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Protistology 17(3): 129-141 (2023) | doi:10.21685/1680-0826-2023-17-3-1
Protistology
Original article
Ultrastructure and molecular phylogeny of Phalansterium foulumis sp. nov. (Amoebozoa: Variosea)
Sergey A. Karpov1,234,*, Lyubov A. Smakova5, Stanislav A. Malavin56, Flemming Ekelund7 and Alexey V. Smirnov1
1 Department ofInvertebrate Zoology, Faculty of Biology, St Petersburg State University, St. Petersburg 199034, Russian Federation
2 Zoological Institute, Russian Academy of Sciences, St Petersburg 199034, Russian Federation
3 I.I. Mechnikov North-Western State Medical University, St. Petersburg 191015, Russian Federation
4 I.D. Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Yaroslavl distr. 152742, Russian Federation
5 Israel Oceanographic and Limnological Research, Tel-Shikmona, 3108000, Haifa, Israel
6 Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boker Campus, 84990, Israel
7 Terrestrial Ecology Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| Submitted July 25, 2023 | Accepted September 14, 2023 |
Summary
Representatives of the genus Phalansterium have single anterior flagellum surrounded by cytoplasmic collar. They belong to the class Variosea, which includes both typical amoeboid organisms (e.g., Flamella, Filamoeba) and some flagellates (e.g., Phalansterium, Multicilia). A recent study revealed Phalansterium arcticum as a very polymorphic species with both sedentary and swimming cells. It also clarified the mode of phagocytosis, and showed a cell division, but the ultrastructure of the cell is still not sufficient. Here, we present for the first time a thorough ultrastructural investigation of the soil flagellate Phalansterium "solitarium" strain Ph185 of SCCAP. We demonstrate that Phalansterium uses pseudopodia to take up bacteria inside its collar, and suggest that the inner cell asymmetry of Phalansterium cytoskeleton is associated with its mode of feeding. On the base of morphology and molecular phylogeny, we describe a new species Phalansterium foulumis with the type strain SCCAP-Ph185.
Key words: Phalansterium, ultrastructure, soil flagellate, flagellar apparatus, taxonomy, molecular phylogeny
https://doi.org/10.21685/1680-0826-2023-17-3-1
Corresponding author: Sergey A. Karpov. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universi-tetskaya Emb. 7/9, 199034 St. Petersburg, Russia; sakarpov4@gmail.com
© 2023 The Author(s)
Protistology © 2023 Protozoological Society Affiliated with RAS
Introduction
The genus Phalansterium includes heterotrophic flagellates with a single flagellum, the proximal part of which is surrounded by a collar-like structure. The genus was erected by Cienkowski (1870) for Monas consociatum Fresenius, 1858. At present, five species are considered valid: colonial P. consociatum (Fresenius, 1858) Cienkowski, 1870 and P. digitatum Stein, 1878, and single-cell P. solitarium Sandon, 1924, P. filosum Cavalier-Smith, 2011 (Smirnov et al., 2011) and P. arcticum Shmakova, Karpov et Smirnov (Shmakova et al., 2018). Hibberd (1983) suggested that P. consociatum and P. digitatum might be conspecific.
A recent study of P. arcticum improved our knowledge offeeding and reproduction of Phalansterium, and revealed polymorphism of sedentary and swimming cells. The latter demonstrated active amoeboid movement, when appeared in thin water film (Shmakova et al., 2018). Electron microscopical studies of P. arcticum revealed the same general cell structure as in P. digitatum. However, the relatively low fixation quality of the material used by Ekelund (2002) and Cavalier-Smith et al. (2004) did not permit finding interspecific ultrastructural differences between these P. solitarium-Mke forms (Shmakova et al., 2018). This demonstrates that our knowledge of the ultrastructural features of the so-called "solitarium" species of Phalansterium remains incomplete.
Therefore, we analyse here a better fixation of the strain Ph185 of the P. "solitarium", comparable to that for P. digitatum by Hibberd (1983), and describe a new species P. foulumis for this strain on the basis ofmorphological characters and molecular phylogeny.
