Keywords: ultrastructure, evolution of eukaryotes, Nibbleridia, cytoskeleton, microtubular roots, 18S rRNA phylogeny

2.1. Establishing clonal cultures

Clonal culture ofNibbleromonas piranha sp. n. (strain Jim-2) was isolated from a sample of coastal marine sediments of the Sea of Japan, Jeodo Island, Republic of Korea, with a salinity of 22‰ on 10 May 2019 (35°03′09.86″N, 128°33′47.24″E) and established usingProcrybtobia sorokini (Zhukov 1975) Frolov et al. 2001 as prey, as described previously [6]. The sample was collected within the framework of the Russian–Korean bilateral cooperation Basic Science Research Program through the National Research Foundation of Korea (NRF) and Russian Foundation for Basic Research.

All studied strains of nibblerids, including Jim-2 and those obtained earlier [6], were cultivated under the same conditions (temperature of 22°C, darkness) in marine Schmalz-Pratt medium (pH 7.2; 22‰) with the same prey concentration. Strains are currently stored in a collection of Live Protozoan Cultures at the Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, and in the University of British Columbia.

2.2. Light microscopy and video

Cells were observed using a Zeiss Axioscope A1 and a 63× water immersion objective with phase contrast or DIC. The images and videos were taken with an MC-20 camera (Lomo-Microsystems, Russia) and an MC-1009/S video camera (AVT Horn, Aalen, Germany).

2.3. Scanning electron microscopy

Cells were centrifuged and fixed with 2.5% glutaraldehyde in marine Schmalz-Pratt medium (pH 7.2) for 30 min at 22°C. Then, the cells were drawn onto a polycarbonate filter (0.8 μm pores). After that, the cells were dehydrated in an ethanol series (30%, 50%, 70%, 96% and 100%) followed by ethanol with propylene oxide (1 to 1 ratio) for 10 min each and 100% propylene oxide (three times for 10 min each). Cells were then incubated overnight in 100% hexamethyldisiloxane and dried. The dry filters were mounted on aluminium stubs, coated with gold and observed with a JSM-6510LV (JEOL, Tokyo, Japan) electron microscope.

2.4. Transmission electron microscopy

For investigation of the ultrathin series, cells were centrifuged for 20 min at 5000×g; 0.5 ml of 4% glutaraldehyde (in 0.1 M cacodylate buffer) was added to 0.5 ml of the resuspended cells and incubated at +4°С for 2 h. The pellet of fixed cells was subsequently embedded in 1% agarose and rinsed twice (10 min each) with cold (4°С) 0.1 M cacodylate buffer. After that, cells were fixed in cold (+4°С) 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h. Then, the pellet was rinsed with 0.1 M cacodylate buffer for 10 min. After dehydration in an alcohol series (30%, 50%, 70%, 96% and 100%) and propylene oxide, the pellet was embedded in Spurr resin (EM 0300 Sigma-Aldrich). Ultrathin sections (60 nm) were prepared with a Leica EMUC6 ultramicrotome (Leica Microsystems, Germany) and observed using a JEM-1011 (JEOL, Japan).

To observe whole-mount preparations, a drop of cell culture was placed on Formvar-coated transmission electron microscopy grids and fixed in vapours of 2% osmium tetroxide for 10 min. After rinsing with distilled water, cells were stained in 1% uranyl acetate (С₄H₆O₆U) for 20 min and rinsed with distilled water again. Whole-mount preparations were observed by using a JEM-1011 (JEOL, Japan).

2.5. 18S rRNA gene sequencing

Cells were harvested from Petri dishes following peak abundance after consuming most of the prey. The cells were collected by centrifugation (1000×g, room temperature) onto the 0.8 µm membrane of a Vivaclear minicolumn (Sartorius Stedim Biotech Gmng, cat. no. VK01P042). Genomic DNA was isolated using the Master Pure Complete DNA and RNA Purification Kit (Epicentre, cat. no. MC85200). The 18S rRNA genes were amplified using the EconoTaq PLUS GREEN 2X Master Mix (Lucigen, cat. no. 30033-1) and universal eukaryotic primers EukA–EukB [19]. Amplified DNA fragments were purified with a QIAquick PCR Purification Kit (Qiagen, cat. no. 433160764). The PCR product was sequenced directly via Sanger dideoxy sequencing. Two additional internal primers, 18SintF (5′-GGTAATTCCAGCTCCAATAGCGTA-3′) and 18SintR (5′-GTTTCAGCCTTGCGACCATACT-3′), were used. The resulting sequences were assembled from four overlapping reads using the Geneious R7 7.0.6 program (https://www.geneious.com).

