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. 2026 Apr 16;73(3):e70080. doi: 10.1111/jeu.70080

Morphology, Ultrastructure, and Classification of Barthelonids (Metamonada Incertae Sedis): With Descriptions of Microbarthelona Gen. Nov., and Parabarthelona Gen. Nov.

Takashi Shiratori 1,✉, Yana Eglit 2,3, Euki Yazaki 4,5, Yuji Inagaki 6, Alastair G B Simpson 2
PMCID: PMC13087550  PMID: 41992732

ABSTRACT

Barthelona is a genus of anaerobic flagellates that forms a sister lineage to Fornicata along with Skoliomonas. Although “Barthelona spp.” are known to separate into three distinct lineages, their detailed morphology has not been examined, and their taxonomic classification is incomplete. In this study, we investigated the morphology, especially ultrastructure, of three cultures of barthelonids, each representing one lineage. These flagellates possess an inconspicuous ventral groove and a highly elongated cytopharynx that originates near the posterior end of the cell and extends toward the anterior end. Their posterior flagellum lacks a vane. The flagellar apparatus shares some features with those of other excavate flagellates in possessing microtubular roots R1, split R2 and R3, but lacks a singlet root, B fiber and discernable C fiber. Notably, R2 splits near the posterior end of the cell. Cell size and the number of microtubules comprising R1 and R2 vary among strains. Based on these observations, we describe two new genera and species, Microbarthelona vorax gen. nov., sp. nov. and Parabarthelona vacuolata gen. nov., sp. nov. Additionally, we propose the following higher taxa for Barthelona, Microbarthelona, and Parabarthelona: Notopharyngea cl. nov., Barthelonida ord. nov., and Barthelonidae fam. nov.

Keywords: Barthelona, electron microscopy, Excavata, flagellar apparatus, Fornicata, Notopharyngea

1. Introduction

Metamonada is one of the major eukaryotic lineages, and consists exclusively of anaerobic protists (Cavalier‐Smith 2003). All metamonads lack typical (aerobic) mitochondria; instead, most possess highly reduced forms, such as hydrogenosomes or mitosomes (Tachezy et al. 2022; Tovar et al. 2003). There are also two probable‐to‐certain cases of complete loss of mitochondria within the group (Karnkowska et al. 2016; Williams et al. 2024). Metamonada was previously classified within a supergroup Excavata, together with Discoba and Malawimonadida, based primarily on morphological similarities such as the ventral feeding groove and a complex flagellar apparatus (Simpson 2003; Simpson and Patterson 1999). However, recent phylogenomic analyses have failed to support the monophyly of Excavata, instead placing these three lineages as two or three separate deep branches in the eukaryotic tree (Brown et al. 2018; Yazaki et al. 2022; Williamson et al. 2025). These phylogenetic insights and comparisons of the flagellar apparatus across major eukaryotic lineages suggest that an excavate‐like ventral feeding groove and a complex flagellar apparatus may be ancestral traits of eukaryotes (Keeling and Burki 2019; Suzuki‐Tellier, Miano, et al. 2024; Williamson et al. 2025); this highlights the importance of excavate lineages, including Metamonada, in reconstructing early eukaryotic cell organization. In parallel, recent molecular phylogenetic analyses using archaeal‐derived genes proposed Metamonada as the earliest‐branching group of eukaryotes (Al Jewari and Baldauf 2023). More recent analyses of genes of bacterial/mitochondrial origin could not place metamonads reliably, and overall support a different position for the eukaryotic root (Williamson et al. 2025); nonetheless, Metamonada clearly remains a key lineage for elucidating eukaryotic evolution.

Metamonada includes three well‐established subgroups, Fornicata, Preaxostyla, and Parabasalia (Simpson 2003), plus the recently proposed phylum Anaeramoebae that includes a single genus, Anaeramoeba (Stairs et al. 2021; Táborský et al. 2017), and two poorly known flagellates, Barthelona and Skoliomonas (see below). Fornicata includes Diplomonadida, a group predominantly composed of parasitic species such as the human parasite Giardia duodenalis (Adam 2021). Most diplomonads possess paired nuclei, each associated with a flagellar apparatus (Adam 2017; Brugerolle and Lee 2000a). Fornicata also contains various free‐living bacterivorous biflagellates known as Carpediemonas‐like organisms (CLOs) (Kolisko et al. 2010), the uniflagellate caviomonads (Vargová et al. 2022), and a small assemblage of intestinal commensals, Retortamonadida (Kulda et al. 2017). Retortamonads and CLOs (excluding caviomonads) retain a typical excavate ventral feeding groove and flagellar apparatus (Simpson 2003). Preaxostyla includes Oxymonadida, a group of morphologically diverse gut symbionts (Brugerolle and Lee 2000b), and the free‐living tetraflagellate groups Trimastix and Paratrimastix with typical excavate features (Hampl 2017). In contrast, Parabasalia lacks a ventral groove and possesses a highly derived flagellar apparatus, making many homologies with other excavates unclear (Simpson 2003). This group mainly consists of parasitic and symbiotic species, including Trichomonas vaginalis, a human pathogen, as well as multiflagellated symbionts of wood‐eating termites (Brugerolle and Lee 2000c; Čepička et al. 2017; Gile 2024).

Barthelona is a genus of free‐living anaerobic biflagellates with a characteristic J‐shaped cytoskeletal feature that largely defines the cell shape (Bernard et al. 2000). The J‐structure gives the cell a subtle ventral groove (rather than a broad and deep groove) while the curve of the J defines a deep furrow in the posterior margin of the cell that terminates in a long, thin cytopharynx (Yazaki et al. 2020; Eglit et al. 2024). Recent studies, which included the first stable cultivations of several “Barthelona spp.” found that they form three distinct lineages and branch as a sister group to Fornicata (Yazaki et al. 2020). Very recently, a sister group for barthelonids, skoliomonads, was discovered; together these two assemblages form a clade informally known as “BaSk” (Eglit et al. 2024; Williams et al. 2024; Yazaki et al. 2025). Despite the genetic diversity of barthelonids, only one species, B. vulgaris, has been formally described, solely from light microscopic observations (Bernard et al. 2000). Thus, the morphological diversity and ultrastructure of barthelonids remain unknown.