Material and methods
Material identification and cultivation
An isolate of Phalansterium sp. was obtained from barley rhizosphere soil collected at the Research Center Foulum (Denmark). Clonal cultures were established as in Ekelund (1996) by repeated dilution and growth of the flagellates in 0.1 g/l Tryptic Soy Broth (Difco BactoR) in Neffs Modified Amoeba Saline (Page, 1988). Cultures were inspected for contaminating organisms several times with repeated negative results. The resulting clonal cultures were kept on 0.01% Cerophyl infusion based on Prescott-
James' medium (Page, 1988) in 50 ml Nunclon flasks (Nunc), 10 ml of liquid per flask, at 18 °C. Under these conditions, the culture can survive for several months without addition of fresh medium. The culture is now deposited in the Scandinavian Culture Centre for Algae and Protozoa as SCCAP Ph185. Dense cultures for light and electron microscopy were obtained by adding the food bacterium, Pseudomonas chlororaphis (ATCC 43928), to a concentration of about 108 cells per ml to the cultures. Cultures were examined and filmed directly in the culture flasks using a Zeiss Axiostar Plus microscope equipped with phase-contrast water dipping objectives, and on slide preparations using a Leica DM2500 microscope with differential interference contrast.
Electron microscopy
For the preparation ofcells for transmission electron microscopy, we used the protocol for fixation of Phalansterium digitatum (Hibberd, 1983). After dehydration in an alcohol series and propylene oxide, a pellet was embedded in Spurr's resin. Blocks were serially sectioned with a diamond knife on a Reichert or LKB ultramicrotome, mounted on formvar-coa-ted slot grids, and post-stained with uranyl acetate and lead citrate. Whole mounts and sections were viewed on a JEM 100CX II electron microscope operating at 80 kV.
SSU rDNA sequencing and molecular phylogeny
Extraction and amplification of the full-length SSU rDNA gene (18S) was performed as in Shmakova et al. (2018). Obtained SSU sequence (GenBank 0R613015) was added to the alignment containing a set ofVariosea sequences, including all available sequences of Phalansterium and a set of other variosean species. The alignment was based on that used in Shmakova et al. (2018) paper. Several sequences of Discosea were added as an outgroup. Sequences were aligned using Mafft 7.4.0.2 (Katoh and Standley, 2013) implemented at CIPRES 3.3 portal (Miller et al., 2010), and the alignment was further manually perfected. The phylogenetic analysis was performed using maximum likelihood method as implemented in RaxML program (Stamatakis, 2014) with GTR + y model; 1370 sites were selected for the analysis, 1000 bootstrap pseudoreplicates were used. Bayesian analysis of the same dataset was performed using MrBayes 3.2.6, 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 5 million generations, by which time they had converged (final average standard deviation of the split frequencies was less than 0.01). The convergence and other parameters of chains were estimated using the built-in MrBayes tools and additionally using the software Tracer 1.6 (Rambaut et al., 2014). Based on the estimates by Tracer, the first 20 % of generations were discarded as burn-in. RaxML and MrBayes programs were run at Cipres 3.3 portal (Miller et al., 2010).
Results
The interphase cells of strain Ph185 had a rounded shape with a short retractable collar and a single long flagellum (Fig. 1, A—D, see also Ekelund, 2002). The surface of the cell appears naked, without any other covering than the cell membrane. Sedentary cells normally produce a thick mucilage layer around them (Fig. 1, A, B). The funnel-shaped flagellar pocket is rather deep, and the flagellum emerges from the center of its bottom (Figs 1, A, B; 2, A). A vesicular nucleus is located laterally, in the middle or anterior part of the cell (Fig. 1, E). A flagellar basal body, or kinetosome, is underlined by a well-developed dictyosome, which separates the kinetosome from the nucleus (Fig. 2, A,C). Mitochondria with branched tubular cristae are scattered throughout the cytoplasm, and microbodies about 0.3 ^m in diameter are likewise present (Figs 1, E; 2, A, B). A contractile vacuole is located posteriorly (Fig. 2, A). One large and, occasionally, several smaller food vacuoles can be seen in the cell. In mature cells, food vacuoles appear to be almost empty, and lipid droplets often occur in the cytoplasm (Figs 1, E; 2, A). Immature (just after division) cells of strain Ph185 are normally only half as long as mature cells (Fig. 2, D compare with 2, A). They are more elongated, have shorter flagella, and their food vacuoles contain many bacteria. On the sections, immature cells are surrounded by dense clumps ofbacteria (Fig. 2, D). The cells occasionally produce short finger-like pseudopodia all over their surface, predominantly posteriorly (Fig. 1, C, E). We suggest that the pseudopodia are used to attach to the substrate, as we have not observed them used for bacterial uptake or for movement.