2.6. Phylogenetic analysis

A previously published dataset of all available provorans and some representative eukaryotic sequences was used for phylogenetic reconstructions [6]. Multiple sequence alignment was performed using the L-INS-i algorithm in MAFFT version v. 7.490 [20], and the sequences were trimmed using an automated trimming heuristic followed by a gap threshold filter of 0.7 in TrimAl version 1.4 [21]. Phylogenetic trees were reconstructed using Bayesian and maximum likelihood (ML) methods. Bayesian analysis was performed in MrBayes v. 5.1.16 [22] using the GTR+GAMMA4+I model to calculate posterior probability. Four independent Metropolis-coupled Markov chains were run for 20 million generations and summarized with a 50% burn-in. ML phylogeny was inferred using IQ-TREE (v. 1.6.12) [23,24] and RAxML-NG (v. 1.0.0) [25]. ML reconstruction with IQ-TREE was performed with 1000 ultrafast bootstrap replicates (phylogeny of provorans and eukaryotes) or nonparametric bootstraps with 1000 replicates (phylogeny of provorans) under the TN+F+R3 model, determined by the in-built ModelFinder. ML reconstruction with RAxML-NG was performed with 20 random starting trees for the best ML tree search based on the GTR+GAMMA4+I model.

3.1. Ultrastructure ofNibbleromonas spp.

All known species of the genusNibbleromonas had similar ultrastructures according to our observations. Most of the data used for the reconstruction of theNibbleromonas spp. cytoskeleton are fromN. quarantinus, whose transmission electron microscopy preparations were characterized by better fixation quality. We also compared data across different strains to create a more comprehensive model (figure 1a,b).

3.2. Main architecture of the cell

The cell surface ofNibbleromonas species consists of a plasmalemma underlined by one to three layers of flattened vesicles. These vesicles are primarily found on the dorsal and lateral sides of the cell, while the ventral and subapical parts are usually covered by the plasmalemma only (figure 2a,d). In relation to the condition of the cell, the number and size of alveoli vary essentially. They often flatten and produce three to seven membrane layers (including the plasma membrane) covering the cell (figure 2a,d). The inner layers of such multimembrane coverings are formed as a result of vesicular transport from the Golgi apparatus and probably promote the formation of food vacuoles during feeding. A dictyosome is located between the flagellar basal bodies (kinetosomes) and the nucleus (figure 2a; electronic supplementary material, figure S4i). A microbody was adjacent to the nucleus, and a single branching mitochondrion was observed (figure 2a,e). Mitochondrial cristae of short tubular or vesicle shapes normally contain a filament (figure 2a,e, inset), which is characteristic of stramenopiles. The nucleus with the central nucleolus is typically located near the cell centre (figure 2a,b) but may move towards the periphery if a large food vacuole is formed (figure 2c). Two flagellar kinetosomes lie subapically at the ventral side of the cell. Two flagella emerge from independent flagellar pockets on the ventral side. Cytoskeleton structures consist of microtubular bands and fibres derived from kinetosomes (figures 1a and2d,f ).

3.3. Extrusomes

Nibblerids have two groups of extrusomes that differ in shape and location. One group of normally five needle-like extrusomes are oriented towards the posterior end of the ventral groove, and called here the cytostomal extrusomes (ces). Another type of extrusive organelles is normally singular, but in rare instances two or three are found, is half the length, ampule-shaped, and lay perpendicular to the other extrusomes, ending between the kinetosomes (figures 2d,f and3a,b,f). This type is referred here as interkinetosomal extrusomes (ies). Most likely, extrusomes can move inside the cell due to cytoplasmic microtubules (figure 3d). One or two cytostomal extrusomes can extend out of the cell by one micron, forming a thorn (figure 3e). The cytostomal and interkinetosomal extrusomes have a similar general morphology of inner structures, but are located in different regions of the cell and differ in shape. The proximal half of the extrusome is filled with electron-dense material forming a basal ampule (figures 1c and3d). A thin cylindrical structure connects the pointed tip of the basal ampule to the base of the small middle ampule, which continues into the less osmiophilic and cross-striated needle tapering at the apical point of the extrusome (figures 1c and3c,e). On the side where the extrusomes make contact with the plasmalemma, a small layer of electron-dense pieces was observed, possibly indicating a special adhesive property in this region (figure 3a, arrows). Considering their unique structure, we propose calling them ampulosomes.

3.4. Flagellar apparatus

The middle and distal parts of the flagellum have a typical organization: an axoneme 9+2 surrounded by a narrow layer of cytoplasm, which is in turn covered by the plasmalemma. Thin hairs were found on the proximal part of the posterior flagellum inside the flagellar pocket (electronic supplementary material, figure S4e). Closer to the base, the posterior flagellum forms two opposite lateral vanes (folds) shown by the arrowheads, and dilation on one side of the flagellum, often containing a fibrillar seal (arrow) (figure 4b,c).

