (1926) described large (up to 30 ^m) irregular crystalline inclusions, which were not noted in any other species described above.
The nuclei of the studied cells have the largest size among "Polychaos fasciculatum-like" species (Table 2). For this strain, it was notable that the nucleus, carried by a fast stream of the cytoplasm, can be deformed (Fig. 2, K): it was flattening from the sides and taking on a more elongated shape (wherein the shape of nucleolus did not change). This fact was not noted for P. annulatum, while for M. gratum Schaeffer (1926) claimed that the nucleus was not deformed as it was carried along the endoplasmic stream. The nucleus of P. fasciculatum can also be deformed while moved by the cytoplasmic stream (Penard, 1902; this study, Fig. 5, E). Another remarkable feature of this isolate is the extremely close location of the nucleolus to the nuclear envelope (Fig. 2, I). All other amoebae mentioned above have considerable distance between the nucleolus and the nuclear envelope (Schaeffer, 1926; Page and Baldock, 1980; Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019).
Metachaosgratum, P. annulatum, and amoebae ofthe present isolate have nucleolar material, which is organized generally in a similar way. Depending on the position ofthe optical section, it is possible to see something resembling an interrupted or completely closed ring, often with broad lobes (present study, Fig. 2, D, G-I, K; Schaeffer, 1926, Plate 3, Fig. 3; Kamyshatskaya et al., 2019, Fig. 2, A,B, D,E). The lobes of the ring in the studied strain always look more uneven than in Polychaos annulatum, and never have the shape of a crescent or a horseshoe. The nucleolar material of P. fasciculatum usually is arranged "in a single band with lobes and broader regions" (Page and Baldock, 1980, p. 225). Its lobes are often more rounded but never crescent-shaped. Polychaos fasciculatum usually possesses a nucleolus, which looks like two approximately equal lobes connected by the poorly visible string or like an open ring (Page and Baldock, 1980, Fig. 10-15). A variety of additional configurations of the nucleolus (Fig. 5, A-E) suggests the presence of nucleolar polymorphism, which is probably most pronounced in this species, in comparison with other "Polychaos fasciculatum-like" species. The central body localized within a hollow sphere of nucleolar material (Fig. 2, A, D, J, K) was observed in P. an-
nulatum (Kamyshatskaya et al., 2019, Fig. 2, A, noted as g, Fig. 5, A-C), P. fasciculatum (present study, Fig. 5, A, C, D; Page and Baldock,1980, Fig. 12), and also mentioned in the description of M. gratum (Schaeffer, 1926).
Unlike in other " Polychaos fasciculatum-like" species, nuclei of the strain Ostrov7II have a unique ultrastructural feature - a layer of curved microfilaments in the karyoplasm. On the tangential sections (Fig. 4, A-D), it partly resembles the nuclear honeycomb-like lamina, which is a remarkable character of P. dubium, the genera Amoeba and Chaos. We have never observed direct contact of this layer with the inner membrane of the nuclear envelope, so this layer cannot be formally termed as a "nuclear lamina" (Stuurman et al., 1998; Goldman et al., 2002; Bridger et al., 2007).
Like all other amoebae of the genus Polychaos, the studied cells have filamentous glycocalyx, in which the filamentous layer extends from the basal amorphous layer (Page and Baldock, 1980; Page, 1986; Smirnov and Goodkov, 1998; Kamyshatskaya et al., 2019, 2020). Filaments of the present isolate are more similar to the filaments of P. annulatum in size (Table 2): they are thicker (4-5 nm versus 2 nm) and longer (180-300 nm against 80-280 nm) than filaments of the P. fasciculatum. They are also less regular in the thickness throughout their entire length than the filaments of P. fasciculatum. Moreover, in comparison with the latter and similarly to the filaments of P. annulatum, they are less densely located (Fig. 3, A).
To summarize the above considerations, we suggest that the investigated strain represents a new species of the genus Polychaos (Schaeffer, 1926) -Polychaos insularis.
The final point confirming our suggestion is the result of the molecular phylogenetic analysis. In the phylogenetic tree, amoebae of the strain Ostrov7II grouped with P. annulatum sequence as a sister clade to P. centronucleolus within the fully supported genus Polychaos (Fig. 6). The level ofidentity confirms the relation of this strain with the species P. annulatum, shown by the phylogenetic tree (Table 1). At the same time, this level of sequence divergence and differences in the 18S rRNA gene structure (numerous indels and serious structure differences in variable regions) are sufficient to be confident in the independent species status of the present isolate.
Taxonomic summary
Polychaos insularis N. SP.