Flagellar apparatus structure
The free part of the flagellum outside the collar is smooth and has an axoneme with the usual 9+2 structure. The proximal part of the flagellum has a thin filamentous coat (Figs 3, B; 4, A, B). Inside the distal part of the collar, there is only one central microtubule in the axoneme (Fig. 3, A—C). This microtubule continues towards the cell, and then enters the so-called plug (Figs 3, D; 4, B), i.e. the amorphous material, which fills the inner space of the axoneme in the flagellar transition zone. In most cells, the central microtubule cannot be traced beyond the distal part of the plug; however, in some cells the central part of the plug is translucent, and here, the central microtubule can be traced inside the proximal part of the plug. The proximal part of the central microtubule curves slightly away from the longitudinal axis of axoneme.
Above the plug, the flagellum is dilated for a short distance (Fig. 4, B). The plug extends 300—350 nm above the cell surface and has a length of about 350—500 nm and a diameter of about 150 nm. Immediately below the plug, a transitional cylinder, which is about 90 nm at its most narrow part, connects to the peripheral doublets by thin filaments (Fig. 3, E). The cylinder further continues along the transition zone into the kinetosome, becoming wider and closer to the peripheral microtubules (Fig. 3, F). The connecting filaments become shorter on the direction to the cell surface.
An asymmetric filamentous circle is present at the distal end ofthe kinetosome (Fig. 3, F); it crosses the transitional cylinder and connects the doublet to the opposite triplet. Such a structure, called an acorn, has been described in the flagellar apparatus of Chlamydomonas, and is also found at the distal part of kinetosome in many other eukaryotes (Geimer and Melkonian, 2004).
The single kinetosome is 250—375 nm long (Fig. 2, C). Its distal end is connected to the cell membrane with transitional fibers (Fig. 3, F, G), and its proximal part is surrounded by a fibrillar circle (the Z-circle, according to the terminology of Hibberd, 1983), which produces a net of thin filaments that form a 80-90 nm broad zone around the kinetosome (Figs 2, C; 3, G—I). The fibrillar zone is highest at its proximal end at the Z-circle, and tapers towards its distal end at the Y-circle composed ofthree dense circles (Figs 2, C; 3, G—I). On the LS, this structure looks like a foot derivate,
Fig. 1. Light microscopic (A-D) and TEM (E) images of Phalansterium foulumis sp. nov. A,B — DIC images of typical vegetative cells with thick mucilage (square bracket) around; C, D — Phase contrast images showing posterior filopodial projections (arrows) and newly formed food cap including bacterium (arrowhead); E — longitudinal section (LS) showing nucleus and organelle disposition, and pseudopodia in posterior and middle parts of the cell. Abbreviations: c — collar, cp — coated pit, f — filopodium, fl — flagellum, fp — flagellar pocket, fv — food vacuole, m — mitochondria, n — nucleus. Scale bars: A-D — 5 ^m, E — 1 ^m.
which is broad at the base (Z-circle) and pointed at the distal end (Y-circle). The more distal X-circle forms a circle, ca. 700—800 nm in diameter, around the distal part of the kinetosome (Fig.3, H ,I). It consists of two separate concentric structures, of which the outer is the narrowest and produces radial microtubules (Fig. 3, F—I). Regular filaments, 30—35 nm in length, and with a mutual distance of 30—35 nm, radiate from the Y-circle anteriorly and connect the Y-circle to the X-circle (Figs 3, H, I; 4, B). The X-circle produces an umbrella-like cone of approximately 50 single radiating microtubules, which are not connected to the nucleus (Figs 3, G—I; 4, A—C). The majority of the microtubules radiate laterally, but at one side of the kinetosome they pass almost perpendicular to the cell surface into the cell interior (Fig. 3, F—I). This asymmetry is, probably, associated with the mode of feeding in Phalansterium (see below).