The transition zones of both flagella are similar. The central pair (cp) of microtubules starts from the axosome (ax), which is located on the concave transition plate (figure 4a,d–f). The base of cp is surrounded by a transitional cylinder (tc). The distal ends of kinetosomes are connected to the plasmalemma by transition fibrils (tf).

To clearly describe the relative positions of each root and their derivatives, all images of the flagellar apparatus were oriented as follows: the anterior end of the cell is directed upwards, and the observer looks at the ventral side of the cell from the outside. This approach is taken because the triplets of kinetosomes are rotated counterclockwise (figure 4f–h); therefore, we look at the kinetosome from the flagellum tip to the base. In this case, a broad cross-striated croissant-like fibrillar bridge (br) connects the kinetosomes to each other from their right side (figure 2a, inset). If the triplets are not visible in the images, we use this bridge for correct cell orientation. The angle between the kinetosomes ranged from 0° to 60° (figures 1a,b,2c and4h).

According to the broadly accepted numbering of kinetosomes and their roots [2], the kinetosome of the anterior flagellum is k2, and the kinetosome of the posterior flagellum is k1.

K1 produces two microtubular roots: root 1 (r1) and root 2 (r2). R1 begins from the point where k1 is connected to the bridge as two microtubules (figures 1a,b and4g; electronic supplementary material, figure S2, S4c) and continues between the kinetosomes inside the adjacent wall of the flagellar pockets, increasing to four microtubules (figures 1a,2f,4d–f,g,i and4d, inset). R1 reinforces the left side of the ventral groove (‘left jaw’) (figures 1a,b,2f; electronic supplementary material, figure S4b−e). The dense fibril originates from the br and is connected to r1 along its entire visible length (figure 1a; electronic supplementary material, figure S1e,f).

R2 is the main feeding root, which originates from the posterior surface of k1. It consists of three microtubules at its origin and is associated with the fibril plate at the bridge (figures 1a and3b; electronic supplementary material, figure S3).

R2 lines the right side of the cytostome (the right ‘jaw’) along the ventral surface of the cell towards the distal end, and the thorn with needle-like ampulosomes (figures 1a,b and2d,f; electronic supplementary material, figure S1,4e–h). The number of microtubules increases to 12. The plate at the base of r2 is connected to the bridge by a network of thin filaments, which extend along this r2 branch to the distal end. The filaments connect with the plasmalemma at the posterior-ventral end of the cell (figure 3c). The long cytostomal fibre originates from the bridge and extends from the distal end of k2 along the entire length of r2 (figures 1a and2d; electronic supplementary material, figure S4e).

The singlet root (sr) passing from the left side of k1 produces few secondary microtubules underlying the cytostome (figures 1a,b andfigure 4f-i; electronic supplementary material, figures S1a,b and S3b–g). The arrangement of r1 and sr in reinforcing the left ‘jaw’ involves each root passing along different sides of the adjacent pocket wall (figure 1a,b; electronic supplementary material, figures S1−S4).

K2 produces root 3 (r3) and root 4 (r4) microtubular roots. R3 originates from the right side of k2, gives a short branch that links to the bridge, and passes anterior-ventral as a band of three microtubules, which produce many secondary microtubules under the dorsal cell surface (figure 1a,2f and4g,h, electronic supplementary material, figures S1a–e, S2a–f, S3a and S4a). R4 is a singlet root that originates from the left side of k2. It is associated with a thin fibril and lengthens parallel to r3 toward the apical end of the cell (figure 4f-h; electronic supplementary material S1a–d and S2a–f).