In this study, we performed light and electron microscopic observations on three cultured isolates originally reported as “Barthelona spp.”, each representing one of the three distinct lineages identified in Yazaki et al. (2020). Our results show that the flagellar and feeding apparatuses of “Barthelona spp.” differ markedly from those of other excavate flagellates and also exhibit some variation among isolates. Based on our observations, we propose a revised taxonomic classification of barthelonids and discuss character evolution within Metamonada.

2. Materials and Methods

2.1. Small Subunit Ribosomal DNA Phylogeny

To illustrate the phylogenetic relationships among barthelonids, we conducted phylogenetic analyses using small subunit ribosomal DNA (SSU rDNA). To construct the SSU rDNA dataset, we collected sequences of barthelonids (LC506386.1–LC506390.1) and representative other metamonads from the NCBI database. In addition, we recovered the SSU rDNA sequence of Barthelona sp. PAP514 (Yazaki et al. 2025) from the transcriptome assembly by BLASTn search using barthelonids sequences as queries. To incorporate environmental sequences of barthelonids, we performed BLASTn searches against the EukBank global dataset (Berney et al. 2023) using barthelonids sequences as queries, applying the following criteria: E‐value ≤ 1e−30, sequence length > 300 bp, and identity > 90%. All SSU rDNA sequences were aligned using MAFFT v.7520 (Katoh and Standley 2013) with the linsi option, and ambiguously aligned positions were removed with BMGE v.1.12 (Criscuolo and Gribaldo 2010) with default settings. Then, the alignments were further inspected by eye and the trimming adjusted manually. The final dataset comprised 126 OTUs, including 14 barthelonid sequences, with 1258 nucleotide positions. This dataset was subjected to maximum‐likelihood (ML) phylogenetic analysis with IQ‐TREE v.3.0.1 (Wong et al. 2025). The best‐fit substitution model (TIM2 + F + R6) was determined by ModelFinder implemented in IQ‐TREE. Branch support was assessed with a nonparametric ML bootstrap method (100 replicates) and SH‐aLRT (1000 replicates). Bayesian phylogenetic analyses were performed using MrBayes v.3.2.7a (Ronquist and Huelsenbeck 2003). Two parallel MCMCMC runs (one cold and three heated chains) were executed for 10,000,000 generations under the GTR + Γ model, sampling every 1000 generations. A flat Dirichlet prior was applied to nucleotide frequencies. Convergence was monitored using the built‐in stopping rule (ASDSF < 0.01), and the first 25% of samples were discarded as burn‐in. The remaining trees from both runs were summarized to obtain Bayesian posterior probabilities.

2.2. Light and Electron Microscopy

The cultivation of isolates EYP1702, PAP020, and PCE was reported in Yazaki et al. (2020). In brief, PAP020 and EYP1702 were isolated from anaerobic mangrove sediments collected in a marine lake in the Republic of Palau in November 2011 and October 2017, respectively. PCE was isolated from intertidal sediment near Cavendish, PEI, Canada, in July 2016. Monoprotistan cultures of barthelonids with a mixed bacterial community derived from the original samples were maintained in 5% mTYGM‐9 medium (https://mcc.nies.go.jp/02medium.html#mtygm9) prepared with sterile natural seawater at 20°C.

For light microscopy of EYP1702 and PAP020, cells were mounted on glass slides and observed using an Axio Imager A2 microscope (Zeiss) equipped with a DP73 CCD camera (Olympus). Cells of isolate PCE were incubated in a Vaseline‐sealed chamber slide overnight and imaged on a Zeiss AxioVert 200 M inverted microscope using DIC optics and a 100× objective, with a Zeiss Axiocam HRc camera (Zeiss).

For scanning electron microscopy (SEM), cells of PAP020 were prefixed using 2% (w/v) glutaraldehyde in autoclaved natural seawater for 1 h at room temperature. During this fixation they were allowed to settle on 8.5‐mm diameter glass SEM plates (Okenshoji Co.) previously coated with 0.1% (w/v) poly‐L‐lysine (Sigma Chemical Co.) for 1 h at room temperature. Cells on the glass plate were subsequently postfixed with 1% (w/v) OsO4 for 1 h at room temperature. Cells were then gradually dehydrated through an ethanol series of 15%–100% ethanol. After dehydration, the specimen was placed in a 1:1 mixture of 100% ethanol and 100% t‐butyl alcohol, which was subsequently replaced with 100% t‐butyl alcohol. The specimen was then frozen in a freezer and freeze‐dried using a VFD‐21S freeze‐drier (SHINKU‐DEVICE). The specimens were coated with platinum–palladium using a Hitachi E1045 (Hitachi High‐Technologies Corp.) and observed under a JSM‐6360F field emission SEM (JEOL).

For transmission electron microscopy (TEM), we used specimens of PAP020 prepared in Yazaki et al. (2020). Specimens of EYP1702 and PCE were newly prepared according to the same protocol described by Yazaki et al. (2020). Ultrathin sections (80 nm thick) of each specimen were cut on a Reichert Ultracut S ultramicrotome (Leica), double stained with 2% (w/v) uranyl acetate and lead citrate, and observed using a Hitachi H‐7650 electron microscope (Hitachi High‐Technologies Corp.) equipped with a Veleta TEM CCD camera (Olympus).