The fibrillar root originates from the proximal end of the kinetosome (Fig. 4, C—E). It starts from the center of a fibrillar cone connected with proximal end of the kinetosome (Fig. 4, C) and continues for approximately 1.2 ^m as a thick fiber along with underlining cisterns of the Golgi apparatus (Fig. 4, A—E). The root is oriented opposite to the vertical microtubules (Fig. 4, D). The main fibrillar root is surrounded by lateral amorphous fibrillar material. The total width of the root and amorphous material is up to 200 nm (Fig. 4, C—E). The fibrillar root is accompanied by several single microtubules, which originate either at the Z-circle or the proximal end of the kinetosome (Fig. 4, C—D). Some of these microtubules pass laterally from the Z-circle in the same direction as the cone microtubules. A few short fibrillar feet emanate from the proximal end of the kinetosome and into the cytoplasm (Fig. 4, A, D), producing single microtubules in different directions.
Fig. 2. General ultrastructure of Phalansterium foulumis sp. nov. A — LS of the mature (adult) cell; B — structure of nucleus, mitochondria and microbodies; C — LS of flagellar apparatus. Arrowheads show 3 layers in Y; Z is, in fact, a broad base of the foot tapering at the Y-circle. D — LS of immature (young) cell. Abbreviations: b — bacterium, bb — flagellar basal body (kinetosome), d — dictyosome of Golgi apparatus, ff — fibrillar feet, l — lipid globule, lf — lateral fibrillar material, mb — microbody, mt — root microtubules, nu — nucleolus, x, y, z — dense fibrillar circles around the kinetosome; other abbreviations as in Fig. 1. Scale bars: A — 1 um, B — 0.6 Um, C — 0.2 um, D — 1 ^m.
Feeding
We have observed feeding in strain Ph185 several times using light microscopy. The cell uses its flagellum to collect bacteria above the opening of the collar. During this process, the distal part of the flagellum beats fast, whereas the proximal part of the flagellum is held almost stiff. The result is that 10 to 20 bacteria accumulate at a distance of approximately
4—5 ^m from the collar. Single bacteria then move slowly along the flagellum towards the cell and into the interior part of the collar (Figs 1, C; 3, A; Video S1). Then the bacteria settle down on the bottom of flagellar pocket where the cell forms a food cap (Fig. 1, D; Video S1). Pseudopodial protrusions are not visible outside the collar during bacterial uptake, but electron microscopy shows pseudopodia in its interior part (Fig. 3, F). The pseudopodia attach
Fig. 3. Flagellar apparatus structure of Phalansterium foulumis sp. nov. Selected serial sections from the tip of flagellum to the base; view from tip to base. A — A central microtubule at the distal part of the collar; B, C — central microtubule enters the plug (D); E — cylinder connected to partition (arrow) from doublets; F — pseudopodium at the level of kinetosome distal end; G-I — consecutive sections through the kinetosome, showing the asymmetry in microtubules orientation: "ventral" microtubules (bottom right of the figures) pass almost vertically into the cell comparatively with "dorsal" microtubules (upper left); H — zone ofthin filaments is visible between y and z circles. Abbreviations: ac — acorn, cf — concentric fiber, cm — central microtubule in flagellar axoneme, cy — cylinder in transition zone and kinetosome of flagellum, pl — plug in flagellar transition zone, ps — pseudopodium, tf — transitional fibers; other abbreviations as in Figs 1 and 2. Scale bars: A — 0.4 ^m, B-I — 0.2 ^m.
to the bacteria and move them inside the flagellar pocket to the base of flagellum (Fig. 3, F). Here, the radiating microtubules are oriented vertically descending into the cytoplasm (Fig. 3, F—I). The
vertical microtubules are probably needed to guide the food vacuole with ingested bacteria in the cytoplasm; therefore, the fibrillar root, the Golgi apparatus and the nucleus are located at the opposite side of cell.
Fig. 4. Flagellar apparatus structure of Phalansterium foulumis sp. nov. A, B — Consecutive LSs of flagellar apparatus; C — oblique section of flagellar kinetosome (arrows show microtubules from Z-circle and proximal end of kinetosome); D, E — consecutive sections almost perpendicular to the longitudinal cell axis. Arrowheads show the microtubules from fibrillar feet and Z-circle. Abbreviations: fr — fibrillar root, lf — lateral fibrillar material; other abbreviations as in Figs 1-3. Scale bars: A, C — 0.25 ^m; B, D, E — 0.35 ^m.