3.5. External morphology and behaviour ofNibbleromnas piranha sp. nov.

The predatory eukaryovorous flagellatesNibbleromonas piranha sp. nov. have crescent-shaped cells (figures 5b and6a). The cell length is 3.2–5.6 µm, and the cell width is 2.7–4.9 µm. Two heterokont acronematic flagella emerge from independent flagellar pockets, which are separated by a cytoplasmic protrusion (figures 5e and6a,b). The ventral feeding groove is located distal to the flagellar pocket of the posterior flagellum (figure 6a). There were no mastigonemes on the surface of the flagella (figure 6a–c,e). The posterior flagellum is approximately three times longer than the cell body and one and a half times longer than the anterior flagellum (figure 6a–c). Both flagella have two keel-like folds that extend from the proximal quarter of the posterior flagellum and distal third of the anterior flagellum. Folds are located ventrally and dorsally relative to the cell body (figure 6a–c). There was a clear dimorphism of well-fed and starving cells (figure 5b,c). The size of the cell can increase up to twofold after feeding. Starving cells impulsively spin around their axis, staying in one place or floating with the apical part forward, changing the direction of movement situationally. Well-fed cells lie at the bottom of a Petri dish or move quickly but more smoothly and do not abruptly change the direction of movement. Small starving cells are not able to consume an entireP. sorokini cell completely, so they bite off most of the cell of the prey (video 1;figure 6f), like other species ofNibbleromonas (figure 5t–y) or, sometimes, feed jointly (figure 5f–l).Nibbleromonas attacksP. sorokini cell by attaching to it with a distal thorn and then apparently immobilizing the prey using ampulosomes located inside the thorn. The prey becomes rounded and stops moving, at which time the ventral part of the predator cell comes into closer contact with the prey (figures 5f and6d).N. piranha sp. nov. stretches over the prey and opens the cytostome (figures 5s and6d). Both flagella wrap around the prey body, helping to hold and engulf it. Sometimes, one of the flagella is attached to the bottom of the Petri dish (video 2). After this, the size of theNibbleromonas cells increased significantly. These cells continue to feed and can engulfP. sorokini cells entirely, forming a large food vacuole (figure 5c). Well-fed cells do not have a thorn in the distal part and are pear-shaped (figure 5c; videos 1,3). Cysts were not observed in the life cycle. The flagella became shorter and duplicated at the first stage of cell division (figure 5d; videohttps://figshare.com/s/e208322e6cb09b969c8a4). Then, a pair of flagella moves to the posterior end of the cell, and cytokinesis occurs. At the end of cytokinesis, the two daughter cells are located upside down to each other. One daughter cell inherits a large food vacuole (figure 5d), and another, a slightly smaller cell inherits a thorn (video4).

Joint feeding occurs when predators attach to an immobilized prey that is already being eaten by another cell ofNibbleromonas piranha sp. nov. In this case, predators consume the prey from different sides, with one of them engulfing most of prey’s cell (or whole cell), while the others competing for prey and attacking each other with ampulosomes (figure 5f–l). When twoN. piranha sp. nov. cells ate the same prey from different sides, one of them always eventually stopped feeding and slipped away. Approximately, 10 cells were found trying to eat a singleP. sorokini cell, and some of them demonstrated cannibalism (video 3,figure 5f–s). The feeding behaviour ofN. piranha sp. nov. differs from that of other knownNibbleromonas species because nibbling of the prey is less frequent compared with eating whole prey, and competition for the same prey cell with other individuals is more common.

3.6. 18S rRNA gene phylogeny

We carried out phylogenetic analysis with all available provorans 18S rRNA sequences. Provora has been clearly divided into two groups, Nibbleridia and Nebulidia, with full BI and ML support (figure 7).Ubyssea and three related environmental sequences form a separate lineage from the genusNibbleromonas with nearly full support (figure 7).N. piranha sp. nov. belongs to a clade containingNibbleromonas species and the OBEP010720414 environmental sequence (figure 7).N. piranha sp. nov. was most closely related toOP101999N. arcticus, which was fully supported in Bayesian analysis (figure 7) and highly supported in the RAxML and IQ-TREE reconstructions (figure 7).

In addition, we reconstructed an unrooted tree of Provora with short-read environmental 18S rRNA sequences. Even with short reads the high support in the main nodes was maintained, the branching order remained basically unchanged compared with trees based on longer sequences (figures 7–9).N. kosolapovi,N. arcticus,N. quarantinus,N. curacaus andN. piranha sp. nov. all branched within clades with several marine environmental sequences with high support (figures 8 and9). The relationships between our newly discovered strain and the uncultured sequences have not yet been fully determined, but we found that they were placed in a separate lineage fromN. arcticus with high support in Bayesian analysis (0.99) and with moderate support (73%) in the IQ-TREE reconstruction (figures 8 and9).

The phylogenetic analysis with wide sampling of eukaryotic taxa showed thatN. piranha sp. nov. was placed within theNibbleromonas clade, with high support. Similarly to the unrooted trees, Provora was divided into two monophyletic groups: Nibbleridia and Nebulidia, albeit with low support (electronic supplementary material, figure S5).

4.1. The ventral feeding groove of nibblerids reinforced by r2 and r1

The r2 is similar to that of malawimonads, metamonads, jakobids [13,28,33] and some early divergent stramenopiles [40,41], if we assume that Bicosoecida is one of the deep branches of the Stramenopile tree [42–44]. The wide part of r2 has a fibril plate with a fibrous connective with a fibril bridge, which appears similar to the architecture of the I-fibre and fibrous material of malawimonads [32, fig. 3d,g], metamonads [34, fig. 28;33, fig. 4a,b;29, fig. 7] and jacobids [28, fig. 5]. These structures could be homologues to the electron-dense material of R2 [41, fig. 5a] and filamentous connective of R2 [41, fig. 4d] seen in some phagotrophic stramenopiles, while the deepest branching stramenopile do not have any fibrils [40]; however, figure 4 of [41] suggests the I-fibre may lay near R2.Platisulcus tardus has a ventral groove and four main flagellar roots +S tubule. The R2 ofP. tardus is split into inner and outer parts. TheNibbleromonas r2 also has a secondary microtubular band, which in turn creates another secondary band on the left side of the cytostome, which is only visible during the feeding process. Notably, sr is an atypical case, as it provides secondary microtubules and could be the right side of r2 or its functional equivalent. Unlike that of nibblerids, the R2 ofP. tardus dissipates towards the distal end of the cell. By contrast, inNibbleromonas, the number of microtubules increases from the apical end to the distal end. Some bicosoecid flagellates likely have wide strong feeding roots (arranged in a ventral groove), but they are associated with the younger kinetosome of the anterior flagellum (k2) or its fibres [30]. However, the filamentous connectives of fp (the fibrillar bridge between kinetosomes) associated with r3 ofRegin rotiferus look similar to the fibrillar plate and r2 ofNibbleromonas [30, fig. 3]. Additionally, the I-fibre can be found in deep-branching alveolates [15;16, fig. 5B].