3. Results

3.1. Molecular Phylogenetic Analysis

In the SSU rDNA tree of Metamonada, barthelonids formed a clade with a SH‐aLRT support value of 99.6% and an ML bootstrap support value of 79%. In the Barthelonida clade, we identified three robust subclades (Figure 1, Figure S1). (i) Parabarthelona vacuolata gen. nov., sp. nov. PAP020, two undescribed strains (LM2 and FB11 isolated from mud of a defunct saltern and intertidal mud flat, respectively), and three metabarcoding sequences found in EukBank grouped together. (ii) Barthelona vulgaris EYP1702 clustered with a strain PAP514 isolated from the sediment of a marine lake and four metabarcoding sequences. (iii) Microbarthelona vorax gen. nov., sp. nov. PCE and one metabarcoding sequence formed the third subclade, distinct from the former two described above. The subclade represented by P. vauolata and that represented by M. vorax formed a very weakly supported clade (78.4% SH‐aLRT support; 54% ML bootstrap support), excluding Barthelona strains and their related metabarcoding sequences. Based on the SSU rDNA phylogeny, we could not resolve the relationship among the three subclades in the Barthelonida clade. All the metabarcoding sequences that showed close affinities to the three representative barthelonids were derived from various marine sediments collected from 1 to 2000 m depth (Table S1). Barthelonids as a whole formed a clade with skoliomonads (previously “BaSk,” now Notopharyngea cl. nov., see discussion) with a SH‐aLRT support value of 84.5% and an ML bootstrap support value of 52% (Figure 1, Figure S1). This same clade is much more strongly supported in phylogenomic analyses (see discussion).

FIGURE 1.

FIGURE 1

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Maximum‐likelihood (ML) tree of a small subunit (SSU) rDNA gene alignment. The ML tree was inferred from 126 OTUs, including 14 barthelonid sequences, with 1258 nucleotide positions. SH‐aLRT values and ML bootstrap values are presented for nodes within Notopharyngea cl. nov. For other clades, SH‐aLRT values or ML bootstrap values (MLBPs) ≤ 80% or Bayesian posterior probabilities (BPPs) ≤ 0.8 are omitted. Nodes marked with open circles indicate SH‐aLRT values = 100%, MLBPs = 100% and BPPs = 1.00. Nodes marked with filled circles indicate SH‐aLRT values > 80%, MLBPs > 80%, and BPPs > 0.8.

3.2. Light Microscopy

Cells of P. vacuolata PAP020, M. vorax PCE, and B. vulgaris EYP1702 are oval with slightly pointed posterior ends (Figure 2). P. vacuolata PAP020 cells measure 7.1 ± 1.0 μm (5.5–9.0 μm) in length and 4.7 ± 0.9 μm (3.4–6.8 μm) in width (n = 34); M. vorax PCE cells measure 4.7 ± 0.5 μm (3.8–5.5 μm) in length and 2.9 ± 0.3 μm (2.3–3.6 μm) in width (n = 24); and B. vulgaris EYP1702 cells measure 7.6 ± 0.5 μm (6.9–8.3 μm) in length and 5.0 ± 0.2 μm (4.7–5.3 μm) in width (n = 7). All isolates have two unequal heterodynamic flagella emerging subapically from the ventral side of the cell (Figure 2A,D,G,I,J,L). The short anterior flagellum is approximately equal to or slightly longer than the cell length, while the long posterior flagellum is 1.5–4 times, 2.1–3.7 times, and 2.5–4 times the cell length in P. vacuolata PAP020, M. vorax PCE, and B. vulgaris EYP1702 respectively (Figure 2A,D,G,K–N). Cells swim with a vibrating motion or attach to substrates using the posterior flagellum. A narrow inconspicuous groove is present on the ventral side, extending from the flagellar insertion to the posterior portion of the cell (Figure 2A,I,J,N). The ventral groove continues as a posterior furrow, which sharply divides the posterior end of the cell (Figure 2A,C,D,H,I,L,M). The groove and posterior furrow can be observed as a J‐shaped structure in lateral view. The dorsal end of the posterior furrow continues into the cell as an elongated cytopharynx that extends anteriorly along the dorsal side, reaching nearly the anterior end (Figure 2D,E,H,I,L–N). Cells contain several food vacuoles (Figure 2A,B,F,L). Parabarthelona vacuolata PAP020 has large spherical food vacuoles that are mostly filled with fluid and contain only sparse bacterial material (Figure 2B,D,E), whereas those of M. vorax PCE and B. vulgaris EYP1702 are more densely packed.

FIGURE 2.

FIGURE 2

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Light micrographs of Barthelonids. (A–E) Parabarthelona vacuolata PAP020. (F–J) Microbarthelona vorax PCE. (K–O) Barthelona vulgaris EYP1702. Scale bars = 5 μm. Arrowheads indicate food vacuoles. AF, anterior flagellum; Cy, cytopharynx; F, posterior furrow; PF, posterior flagellum; VG, ventral groove.

3.3. Scanning Electron Microscopy

Scanning electron microscopy of P. vacuolata PAP020 showed that the flagella emerge on the right side of the ventral face of the cell (Figure 3A). A cytoskeletal band running just beneath the ventral surface of the cell was also observed. It originates at the base of the posterior flagellum and extends posteriorly (Figure 3A). An arc‐shaped ridge arises between the base of the anterior and posterior flagella and extends posteriorly (Figure 3A). Transmission electron microscopy suggests that the cytoskeletal band and the arc‐shaped ridge correspond to microtubular roots R2 and R1, respectively (see below). The posterior end of the cell splits into a larger left and smaller right portion, with the division representing the posterior furrow (Figure 3A). Both flagella are naked and lack vanes or other appendages (Figure 3A).

FIGURE 3.

FIGURE 3

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Scanning and transmission electron micrographs of Parabarthelona vacuolata PAP020. (A, B) Scanning electron micrograph of whole cells. (C) TEM of a mitochondrion‐related organelle (MRO). (D) Transmission electron micrograph (TEM) of a whole cell. (E) TEM of the two basal bodies. (F–H) TEM of selected serial sections showing B1 and associated microtubular roots and fibers. (I) TEM of cytopharynx reaching the anterior end of the cell. (J) TEM of Golgi region located just beneath R2. (K) TEM of distal end of R2 showing microtubules linked to each other by bridges, and its proximity to basal bodies. The image also shows proximal section of R1. A, A fiber; AF, anterior flagellum; Cy, cytopharynx; F, posterior furrow; FV, food vacuole; G, Golgi‐like structure; I, I fiber; N, nucleus; PF, posterior flagellum. Black arrow indicates an axosome. Black double arrowhead indicates MRO. White double arrowheads indicate a cytoskeletal band. White pointed arrowheads indicate an arc‐shaped ridge. Scale bars: (A, B, I) = 1 μm, (C, D, G, H, J) = 500 nm.