Molecular phylogeny
In our analysis, all the clades revealed by Berney et al. (2015) and Shmakova et al. (2018) appeared with high support, but higher-level branching had low support and clade relationships differed from those recovered by Berney et al. (2015). Our results demonstrated a robust grouping of the present isolate
with other species ofthe genus Phalansterium (Fig. 5). P. foulumis appeared sister to P. filosum SR1-9H (Smirnov et al., 2011), and their joint clade was the neighbour to the clade consisting of three other Phalansterium sequences. The whole Phalansterium clade was sister to the cluster of amoebozoans that included members of the genera Dictyamoeba, Multicila and the members of the "clade nine" sensu Berney et al. (2015).
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EU 186022 Ftamelia arnhemetiiif GUI86021 Flamellaaegyptk -EU 186024 FtunteUa fluvlatiils
-KP219428 Ftamelia beringiania ■ k PS!64049 TflaepOlella sp. — EU273440 Telaepoleltn ittbasferens
- AY71436') Pilatnoeba stm-iisis AY7I436S FilamoL'ba nn'onii 49/0.75
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KP864ÔE3 Angulamoeba micracysSvorans
-FJ766480 Soliformovum irregular*
- FJ766479 Soliformovum expsUsuni
- GQ43K742 Crcllamoeha mhi/.ua
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KP864093 hchmitooebd sp. KP864092 fschnamoeba nitmiuna KP86408S Durbyshmiia sp. KP8640S7 Darbvshirella terres!ris
. FJ544418 Schizoplasmndium eavmaeUmdes
F J544420 NenuttosteHum ovrnum '-FJÎ44419 CeraTiomyxeIUi tahitiènsia 52/0.94 |—EF14396È Phaiansterium fiioium
66/o.9.11 phaiansteriumfouUimis sp, nov.
i-KX844828 Phiilansteritim areticum 93/0.99 | KF539978 Phulnnsteriiim sp. strain JFP 2013
95/1.1)'— AF2S0078 Plmlanswrium cf. wHtarfan ATCC50327 ,— M13435 Acanthamoeha caxtelkmii '— AF477019 Balamuthia numdrittark
-JN247435 PuriMiermtmtoebu levin
--AY294I43 Muyniellu sp.
Phaiansterium
OUTGROUP
Fig. 5. Molecular phylogeny of Phalansterium species based on 18S rRNA gene sequences. New species is in bold. Tree was calculated using 1370 nucleotide positions. Supports: RAxML (GTR+ gamma correction model, 8 rate categories; 1000 BS pseudoreplicates) / MrBayes (GTR model + gamma correction, 8 rate categories + covarion). Black dot means full support by both methods (100/1.0). Supports below 50/0.5 are not indicated.
Discussion
All the "solitarium" species are similar to each other, but the recent paper (Shmakova et al., 2018) partly clarified the situation. Based on some morphological information and the SSU gene differences, we are able to outline several species (Tables 1, 2).
Cavalier-Smith et al. (2004) were not certain that the Phalansterium solitarium strain ATCC 50327 was correctly identified, as they observed that the collar was not permanently visible but only appeared transiently during feeding. In ATCC 50327, the collar is rather long and has narrow walls, but unfortunately Cavalier-Smith et al. (2004) provided neither scale bars on their figures nor size data in the text. We observed all transitional stages between fully intact and completely retracted collars in P. foulumis sp. nov., which suggests that the collar
is a highly variable and contractile structure. The same conclusion has been drawn by Shmakova et al. (2018) for P. arcticum.
Palansterium foulumis sp. nov. and strain ATCC 50327 differ in some other respects. Cavalier-Smith et al. (2004) did not observe a contractile vacuole in ATCC50327, and claim that neither did Sandon (1924) in P. solitarium. However, Sandon (1924) saw a contractile vacuole just behind the center of the body, though stated that it is usually not visible (Sandon, 1924, Fig. 1). The strain ATCC 50327 also has a more developed Golgi apparatus and endoplasmic reticulum than P. foulumis sp. nov. This may be ascribed to different physiological states of the examined cells, or to the cell age, since this character may vary even in the same strain, as we observed in P. foulumis sp. nov. (compare Figs 2, A and 2, D). The star-shaped cyst reported from ATCC
Table 1. Sequence identity levels for 18 rRNA genes among species of the genus Phalansterium calculated using the "Ident and Sim" tool on a 1832 bp unambiguously aligned fragment shared by all sequences. Numbers in parentheses indicate the number of different nucleotide positions.