The r1 consists of two microtubules at its proximal end and is most likely homologous to r1 of chrysophytes and related ochrophytes, which also have two microtubules [45]. The dense fibril of its location and architecture could be equivalent to a C-fibre, which has been observed in many branches of eukaryotes [1].

The r3 shares common features with that ofMalawimonas [27],Carpediemonas [33] andPlatysulcus [40], but their r3 does not split into two parts. The only description of a similar structure is fromGiraudyopsis stellifer [45,46], which was marked as a bypassing rootlet. This is interesting, coupled with assumptions about the appearance of the bypassing band (BB) [15].

It has been proposed that the r4 could potentially arise independently in different lineages [15], similar to the chiral root of the r2, sr or as a modification of the anterior root (AR) of excavates. It could be related to its reduction or, possibly, the transformation of this root into a r4 singlet to provide more successful support for AF. This is also important for Provora, which have fast and mobile movement of cells. However,Platisulcus has both singlet roots R4 and S tubule [40], unlikeRictus, which has only the SR [41, fig. 6].

Overall, the ultrastructure of vanes, the presence of alveoles under the plasmalemma, and the main architecture of the flagellar apparatus suggest that the possible ancestor of such superclusters as TSAR+Haptista and Archaeplastida+Cryptista was an alveolate-like phagotrophic flagellate with a ventral groove and flagellar vanes on both flagella [15,47].

4.2. External morphology and behavioural features ofN. piranha sp. nov.

The typical characteristic of the genusNibbleromonas is the presence of a thorn on the ventro-caudal side of the cell [6]. This structure contains several ampulosomes that help to immobilize and capture prey. All known species ofNibbleromonas exhibit a classic predator‒prey feeding strategy. Additionally,N. piranha sp. nov. demonstrates unique behaviour and attacks individuals of its own species, resulting in cannibalism.

Biting off a larger portion of the prey cell is a distinguishing feature ofN. kosolapovi,N. arcticus andN. quarantinus. As a result, the unconsumed part of the prey separated into a small vesicle, which was seemingly consumed by bacteria rather than by another predator. It seems thatN. piranha sp. nov. rarely employs this feeding strategy. This species consumesP. sorokini completely, or if the predator is starving and much smaller than the prey, several cells feed on a single prey jointly. Joint feeding is also observed in some other flagellates, such as opistokont protists and colpodellids, but this behaviour has never been observed to result in cannibalism or attacks on one another [3,4]. This appears to occur by mistake, leading to cannibalism, a behaviour observed in some other protists as well [48–51].

N. piranha sp. nov. demonstrates a higher frequency of competing feeding than other species ofNibbleromonas. The reason for this and mechanism by whichNibbleromonas cells are attracted to prey are currently unknown. However, it is possible thatNibbleromonas predators, such asN. piranha sp. nov., may excrete signalling molecules during feeding. These molecules could potentially be up-regulated in response to feeding and encoded by genes involved in cell signalling, similar to those in predatory opisthokonts [52] or the extraction of gamons in ciliates [53]. When a predator is already feeding and anotherN. piranha sp. nov. attacks the same prey, it can become attached to the thorn to the first predator, leading to cannibalistic feeding. These mistakes are deleterious evolutionarily, but perhaps the benefit of competitive feeding over nibbling balances favourably against this disadvantage, since it results in the complete consumption of large prey. Further research is needed to explore and understand the specific mechanisms ofNibbleromonas cell attraction.