3.4. Transmission Electron Microscopy

To describe the flagellar apparatus of P. vacuolata PAP020, M. vorax PCE, and B. vulgaris EYP1702, we used the universal terminology for basal bodies and microtubular roots (Moestrup 2000; Yubuki and Leander 2013). Although flagellar transformation in these strains was not confirmed, we identified the anterior basal body as B2 and the posterior basal body as B1, which is the common arrangement in excavate flagellates.

Cells of P. vacuolata PAP020, M. vorax PCE, and B. vulgaris EYP1702 are covered by a plasma membrane, and no extracellular structures were observed (Figures 3B, 5A, 7A). The flagella lack vanes or other appendages (Figures 3F, 5B, 7O,P). In P. vacuolata PAP020, the flagella were occasionally observed embedded within the cytoplasm, likely due to fixation artifacts (Figure 4J–N). Membrane‐bounded, electron‐dense structures that are probably MROs are scattered throughout the cells of P. vacuolata PAP020 and B. vulgaris EYP1702, and are more rarely observed in the cells of M. vorax PCE (Figures 3B,C, 5C, 7M, 8B). A single nucleus is positioned in the anterior region of the cell, and the two basal bodies are located on the right side of the nucleus (Figures 3B,D–G, 5A,E–G, 7A–F). The two basal bodies lie in the same plane at a slightly acute angle (Figures 3G, 4A–C, 5D, 7G,H). Basal bodies are short, with the triplet microtubules terminating at a length of approximately 250 nm (Figure S3). The central pair of flagellar microtubules originates from a dense axosome located slightly below the level of the plasma membrane. Thus, the transitional region is completely embedded within the cytoplasm (Figures 3G, 4A,B, 7E, Figure S3). Obvious transitional fibers were not observed. A poorly structured Golgi region is positioned at the posterior ventral side of the nucleus, just beneath microtubular root R2 (Figures 3I, 5J,K, 7A). An elongated cytopharynx originates at the posterior end of the cell and extends anteriorly through the cytoplasm (Figures 4J–O, 5A, 7A). The cytopharynx continues along the dorsal side of the cell toward the anterior end, terminating close to the basal bodies (Figures 3H, 5A,G,H, 7C, 8I–K). Several food vacuoles that contain bacteria and their digestive residues were observed: they are large, spherical, and predominantly empty in P. vacuolata PAP020 (Figure 3B,H), whereas in M. vorax PCE and B. vulgaris EYP1702, food vacuoles are filled with particulate contents (Figures 5A,H, 7A).

FIGURE 5.

FIGURE 5

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Transmission electron micrographs (TEM) of Microbarthelona vorax PCE. (A) Longitudinal section through whole cell. (B) Two flagella. (C) Mitochondrion‐related organelle (MRO). (D) Two basal bodies with associated microtubular roots and array “internal microtubules” (Imt). (E) Proximal end of R2. (F) Proximal end of R1. (G) Distal end of cytopharynx. (H) Longitudinal section of cytopharynx reaching the anterior end of the cell. (I–L) Selected serial sections showing the anterior portion of cell, including R1, R2, and R3. A, A fiber; AF, anterior flagellum; Cy, cytopharynx; FV, food vacuole; G, Golgi‐like structure; I, I fiber; Imt, internal microtubule; N, nucleus; PF, posterior flagellum. Double arrowhead indicates MRO. Scale bars: (A, H) = 1 μm, (B, D, E, F, G, I) = 500 nm, (C) = 200 nm.

FIGURE 7.

FIGURE 7

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Transmission electron micrographs (TEM) of Barthelona vulgaris EYP1702. (A) Whole cell. (B) Two basal bodies. (C) Distal end of cytopharynx. (D–F) Selected serial sections showing proximal end of R2 and, obliquely, of R1. (G–J) Selected serial sections showing origins of R1, R2, R3 and internal microtubules (Imt); note Golgi region located just beneath R2. (K–P) Selected serial sections of the ventral side of the cell showing paths of R1 and R2, plus R2 splitting into iR2 and oR2. A, A fiber; AF, anterior flagellum; Cy, cytopharynx; FV, food vacuole; G, Golgi‐like structure; I, I fiber; Imt, internal microtubule; N, nucleus; PF, posterior flagellum. Arrow indicates an axosome. Double arrowhead indicates MRO. Scale bars: (A, K) = 1 μm, (B, C, D, G) = 500 nm.

FIGURE 4.

FIGURE 4

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Transmission electron micrographs (TEM) of Parabarthelona vacuolata PAP020. (A–D) Selected serial sections showing two basal bodies and associated microtubular roots. (E) B1 and associated microtubular roots. (F) Dorsal region of cell with ascending iR2 and oR2 microtubules surrounding cytopharynx. (G) Dorsal region with ascending iR2 and oR2 microtubules, but no visible cytopharynx. (H, J–O) Selected serial sections showing approximately transverse view of the posterior region of the cell. (I) High magnification view of (H), showing cytopharynx and supporting oR2‐derived microtubules. AF, anterior flagellum; CF, composite fiber; Imt, internal microtubule; FV, food vacuole; PF, posterior flagellum. Arrows indicate axosomes. Double arrowhead indicates MRO. Scale bars: (A, F, G) = 500 nm, (E, H) = 1 μm, (I) = 200 nm.

FIGURE 8.

FIGURE 8

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Transmission electron micrographs (TEM) of Barthelona vulgaris EYP1702. (A–H) Selected serial sections of an approximately transverse view of the posterior region of the cell, highlighting the origin and structure of the cytopharynx. (I–K) Selected serial sections showing an approximately longitudinal view of the cell. CF, composite fiber; Cy, cytopharynx; FV, food vacuole; N, nucleus. Arrows indicate additional microtubules originating near the CF. Double arrowhead indicates MRO. Scale bars: (A) = 500 nm, (I) = 1 μm.