P. foulumis SCCAP-ph185 P. solitarium ATCC AF280078 Phalansterium sp. JFP KF539978 P. arcticum KX844828
P. solitarium ATCC AF280078 94.33 (104)
Phalansterium sp. JFP KF539978 95.20 (88) 98.36 (30)
P. arcticum KX844828 95.53 (82) 96.40 (66) 97.43 (47)
P. filosum EF143966 95.15 (89) 93.30 (123) 93.85 (113) 94.28 (105)
Table 2. Comparison of the characters of known Phalansterium species.
Species/ characters P. digitatum (Hibberd, 1983) P. filosum (Smirnov et al., 2011) P. arcticum (Shmakova et al., 2018 P. solitarium (Sandon, 1924) P. aff. solitarium ATCC50327 (Cavalier-Smith et al., 2004) P. foulumis (present paper)
colonial + - - - - -
"solitarium" - + + + + +
sedentary cells (|m) 18-25x6-8 FL 36-50 6.3-8.5x5.8-8.8 FL 46 7x3 Fl 12-55 15x10 Fl 30-60 Thick mucilage * 9x7 Fl* 45 8x6 FL 30-50 Thick mucilage
swimmers (|m) - - 7-10x2-3.5 FL 17-35 10-15 - -
flagellated amoebae - +, filopodia + - - +, filopodia
aflagellated amoebae - +, filopodia + - - +, filopodia
cysts shape and size (|m) ND rounded, 5 rounded 5-6 angular angular rounded, 4-5
kinetosome length (nm) 250-300** ND 200 ND ND 250-375
plug in flagellar transition zone length (nm) 250-300 ND ND ND ND 350-500
Designations: Fl - flagellum, ND - no data, - absent, + present, * measured from Fig. S2 in: Smirnov et al. (2011), ** remeasured based on microtubular diameter (25 nm) from Fig. 13 in: Hibberd (1983).
50327 is very different from that of P. foulumis sp. nov. (Ekelund, 2002). Cysts of P. foulumis sp. nov. (Ekelund, 2002) are rounded, have thick envelopes, and look similar to the cysts of P. filosum.
According to molecular phylogeny (Fig. 5), P. foulumis sp. nov. is sister to P. filosum, although with low support. The raw distance of P. filosum to P. foulumis sp. nov. is nearly 5%—high enough for interspecies level (Shmakova et al., 2018).
Phalansterium filosum and P. foulumis sp. nov. have several characters in common, i.e., the same cell and flagellum dimensions, the presence of both flagellated and aflagellated forms that produce filopodial projections, and the same dimensions of rounded cysts (Table 2). At the same time, both
species differ in some respect. The sedentary cells of P. foulumis sp. nov. are surrounded with thick mucilage (gelatinous sheath), which is absent in P. filosum, and have much shorter filopodia serving for the cell attachment and not for movement.
On the basis of molecular phylogeny, light microscopical, and ultrastructural data, we propose that the strain SCCAP Ph185 should be assigned to a new species Phalansterium foulumis.
Phylum Amoebozoa Lühe, 1913 Class Variosea Cavalier-Smith, 2004 Order Phalansteriida Hibberd, 1983 sensu Cavalier-Smith et al. (2016)
Family Phalansteriidae Saville Kent, 1880-1881
Genus Phalansterium Cienkowski, 1870
Phalansterium foulumis Karpov et Smirnov, sp. nov., present paper.
Diagnosis: Solitary species able to form spherical flagellated and aflagellated cells 6—8 ^m in diameter. Cylindrical retractable cytoplasmic collar, 1 ^m long, around the base of flagellum (12—55 ^m long) emerging from the deep bulb-shaped flagellar pocket. Flagellated and nonflagellated amoeboid cells produce filopodia. Sedentary cells are surrounded by a layer ofmucus with a thickness from halfto one cell diameter. Rounded single-walled cysts 4—5 ^m in diameter.
Type figures: Fig. 1 A-C in: Ekelund (2002); Fig. 1-4, present paper.
Type strain: Ph185 kept in the SCCAP.
Hapantotype is in the resin blocks Ph185 in SCCAP.
Sequence of the 18S rRNA gene of a type strain:
GenBank OR613015.
Type locality: strain Ph185 was isolated from the sample of barley rhizosphere soil collected at Research Center Foulum (Copenhagen, Denmark).