• 1.Yubuki N, Leander BS. 2013. Evolution of microtubule organizing centers across the tree of eukaryotes. Plant J.75, 230–244. ( 10.1111/tpj.12145) [DOI] [PubMed] [Google Scholar]
• 2.Moestrup Ø. 2000. The flagellar cytoskeleton: introduction of general terminology for microtubular flagellar roots in protists. In The Flagellates: Unity, Diversity and Evolution (eds Green JC, Leadbeater BSC), pp. 69–94. London: Taylor & Francis. [Google Scholar]
• 3.Brugerolle G. 2002. Colpodella vorax: ultrastructure, predation, life-cycle, mitosis, and phylogenetic relationships. Eur. J. Protistol.38, 113–125. ( 10.1078/0932-4739-00864) [DOI] [Google Scholar]
• 4.Tikhonenkov DV, et al. 2020. New lineage of microbial predators adds complexity to reconstructing the evolutionary origin of animals. Curr. Biol.30, 4500–4509. ( 10.2139/ssrn.3606769) [DOI] [PubMed] [Google Scholar]
• 5.Cavalier-Smith T. 2022. Ciliary transition zone evolution and the root of the eukaryote tree: implications for opisthokont origin and classification of kingdoms protozoa, plantae, and fungi. Protoplasma259, 487–593. ( 10.1007/s00709-021-01665-7) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Tikhonenkov DV, et al. 2022. Microbial predators form a new supergroup of eukaryotes. Nature612, 714–719. ( 10.1038/s41586-022-05511-5) [DOI] [PubMed] [Google Scholar]
• 7.Tikhonenkov DV, et al. 2023. Provora. Curr. Biol.33, R790–R791. ( 10.1016/j.cub.2023.06.004) [DOI] [PubMed] [Google Scholar]
• 8.Arndt H, et al. 2000. Functional diversity of heterotrophic flagellates in aquatic ecosystems. In The Flagellates: Unity, Diversity and Evolution (eds Green JC, Leadbitter BSC), pp. 240–268. London: Taylor & Francis. [Google Scholar]
• 9.Cavalier-Smith T, Chao EE. 2004. Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.). Eur. J. Protistol.40, 185–212. ( 10.1016/j.ejop.2004.01.002) [DOI] [Google Scholar]
• 10.Vickerman K, et al. 2005. Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa. Protist156, 335–354. ( 10.1016/j.protis.2005.07.003) [DOI] [PubMed] [Google Scholar]
• 11.Brown T. 1979. Observations by immunofluorescence microscopy and electron microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures. J. Med. Microbiol.12, 363–371. ( 10.1099/00222615-12-3-363) [DOI] [PubMed] [Google Scholar]
• 12.O’Kelly CJ. 1993. The Jakobid flagellates: structural features of Jakoba, Reclinomonas and Histiona and implications for the early diversification of eukaryotes. J. Eukaryot. Microbiol.40, 627–636. ( 10.1111/j.1550-7408.1993.tb06120.x) [DOI] [Google Scholar]
• 13.Simpson AGB. 2003. Cytoskeletal organization, phylogenetic affinities and systematics in the contentious taxon Excavata (Eukaryota). Int. J. Syst. Evol. Microbiol.53, 1759–1777. ( 10.1099/ijs.0.02578-0) [DOI] [PubMed] [Google Scholar]
• 14.Cavalier-Smith T. 2013. Early evolution of eukaryote feeding modes, cell structural diversity, and classification of the protozoan phyla Loukozoa, Sulcozoa, and Choanozoa. Eur. J. Protistol.49, 115–178. ( 10.1016/j.ejop.2012.06.001) [DOI] [PubMed] [Google Scholar]
• 15.Cavalier-Smith T. 2018. Kingdom Chromista and its eight phyla: a new synthesis emphasising periplastid protein targeting, cytoskeletal and periplastid evolution, and ancient divergences. Protoplasma255, 297–357. ( 10.1007/s00709-017-1147-3) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Tikhonenkov DV, et al. 2014. Description of Colponema vietnamica sp.n. and Acavomonas peruviana n. gen. n. sp., two new alveolate phyla (Colponemidia nom. nov. and Acavomonidia nom. nov.) and their contributions to reconstructing the ancestral state of alveolates and eukaryotes. PLoS ONE9, e95467. ( 10.1371/journal.pone.0095467) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.Schweikert M, Schnepf E. 1997. Light and electron microscopical observations on Pirsonia punctigerae spec. nov., a nanoflagellate feeding on the marine centric diatom Thalassiosira punctigera. Eur. J. Protistol.33, 168–177. ( 10.1016/S0932-4739(97)80033-8) [DOI] [Google Scholar]