All three isolates possess three microtubular roots: R1, R2, and R3. R1 consists of two microtubules in P. vacuolata PAP020 and M. vorax PCE, but only a single microtubule in B. vulgaris EYP1702 (Figures 3J, 5F, 7D–F). It originates near the anterior‐ and left‐most side of B1 (Figures 3D, 5E,F, 7D–F). In P. vacuolata PAP020 and M. vorax PCE, R1 initially curves leftward before extending posteriorly along the ventral surface of the cell, where it merges with iR2 (see below) and runs alongside it before terminating at the posterior end of the cell (Figures 4E,H–M, 5I–L, 6A–J). In B. vulgaris EYP1702, R1 curves leftward and terminates at the intersection with iR2 about halfway down the cell (Figure 7L–O).

FIGURE 6.

FIGURE 6

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Transmission electron micrographs (TEM) of Microbarthelona vorax PCE. (A–D) Selected serial sections of the ventral side of the cell showing R2 splitting into iR2 and oR2. (E–H) Selected serial sections of the posterior end of the cell showing the proximal end of the cytopharynx. (I–L) Selected serial sections of the posterior end of the cell showing the proximal end of the cytopharynx. (M–O) Selected serial sections of the posterior region of the cell, cut longitudinally. CF, composite fiber; Cy, cytopharynx; FV, food vacuole. Scale bars: (A, E, I, M) = 500 nm.

R2 originates alongside the posterior‐most side of B1 and immediately after its origin consists of 7–8 (PAP020) or 5–6 (PCE and EYP1702) microtubules (Figures 3D–F, 5E, 7D–F). Two distinct nonmicrotubular elements, the A fiber and I fiber, are associated with R2 at its proximal end on the inner (convex) and outer (concave) faces of R2 respectively (Figures 3D,E, 5E, 7E,F). Both fibers are connected to the posterior side of B1 (Figures 3D, 5E, 7E). R2 extends posteriorly along the ventral surface of the cell (Figures 4E, 5J–L, 7A,H–J). Toward the posterior end of the cell, R2 splits into iR2 and oR2. The iR2 consists of four (PAP020 and EYP1702) or two (PCE) microtubules on the left side of R2, whereas oR2 is composed of the remaining microtubules (Figures 4H,J, 6A–D, 7K–P). At the posterior end of the cell, both iR2 and oR2 curve toward the dorsal side along the cell surface (Figures 4H–O, 6E–H, 8A–H). At this point, oR2 increases in microtubule number and spreads out, curving gently to the right, and the posterior furrow is formed between iR2 and oR2 (Figures 4H–O, 6E–H, 8A–H). The iR2 and oR2 converge again at the beginning of the cytopharynx and run parallel to the cytopharynx (Figures 4H–O, 6E–H, 8A–H). The oR2 encircles the cytopharynx and each microtubule is connected by bridges (Figure 4I). In P. vacuolata PAP020, iR2 is positioned interior to the cytopharynx (Figure 4K), whereas in M. vorax PCE and B. vulgaris EYP1702, iR2 is also positioned interiorly, but more distant from the cytopharynx (Figures 6L, 8B). The spacing between oR2 microtubules encircling the cytopharynx varies among cells (Figure 4F,I). Some cells lack a cytopharynx, with only the supporting microtubules observed (Figure 4G). At the beginning of the cytopharynx, an arched structure that we provisionally identify as a composite fiber (CF) is associated with oR2 and iR2 (Figures 4F,L–O, 6G,H,K,L, 8G). The CF supports part of the outer margin of the cytopharyngeal opening (Figure 6K,M–O; 8G, H). In P. vacuolata PAP020 and M. vorax PCE, the CF shows a faint striated pattern, whereas in B. vulgaris EYP1702, it is well‐developed with distinct striations. In P. vacuolata PAP020 and M. vorax PCE, R1 runs together with iR2 at the posterior portion of the cell but terminates near the opening of the cytopharynx. In B. vulgaris EYP1702, additional microtubules originate from the CF and run along the cytopharynx (Figure 8A–F).

R3 is a short root consisting of two microtubules, originating from the dorsal side of B2 and extending anteriorly (Figures 3G, 4B, 5D,G, 7H,I). Eight or more short “internal” microtubules originate from the region near the proximal ends of the basal bodies, especially B2 (Figures 4A,B, 5D,L, 7J). The flagellar apparatus of P. vacuolata PAP020 is illustrated in Figure 9. In P. vacuolata PAP020, cells with four basal bodies were occasionally observed, which was considered to represent basal body duplication prior to cell division. These cells had R2 with an additional band of secondary microtubules and extended internal microtubules (Figure S2).

FIGURE 9.

FIGURE 9

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Illustration of the flagellar apparatus of Parabarthelona vacuolata PAP020. (A) Proximal part of the flagellar apparatus, viewed from the right dorsal side. (B) Overview of the cell cytoskeleton, viewed from the right ventral side. Green and red area indicate left and right portion of the cell respectively. A, A fiber; CF, composite fiber; Cy, cytopharynx; I, I fiber; Imt, internal microtubule.

4. Discussion

Barthelona is a genus of anaerobic flagellates that currently comprises a single species, B. vulgaris , which was described based on light microscopy of mixed cultures derived from marine sediment collected in Australia (Bernard et al. 2000). Molecular phylogenetic analyses by Yazaki et al. (2020) revealed that five isolates of “Barthelona spp.” belong to Metamonada as a sister lineage to Fornicata and form three distinct lineages in SSU rDNA trees. More recently, skoliomonads, a group of anaerobic flagellates isolated from alkaline lakes, were shown to be closely related to barthelonids, and together these two groups form a robust clade in phylogenomic analyses that was referred to as “BaSk” (Eglit et al. 2024; Williams et al. 2024; Yazaki et al. 2025). Our phylogenetic analysis of SSU rDNA sequences also recovered the three subclades of barthelonids, as well as the sister relationship to skoliomonads; the weak support for the latter is consistent with previous analyses with this single marker (Eglit et al. 2024). The survey of barthelonid metabarcoding sequences in the EukBank database suggested that barthelonids are globally distributed in saline mud and sediments, ranging from tidal flats to the deep sea (Table S1). Based on molecular phylogenetic analyses and the morphological similarities between barthelonids and skoliomonads, such as the cytopharynx extending back up the dorsal side of the cell (Eglit et al. 2024), we propose a new class, Notopharyngea cl. nov., for the BaSk clade.