Etymology: foulumis (L) — from the name ofthe place of sample collection: Foulum (Copenhagen, Denmark).
Note. Differs from the closest related P. filo-sum by shorter filopodia, thick mucilage around vegetative cell, and 89 nucleotide positions in the complete SSU rDNA sequence.
Other species: P. filosum, P. arcticum, P. digi-tatum, P. consociatum.
General ultrastructure
For the discussion of Phalansterium spp. ultrastructure, we mainly focus on the data of P. digitatum (Hibberd, 1983) and P. arcticum (Shmakova et al., 2018), as these are most comparable with our results.
We suggest that P. foulumis uses filopodia usually produced from the posterior cell end (Fig. 1, B, E) to attach to the substrate. During preparation of culture material for EM, the cells were detached from the substrate, and they presumably produced filopodia at any side of the cell (Fig. 1, E) in order to regain contact with the substrate. Phalansterium digitatum contains more and larger microbodies (Hibberd, 1983) than P. arcticum and P. foulumis, which may be associated with the production ofiron-containing granules in the spherule-producing vesicles that are absent in the latter species.
Flagellar apparatus
In both, P. foulumis and P. digitatum, the proximal part ofthe flagellum has a thin fibrillar coat and a proximal dilation, which has somewhat intermediate width between P. digitatum and P. arcticum where it is nearly absent (Shmakova et al., 2018). All three species have a plug in the flagellar transition zone and a concentric fibre under the plug. The plug of P. foulumis has the same thickness as that of P. digitatum, but is much longer, i.e., 350—500 nm vs. 250—300 nm. The central microtubule ofboth strains forms a slightly oblique angle to the longitudinal axis of the axoneme when it enters the plug. The kinetosome in P. digitatum is approximately of the same length as in P. foulumis, 250—300 and 250—375 nm, correspondingly (Table 2).
The prominent plug, an electron dense column, above the transitional cylinder in the flagellar transition zone were found in the pelobiontids (Arch-amoebae), i.e., Mastigamoeba spp. (Simpson et al., 1997; Walker et al., 2017).
The organisation of the microtubular complex in P. foulumis is very similar to that of P. digitatum. The composite structure of the Y-circle consisting of three dense circles is also clearly visible on Fig. 18 in Hibberd (1983). The thin fibrillar material between the concentric structures is not very conspicuous on the pictures in Hibberd (1983), but is obviously present. Hibberd (1983) reported that 60 microtubules surrounded the kinetosome of P. digitatum, whereas we only counted up to 50 in P. foulumis and 50 in P. arcticum (Shmakova et al., 2018). This difference is, probably, not essential, as it may be ascribed to different age of the examined cells.
The fibrillar root in P. foulumis is obviously more conspicuous than that of P. digitatum because it is associated with more lateral fibrillar material. Unlike P. digitatum, P. arcticum has a shorter and less conspicuous fibrillar root originating from a special plate, which is also present in P. foulumis. In P. digitatum, single microtubules originating from the proximal part of the feet were not observed.
While the Z-circle in P. foulumis and P. digitatum looks like a ring around the proximal part of kine-tosome, in P. arcticum it is fragmented and each microtubular triplet has its own Z sector (Shmakova et al., 2018). We can suggest, that the Z-circle in P. foulumis and P. digitatum is represented by fused nine Z sectors, or feet. While single microtubules originating from the proximal end of kinetosome
were not observed in P. digitatum, in P. foulumis we found microtubules passing both, from the Z-circle and short fibrillar feet.
The unikont kinetid of Phalansterium has a very unusual microtubular root system. The radiating microtubules originating distantly from the single kinetosome is a unique feature. They form a cone around the kinetosome descending within the cytoplasm but do not connect to the nucleus, as in many pelobiontids. A single kinetosome of archamoebae produces radial microtubules from dense sheath around the kinetosome (similar to Z-circle in Phalansterium), a lateral band of microtubules, and microtubules ofthe nuclear cone, starting from a separate foot at the proximal end of kinetosome (Walker et al., 2017). From this set of roots, probably, the nuclear cone microtubules are homologous to single microtubules of fibrillar feet in Phalansterium. Single microtubules from Z-circle may be homologous to radial microtubules of archamoebae.