• 18.Aleoshin VV, et al. Heterokont predator Develorapax marinus gen. et sp. nov.—a model of the ochrophyte ancestor. Frontiers Microbiol.7, 1194. ( 10.3389/fmicb.2016.01194) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Medlin L, et al. 1988. The characterization of enzymatically amplified eukaryotic 16s-like rRNA-coding regions. Gene71, 491–499. ( 10.1016/0378-1119(88)90066-2) [DOI] [PubMed] [Google Scholar]
• 20.Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol.30, 772–780. ( 10.1093/molbev/mst010) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Capella-Gutiérrez S, et al. 2009. TrimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics25, 1972–1973. ( 10.1093/bioinformatics/btp348) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Ronquist F, et al. 2012. MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol.61, 539–542. ( 10.1093/sysbio/sys029) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Nguyen LT, et al. 2015. IQ-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol.32, 268–274. ( 10.1093/molbev/msu300) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics30, 1312–1313. ( 10.1093/bioinformatics/btu033) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Kozlov AM, et al. 2019. RAxML-ng: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics35, 4453–4455. ( 10.1093/bioinformatics/btz305) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Cavalier-Smith T. 2000. Flagellate megaevolution: the basis for eukaryote diversification. In The Flagellates: Unity, Diversity and Evolution (eds Green JC, Leadbeater BSC), pp. 361–390. London, UK: Taylor & Francis. [Google Scholar]
• 27.O’Kelly CJ, Nerad TA. 1999. Malawimonas jakobiformis n. gen., n. sp. (Malawimonadidae n. fam.): a Jakoba‐like heterotrophic nanoflagellate with discoidal mitochondrial cristae. J. Eukaryot. Microbiol.46, 522–531. ( 10.1111/j.1550-7408.1999.tb06070.x) [DOI] [Google Scholar]
• 28.Simpson AGB, Patterson DJ. 2001. On core jakobids and excavate taxa: the ultrastructure of Jakoba incarcerata. J. Eukaryot. Microbiol.48, 480–492. ( 10.1111/j.1550-7408.2001.tb00183.x) [DOI] [PubMed] [Google Scholar]
• 29.Yubuki N, et al. 2013. Comprehensive ultrastructure of Kipferlia bialata provides evidence for character evolution within the Fornicata (Excavata). Protist164, 423–439. ( 10.1016/j.protis.2013.02.002) [DOI] [PubMed] [Google Scholar]
• 30.Harder CB, et al. 2014. Ultrastructure and phylogenetic position of Regin rotiferus and Otto terricolus genera et species novae (Bicosoecida, Heterokonta/Stramenopiles). Protist165, 144–160. ( 10.1016/j.protis.2014.01.004) [DOI] [PubMed] [Google Scholar]
• 31.Heiss AA, et al. 2021. Description of Imasa heleensis, gen. nov., sp. nov. (Imasidae, fam. nov.), a deep‐branching marine malawimonad and possible key taxon in understanding early eukaryotic evolution. J. Eukaryot. Microbiol.68, e12837. ( 10.1111/jeu.12837) [DOI] [PubMed] [Google Scholar]
• 32.Heiss AA, et al. 2018. Combined morphological and phylogenomic re-examination of malawimonads, a critical taxon for inferring the evolutionary history of eukaryotes. R. Soc. Open Sci.5, 171707. ( 10.1098/rsos.171707) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Simpson AGB, Patterson DJ. 1999. The ultrastructure of Carpediemonas membranifera (Eukaryota) with reference to the 'excavate hypothesis'.Eur. J. Protistol.35, 353–370. ( 10.1016/S0932-4739(99)80044-3) [DOI] [Google Scholar]
• 34.Yubuki N, et al. 2007. Ultrastructure and ribosomal RNA phylogeny of the free-living heterotrophic flagellate Dysnectes brevis n. gen., n. sp., a new member of the Fornicata. J. Eukaryot. Microbiol.54, 191–200. ( 10.1111/j.1550-7408.2007.00252.x) [DOI] [PubMed] [Google Scholar]
• 35.Yabuki A, et al. 2018. Ophirina amphinema n. gen., n. sp., a new deeply branching discobid with phylogenetic affinity to jakobids. Sci. Rep.8, 16219. ( 10.1038/s41598-018-34504-6) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Janouškovec J, et al. 2017. A new lineage of eukaryotes illuminates early mitochondrial genome reduction. Curr. Biol.27, 3717–3724. ( 10.1016/j.cub.2017.10.051) [DOI] [PubMed] [Google Scholar]
• 37.Foissner W, et al. 1988. The Hemimastigophora (Hemimastix amphikineta nov. gen., nov. spec.), a new protistan phylum from gondwanian soils. Eur. J. Protistol.23, 361–383. ( 10.1016/S0932-4739(88)80027-0) [DOI] [PubMed] [Google Scholar]