In this study, we performed light and electron microscopy observations on three strains previously referred to as “Barthelona spp.” (PAP020, PCE, and EYP1702), each representing one of the three subclades. These three strains share a characteristic structure referred to as the “J‐shaped cytoskeleton,” which is the most distinctive morphological feature of barthelonids. Our microscopic observations revealed that this “J‐shaped cytoskeleton” corresponds to a series of structures supporting the subtle groove on the ventral side of the cell, the posterior depression, and the origin of the cytopharynx; this organization is based primarily on the R2 microtubular root that, interestingly, splits into two parts near the posterior end of the cell. PAP020 and EYP1702 are morphologically similar to the original description of B. vulgaris in terms of cell size and flagellar length. A key difference between these two strains is in the appearance of their food vacuoles under light microscopy: EYP1702 possesses food vacuoles filled with particulate contents, resembling the refractile granules reported for B. vulgaris (Bernard et al. 2000), whereas PAP020 has larger, spherical food vacuoles containing a large volume of fluid as well as enclosed particles. Based on these morphological features, we identified EYP1702 as an isolate of B. vulgaris , and PAP020 as a new taxon (see below). EYP1702 also has some ultrastructural differences from PAP020 and PCE; it possesses a singlet R1 and a well‐developed composite fiber with a distinct striated pattern. PAP020 differs from both EYP1702 and PCE in possessing the large spherical food vacuoles mentioned above and a greater number of microtubules in R2. PCE is the smallest of the three strains, differing ultrastructurally from EYP1702 in the number of microtubules in R1, and from PAP020 in having fewer microtubules in R2. Based on these morphological and ultrastructural differences, in addition to the SSU rDNA data and trees, we propose the establishment of two new genera and species, Parabarthelona vacuolata gen. nov., sp. nov. for PAP020 and Microbarthelona vorax gen. nov., sp. nov. for PCE. Additionally, we propose new higher‐level taxa, Barthelonida ord. nov., and Barthelonidae fam. nov., to accommodate Barthelona, Microbarthelona, and Parabarthelona (see Taxonomic summary below).

The ventral groove is a feeding structure found in many excavate flagellates. It is positioned longitudinally along the ventral surface of the cell and is supported by multiple microtubular roots (Simpson 2003). In typical excavates, this structure facilitates food uptake primarily by increasing the feeding current generated by a vane‐bearing posterior flagellum, with bacteria captured within the groove and transported toward the cytopharynx (sensu lato) at the posterior end (Suzuki‐Tellier, Kiørboe, and Simpson 2024). Ventral grooves are widespread among Metamonada (Fornicata, Preaxostyla, and skoliomonads), yet barthelonids exhibit a much less distinct ventral groove and lack a flagellar vane. Since Skoliomonas possesses a conspicuous ventral groove (and a broad flagellar vane), the ventral groove in barthelonids likely represents a secondary reduction.

Compared to typical excavate flagellates, barthelonids also lack several subcomponents of the flagellar apparatus, including the B fiber. The B fiber associates mainly with R2, but also forms an arch with R1 at its origin in deep‐branching Fornicates, specifically CLOs and retortamonads (Table 1). The R1‐associated arched B fiber is considered a synapomorphy of Fornicata. In typical excavates, including CLOs, the B fiber directly supports the right wall of the most anterior part of the ventral groove, while R2 runs more interiorly at this level; only more posteriorly does oR2 directly support the plasma membrane of the right groove wall (Simpson and Patterson 1999; Park et al. 2009, 2010). Barthelonids also differ in the size of R2: R2 consists of up to eight microtubules, whereas in other groove‐bearing fornicates and preaxostylans, R2 contains over a dozen microtubules, sometimes several dozen (Yubuki et al. 2016; Zhang et al. 2015). At least one clade among CLOs, caviomonads, has secondarily lost the ventral groove, and these flagellates also exhibit a highly reduced flagellar apparatus (Vargová et al. 2022; Yubuki et al. 2017). The reduced flagellar apparatus in barthelonids may similarly reflect secondary reduction of their ventral groove. For example, the absence of the B fiber in barthelonids is consistent with the apparent function of the B fiber in helping to form a deep‐walled groove (see above). Although barthelonids lack the B fiber, further ultrastructural studies of skoliomonads, which possess prominent ventral grooves, are necessary to clarify the appropriate taxonomic placement of Notopharyngea within Metamonada.

TABLE 1.

Morphological and ultrastructural characteristics of barthelonids and other metamonads.