Feeding
Sandon (1927) and Hibberd (1983) both suggested that Phalansterium feeds on dissolved organic matter, whereas Ekelund (2002) and Cavalier-Smith et al. (2004) observed bacteria in the interior part of the collar. Cavalier-Smith has presented the most comprehensive description of feeding for P. filosum (Smirnov et al., 2011). Individual bacteria are ingested in the pocket after passing through the space within the collar. The Figure 7I-K in the above cited paper illustrates extending asymmetrical lamellipodium along external side of collar. However, this lamellipodium seems not to be related with the engulfment of bacteria, since no visible bacteria are present in the collar region. Other authors have not observed the formation of lamellipodia during feeding (Shmakova et al., 2018). However, Figure 7 M-R in Smirnov et al. (2011) definitely demonstrate engulfment of a bacterium inside the collar and further transport of the food vacuole in the cytoplasm opposite to the nucleus. This is in full agreement with our observations suggesting the asymmetry in the nucleus position due to feeding.
In P. arcticum, Shmakova et al. (2018) observed two bacteria inside a newly formed food vacuole, and concluded that Phalansterium uptakes individual bacteria in the collar one by one, but engulfs several ofthem at once. Collection of bacteria at the bottom of the flagellar pocket near the base of the flagellum
is also illustrated on Fig. 3, F and on the Video S1 in the present paper.
The asymmetrical position of the base of the flagellum in P. arcticum leads to the fact that the absorption of food occurs constantly from one side of the cell along the descending part of the micro-tubular cone, for the convenience — on the "ventral" side. Similar asymmetry of the flagellar base, the dictyosome, and the nucleus is also present in P. digitatum (Hibberd, 1983, Figs 6-8). Probably, the colonial P. digitatum is also able to ingest bacteria. We do not know whether the site ofbacterial engulfment and, consequently, the cell structure asymmetry in Phalansterium is permanent, but if so, it will make possible to define a ventral and dorsal side ofits cell, correspondingly, even though the asymmetry is not reflected in the external morphology. Finally, we cannot exclude that dissolved organic material is also a part of the Phalansterium diet, as coated pits have been noted in P. digitatum (Hibberd, 1983), P. arcticum (Shmakova et al., 2018), and P. foulumis (Fig. 1, E in present paper). This indicates that endocytosis takes place in all these species.
The presence of the acorn suggests an initially
BIFLAGELLATED STATE OF PHALANSTERIUM LINEAGE
in this study, we demonstrated the presence of asymmetrical acorn in the distal part of the kine-tosome (Fig. 3, F), and also discovered traces of it in P. digitatum (Hibberd, 1983; Fig. 10). The acorn is probably a structural equivalent of the cartwheel at the distal end of the kinetosome in biflagellated protists, which provides their rotational symmetry (Geimer and Melkonian, 2004). The presence ofthis structure is believed to be a shared feature in many eukaryotic lineages and suggests that the ancestral eukaryotic cell had paired kinetosomes (Mitchel 2007; Roger and Simpson, 2009; Cavalier-Smith, 2021). Therefore, the presence of an acorn in Pha-lansterium suggests that its unikont condition is a derived character. Modern phylogenomic studies do not support the basal split of all Amoebozoa into Lobosa and Conosa clades (Lobosa/Conosa dichotomy sensu Cavalier-Smith, 1998). Instead, it suggests the subsequent branching of all lineages in Amoebozoa tree, with the basal Discosea or Tubulinea lineage, depending on the analysis (Kang et al., 2017; Tekle et al., 2022). The presence of acorn further supports the origin of Amoebozoa from a biflagellated ancestor, although the exact time of loss of the second kinetosome is unclear.
Among Variosea, there are species with multiple uniflagellate kinetids (e.g., Multicilia), but none with a typical paired kinetosome. So, the loss of the second kinetosome should be an ancient event in the evolution of this group of Amoebozoa.
Acknowledgements
The authors are grateful to B.S.C. Leadbeater for manuscript review and language correction. SAK thanks the RSF grant No 23-14-00280, https://rscf. ru/project/23-14-00280/ for supporting ultrastructural study, general data analysis and writing the manuscript. AS acknowledges the support ofthe RSF project 23-74-00050 (molecular phylogenetic analysis).
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Supplementary material
Video S1. Bacteria engulfment and food vacuole formation by sedentary cell of Phalansteriumfoulumis.