• 38.Foissner I, Foissner W. 1993. Revision of the family Spironemidae Doflein (Protista, Hemimastigophora), with description of two new species, Spironema terricola n. sp. and Stereonema geiseri n. g., n. sp. J. Eukaryot. Microbiol.40, 422–438. ( 10.1111/j.1550-7408.1993.tb04936.x) [DOI] [Google Scholar]
• 39.Cooney EC, et al. 2023. Photosystems in the eye-like organelles of heterotrophic warnowiid dinoflagellates. Curr. Biol.33, 4252–4260. ( 10.1016/j.cub.2023.08.052) [DOI] [PubMed] [Google Scholar]
• 40.Shiratori T, et al. 2015. A new deep-branching Stramenopile, Platysulcus tardus gen. nov., sp. nov. Protist166, 337–348. ( 10.1016/j.protis.2015.05.001) [DOI] [PubMed] [Google Scholar]
• 41.Yubuki N, et al. 2010. Ultrastructure and molecular phylogenetic position of a novel phagotrophic stramenopile from low oxygen environments: Rictus lutensis gen. et sp. nov. (Bicosoecida, incertae sedis). Protist161, 264–278. ( 10.1016/j.protis.2009.10.004) [DOI] [PubMed] [Google Scholar]
• 42.Keeling PJ, Burki F. 2019. Progress towards the tree of eukaryotes. Curr. Biol.29, R808–R817. ( 10.1016/j.cub.2019.07.031) [DOI] [PubMed] [Google Scholar]
• 43.Cho A, et al. 2022. Monophyly of diverse bigyromonadea and their impact on phylogenomic relationships within Stramenopiles. Mol. Phylogenet. Evol.171, 107468. ( 10.1016/j.ympev.2022.107468) [DOI] [PubMed] [Google Scholar]
• 44.Cho A, et al. 2024. Phylogenomic position of genetically diverse phagotrophic stramenopile flagellates in the sediment-associated MAST-6 lineage and a potentially halotolerant placididean. Mol. Phylogenet. Evol.190, 107964. ( 10.1016/j.ympev.2023.107964) [DOI] [PubMed] [Google Scholar]
• 45.Andersen RA. 1991. The cytoskeleton of chromophyte algae. Protoplasma164, 143–159. ( 10.1007/BF01320820) [DOI] [Google Scholar]
• 46.O’Kelly CJ, Floyd GL. 1985. Absolute configuration analysis of the flagellar apparatus in Giraudyopsis stellifer (Chrysophyceae, Sarcinochrysidales) zoospores and its significance in the evolution of the Phaeophyceae. Phycologia24, 263–274. ( 10.2216/i0031-8884-24-3-263.1) [DOI] [Google Scholar]
• 47.Takahashi T, et al. 2015. Ultra-high voltage electron microscopy of primitive algae illuminates 3d ultrastructures of the first photosynthetic eukaryote. Sci. Rep.5, 14735. ( 10.1038/srep14735) [DOI] [PMC free article] [PubMed] [Google Scholar]
• 48.Lindberg RE, Bovee EC. 1975. Induction of phagocytosis and 'cannibalism' by the giant ameba, Chaos carolinensis. J. Protozool.23, 349–355. ( 10.1111/j.1550-7408.1976.tb03782.x) [DOI] [Google Scholar]
• 49.Fenchel T. 1987. Ecology of protozoa: the biology of freeliving phagotrophic protists. Berlin: Springer. [Google Scholar]
• 50.Sleigh MA. 2000. Trophic strategies. In The Flagellates: Unity, Diversity and Evolution, pp. 147–165. London: Taylor & Francis. [Google Scholar]
• 51.Cavalier-Smith T, et al. 2009. Helkesimastix marina n. sp. (Cercozoa: Sainouroidea superfam. n.) a gliding zooflagellate of novel ultrastructure and unusual ciliary behaviour. Protist160, 452–479. ( 10.1016/j.protis.2009.03.003) [DOI] [PubMed] [Google Scholar]
• 52.Hehenberger E, et al. 2017. Novel predators reshape holozoan phylogeny and reveal the presence of a two-component signaling system in the ancestor of animals. Curr. Biol.27, 2043–2050. ( 10.1016/j.cub.2017.06.006) [DOI] [PubMed] [Google Scholar]
• 53.Luporini P, et al. 2015. Ciliate pheromone structures and activity: a review. Ital. J. Zool.82, 3–14. ( 10.1080/11250003.2014.976282) [DOI] [Google Scholar]
• 54.Belyaev AO, Karpov SA, Keeling PJ, Tikhonenkov DV. 2024. The nature of ‘jaws’: a new predatory representative of Provora and the ultrastructure of nibbling protists. Figshare ( 10.6084/m9.figshare.25998964) [DOI] [PubMed]
• 55.Belyaev AO, Karpov SA, Keeling PJ, Tikhonenkov DV. 2024. . Supplementary material from: The nature of ‘jaws’: a new predatory representative of Provora and the ultrastructure of nibbling protists. Figshare ( 10.6084/m9.figshare.c.7502975) [DOI] [PubMed]

Data Availability Statement

All data are available in the main text and on Figshare [54].

Zoobank Registration. urn:lsid:zoobank.org:act:47C60B3B-D3AE-485C-AA63-DNA sequences: GenBank accession numberPQ417912.

Supplementary material is available online [55].