Taxon R1 Split R2 R3 Singlet root Dorsal fan A fiber B fiber C fiber I fiber Composite fiber Flagellar vane Ventral groove References
Fornicata Diplomonadida Trepomonas agilis + + + − + − − − + − − + (homology?) Simpson (2003)
Retortamonadida Chilomastix cuspidata + + − + − ? + + + + + + Bernard et al. (1997)
CLOs Kipferlia bialata + + + + + + + + + + + + Yubuki et al. (2013)
Caviomonadidae Caviomonas mobilis ? − − − + − − ? − − − − Yubuki et al. (2017)
Notopharyngea Barthelonida Barthelona vulgaris + + + − − + − − + + − + This study
EYP1702 (1 mt) (5–6 mt) (2 mt)
Microbarthelona vorax + + + − − + − − + + − + This study
PCE (2 mt) (5–6 mt) (2 mt)
Parabarthelona vacuolata + + + − − + − − + + − + This study
PAP020 (2 mt) (7–8 mt) (2 mt)
skoliomonad Skoliomonas litria ? ? ? ? ? ? ? ? ? ? + + Eglit et al. (2024)
Parabasalia Trichomonadida Trichomonas vaginalis − − − − ? (pelta) − − ? (costa) − − − − Simpson (2003)
Preaxostyla Trimastigida Trimastix marina + + + + + + + + + + + + Zhang et al. (2015)
Paratrimastigida Paratrimastix eleionoma + + + + + + + + + + + + Simpson et al. (2000)
Oxymonadida Monocercomonoides hausmanni + − + + ? (pelta) − + + + − − − Simpson (2003)
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R2 is the most conspicuous microtubular root in typical excavate flagellates. In most of these taxa it immediately splits into iR2 and oR2 at the proximal end, supporting the right portion of the ventral groove (Simpson 2003). In barthelonids, R2 also splits into iR2 and oR2. However, it initially extends posteriorly as a single microtubular band and only splits into iR2 and oR2 at a much more posterior position in the cell, after which iR2 and oR2 support the left and right edges of the posterior furrow respectively. This organization of R2 is distinctive for barthelonids. At the dorsal end of this posterior furrow, iR2 and oR2 are associated with the composite fiber, forming the opening of the cytopharynx. Although the composite fiber is consistently associated with iR2 and oR2 at the posterior end of the cell, its precise point of origin could not be determined from the available serial TEM sections. The cytopharynx is surrounded by microtubules and is highly elongated, terminating at the anterior end of the cell after passing back up most of the dorsal side. A similarly elongated and looping cytopharynx is observed in Skoliomonas (Eglit et al. 2024); in this case, the cytopharynx has a wider opening and originates within a broad and high‐sided ventral groove, but nonetheless extends back toward the anterior end of the cell within the dorsal portion of the cytoplasm. This suggests that the common ancestor of barthelonids and Skoliomonas possessed an elongated cytopharynx that extended to the anterior end of the cell. In contrast, most other excavate taxa either lack a distinct cytopharynx or possess a much shorter structure associated with the posterior end of the ventral groove. Among metamonads, retortamonads have the most similar cytopharynx to that of barthelonids and skoliomonads. In retortamonads, the cytopharynx has a large opening positioned at the posterior end of an often‐short ventral groove, curves leftward, and then extends anteriorly (Brugerolle 1973; Kulda et al. 2017), although it can continue to curve around to eventually run more posteriorly again (Bernard et al. 1997). The presence of a highly developed, anteriorly directed cytopharynx surrounded by linked microtubules in both barthelonids and retortamonads could indicate inheritance from a common ancestor of Fornicata. However, this would imply at least four independent losses among other lineages of fornicates (based on the phylogeny of Vargová et al. 2022). Alternatively, the similarity may reflect convergent evolution.

In non‐excavate flagellates, similar arrangements of the feeding apparatus have also been reported among stramenopiles, in which a split R2 supports a cytopharynx (Moestrup and Thomsen 1976; O'Kelly and Nerad 1998). However, in these taxa the cytopharynx is positioned either in the ventral side or near the anterior part of the cell. In addition, the stramenopile cytopharynx is typically supported by a loop of microtubules surrounding the cytopharyngeal opening, rather than by microtubules running parallel to the long axis of the cytopharynx. None of them exhibits a cytopharynx that originates at the extreme posterior end and extends anteriorly along the dorsal side of the cell, as seen in barthelonids. Thus, the organization and placement of the feeding apparatus in barthelonids appear to be unique among both metamonad and non‐metamonad protists. Vaned posterior flagella are widely observed across excavates (Simpson 2003). Within Metamonada, vaned flagella have been reported in Preaxostyla (in Trimastix and Paratrimastix), Fornicata (CLOs and retortamonads) and skoliomonads, suggesting strongly that this trait represents an ancestral character of Metamonada (Eglit et al. 2024; Simpson 2003; Yubuki et al. 2013). Barthelonids, however, prove to lack flagellar vanes, as previously suggested based on light microscopy observations (Eglit et al. 2024). Recent studies indicate that the vaned posterior flagellum efficiently generates the feeding current that moves food particles into the ventral groove, and that the broad groove closely associated with the vaned posterior flagellum is necessary for the vane to be effective (Suzuki‐Tellier, Kiørboe, and Simpson 2024). Indeed, most excavate flagellates with vaned posterior flagella also possess a well‐developed ventral groove (Simpson 2003; Yubuki et al. 2017). In this context, the absence of both a pronounced ventral groove and flagellar vanes in barthelonids suggests the presence of a different feeding mode from that of typical groove bearing excavates. Given that Skoliomonas retains both a well‐developed ventral groove and a vaned posterior flagellum (Eglit et al. 2024), it is plausible that barthelonids lost these structures after diverging from Skoliomonas, and with this they adopted a different mode of feeding than typical excavates.

The flagellar transitional region of barthelonids is positioned entirely beneath the plasma membrane: the central pair of the axoneme originates below the level of the flagellar insertion, and the triplet microtubules of the basal bodies are also unusually short. As a result, a comparatively long transitional region is embedded within the cytoplasm. Although such an embedded transitional region is relatively rare in other flagellates (Moestrup 1982), similar arrangements are common in Fornicata; they can be observed in CLOs (Simpson and Patterson 1999; Yubuki et al. 2013, 2016), Chilomastix (Bernard et al. 1997), and diplomonads (Brugerolle 1991). This feature is also observed in some other excavates, including malawimonads and some, but not all, jakobids (O'Kelly et al. 1999; Yabuki et al. 2018); by contrast the typical excavates Trimastix and Paratrimastix (Metamonada; Preaxostyla) have notably large transitional fibers projecting perpendicularly from the basal body to the cell membrane (Brugerolle and Patterson 1997; Simpson et al. 2000; Zhang et al. 2015), essentially forcing the transitional region to not be embedded. The embedded transitional region may represent an ancestral character associated with excavate morphology, though one that has been lost several times; alternatively, it could be a convergently derived feature, or a fixation artifact associated with this particular cell shape.