Diagnosis: Usually polypodial, from palmate with a fasciculate uroid, during slow locomotion — to elongated, orthotactic in rapid movement, with morulate uroid. Length in locomotion: 146—223 ^m, breadth 48—120 ^m. The L/B ratio varies from 0.8 in palmate cells to up to 3 in orthotactic cells. Spherical nucleus, 20 — 31 ^m in diameter. Nucleolar material arranged in a hollow perforated sphere, 17—25 ^m across, in close proximity to the nuclear envelope. The karyoplasm is differentiated into external and internal parts, separated by a layer of curved filaments. Filamentous glycocalyx. No cysts known.
Type material: Illustrations of the live amoeba cell (Fig. 1, A-D, Suppl. 1) stored in the image collection of the Department of Invertebrate Zoology, St. Petersburg State University. The sequence of the 18S rRNA gene of this strain is deposited in GenBank under the accession number OR947210.
Type location: The bottom sediments of freshwater ditch in the surroundings of the town Ostrov, Pskov Region, Russia (57.335744N, 28.369557E).
Etymology: "insularis" from island (Lat. "insula") due to the name ofthe type location of new species — town Ostrov, that means "island".
ZooBank registration: urn:lsid:zoobank.org:act: 0EC5731F-87B5-4CDA-9CEC-C369FF7709FF
Differential diagnosis. Within the genus Polychaos, this species is distinguished by the organization of nucleolar material in a hollow perforated sphere. Among all "P. fasciculatum-like" species, it has the largest size of nuclei: 20—31 ^m. In contrast to P. annulatum and M. gratum, its nucleus is capable of being deformed, shifting due to fast cytoplasm streaming, and its nucleolus are localized much closer to the nuclear membrane. Unlike P. fasciculatum, the nucleolus of P. insularis has never been observed as a single band with lobes and wide regions. In contrast to P. annulatum, it does not have a monotactic locomotive form. During rapid locomotion, amoebae of this species are orthotactic but never monotactic. The unique feature of this species is the presence of a layer of curved filaments in the karyoplasm. The filaments of the cell coat (ca. 180—300 nm in length) are thicker and longer than those of P. fasciculatum, P. dubium and P. centronucleolus.
Fig. 7. The general nuclear organization in "P.fas-ciculatum-like," species. Abbreviations: ek — external karyoplasm, ik — internal karyoplasm, black arrows — bundles of filaments, ne — nuclear envelope, nu — nucleolar material, g — granulated material.
Structural organization of nucleus in the "Polychaosfasciculatum-like" species
The fine structure of the nucleus of "Polychaos fasciculatum-like" species follows a general principle (Fig. 7): the karyoplasm is divided to the external and the internal parts. The outer part is always denser and finely granulated than the inner part. Although the division of the karyoplasm into different parts was not noted for Polychaos fasciculatum (Page and Baldock, 1980; Page, 1986), it is suggested according to our electron-microscopic images (Fig. 5 F), where the difference between them is slightly visible.
At the border of two parts of karyoplasm, there is a layer of filaments. The presence of this layer has been shown in all species except Polychaos fasciculatum (Page and Baldock 1980). Page (1986, Fig. 32) suggested the presence of "fibrillar material parallel to nuclear envelope", and this was confirmed in our study (Fig. 5, G).
The nucleolar material, organized in a hollow perforated sphere/ellipsoid, is localized in the internal karyoplasm (present study, Fig. 7; Smirnov and Goodkov, 1998, Fig. 12). The walls of this sphere are represented by the heterogeneous nucleolar material of a varying thickness and are perforated
by large holes. Therefore, in the cross sections through the central part ofthe nucleus, the nucleolar material looks as a ring, varying in thickness from hardly distinguishable line to wide lobes (Fig. 4, E, F). These lobes look as separate patches of nucleolar material in sections, which do not pass precisely through the center of the nucleus (Fig. 5, F). Usually, inside the sphere of the nucleolar material, there is a central body (Fig. 4, F; Fig. 5, F-H, Fig. 7). This body contains loosely packed electron-dense granules.
Acknowledgments
The study was supported by the Russian Science Foundation project 23-24-00264. This study utilized equipment of the Core Facility Centers "Biobank", "Development of Molecular and Cell Technologies" and "Culture Collection of Microorganisms" of the Research Park ofSt. Petersburg State University. We are grateful to Victoria Tcvetkova for her help in collecting a sample that was a source of the strain Ostrov7II. Alexey Smirnov is grateful to Bland Finlay, Susan Brown and Ken Clarke for the possibility to study CCAP strains during his work in CEH Windermere in 1999.
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Supplementary materials
Fig. S1. Illustrations of the single amoeba cell of the strain Ostrov7II moving at the bottom of the Petri dish, designated as a type-material of Polychaos insularis n. sp.