This study provides the first ultrastructural insights into the three subclades of barthelonids. Despite their molecular phylogenetic divergence, they share key ultrastructural features, including a reduced flagellar apparatus relative to typical excavates, an inconspicuous ventral groove, an extremely elongated cytopharynx, and a posterior furrow. The presence of an elongated, anteriorly running cytopharynx in both Barthelona and skoliomonads suggests that this structure was present in their last common ancestor. Although barthelonids lack several components of the typical excavate flagellar apparatus, these reductions may be secondary and related to the reduction of the ventral groove. Further ultrastructural studies on skoliomonads will be necessary to elucidate the evolution of ultrastructure and feeding strategies within Notopharyngea, as well as to clarify the taxonomic position of Notopharyngea within Metamonada.

4.1. Taxonomic Summary

Notopharyngea cl. nov.

Diagnosis: Anaerobic flagellates with a highly elongated cytopharynx that originates at the posterior end of the cell and extends anteriorly along the dorsal side. Includes Barthelonida (ord. nov.) and Skoliomonadida (ord. nov.).

Barthelonida ord. nov.

Diagnosis: Anaerobic flagellates with a narrow groove present on the ventral side of the cell. Microtubular roots R1, R2, R3 and internal microtubules are present. R2 splits into iR2 and oR2 at the posterior end of the cell. A and I fibers are associated with the left and right sides of R2 at its proximal end. B and C fibers are absent. The posterior flagellum lacks vanes. Includes Barthelonidae fam. nov.

Barthelonidae fam. nov.

Diagnosis: Anaerobic flagellates with two unequal heterodynamic flagella. The cell is oval with a slightly pointed posterior end. A posterior furrow splits the posterior portion of the cell into left and right sides. The cells swim in the water column with a vibrating motion or attach to substrates by the posterior flagellum.

Type genus: Barthelona (Bernard et al. 2000).

Other included taxa: Parabarthelona gen. nov., Microbarthelona gen. nov.

Barthelona (Bernard et al. 2000).

Emended diagnosis: Anaerobic flagellates with two unequal heterodynamic flagella. The cell is oval with a slightly pointed posterior end. The cells swim in the water column with a vibrating motion or attach to substrates by the posterior flagellum. R1 consists of a single microtubule. R2 consists of 5–6 microtubules. Composite fiber is more developed than in Microbarthelona and Parabarthelona.

Parabarthelona gen. nov.

Diagnosis: Anaerobic flagellates with two unequal heterodynamic flagella. The cell is oval with a slightly pointed posterior end. A narrow inconspicuous ventral groove is present on the ventral side of the cell. A posterior furrow splits the posterior portion of the cell into left and right sides. The cells swim in the water column with a vibrating motion or attach to substrates by the posterior flagellum. R1 consists of two microtubules. R2 consists of 7–8 microtubules. Composite fiber is less conspicuous than in Barthelona.

Type species: Parabarthelona vacuolata.

Etymology: The generic name Parabarthelona is derived from para‐ (Greek: παρα‐), meaning “beside” or “near,” combined with Barthelona, referring to its phylogenetic position. Feminine.

Zoobank LSID : urn:lsid:zoobank.org:act:2265EF6A‐6F02‐4FC5‐850D‐23D5DC256434.

Parabarthelona vacuolata sp. nov.

Diagnosis: Parabarthelona with the cell 7.1 μm (5.5–9.0 μm) in length and 4.7 μm (3.4–6.8 μm) in width. The cell possesses large spherical food vacuoles filled with fluid.

Hapantotype: One EM block (TNS AL‐66034tba) of isolate PAP020, deposited in the herbarium of the National Museum of Nature and Science (TNS), Tsukuba.

Paratype: One EM block (TNS AL‐66034tbb) of isolate PAP020, deposited in TNS. These cells were derived from the same sample as the hapantotype.

Type locality: Marine lake sediment in Ngeruktabel, the Republic of Palau (7°15′20.9″ N, 134°26′44.5″ E).

Collection date: November 5, 2011.

Etymology: The specific epithet vacuolata is derived from the Latin vacuolatus, meaning “vacuolated” referring to the presence of large, spherical food vacuoles in the cells.

Gene sequence: The partial SSU rDNA sequence of isolate PAP020 was previously accessioned in GenBank as LC506386.

Zoobank LSID: urn:lsid:zoobank.org:act:C1D5A641‐6402‐41BE‐B606‐57E77415755C.

Microbarthelona gen. nov.

Diagnosis: Anaerobic flagellates with two unequal heterodynamic flagella. The cell is oval with a slightly pointed posterior end. A narrow inconspicuous ventral groove is present on the ventral side of the cell. A posterior furrow splits the posterior portion of the cell into left and right sides. The cells swim in the water column with a vibrating motion or attach to substrates by the posterior flagellum. R1 consists of two microtubules. R2 consists of 5–6 microtubules. The cell size is smaller than Barthelona vulgaris and Parabarthelona vacuolata.

Type species: Microbarthelona vorax.

Etymology: The generic name Microbarthelona is derived from micro‐ (Greek: mikros), meaning “small,” combined with Barthelona, referring to its smaller cell size. Feminine.

Zoobank LSID: urn:lsid:zoobank.org:act:0E9AF262‐2F4F‐4080‐9FDE‐28512F59F9AA.

Microbarthelona vorax sp. nov.

Diagnosis: Microbarthelona with the cell 4.7 μm (3.8–5.5 μm) in length and 2.9 μm (2.3–3.6 μm) in width.

Hapantotype: One EM block (TNS AL‐66033tba) of isolate PCE, deposited in the National Museum of Nature and Science (TNS), Tsukuba.

Paratype: One EM block (TNS AL‐66033tbb) of isolate PCE, deposited in TNS. These cells were derived from the same sample as the hapantotype.

Type locality: Intertidal sediment near Cavendish, PEI, Canada (46°29′33.1″ N, 63°25′45.2″ W).

Collection date: July 2016.

Etymology: The specific epithet vorax is derived from Latin vorax, meaning “swallowing greedily” referring to the presence of many phagocytosed bacteria in the cell.

Gene sequence: The partial SSU rDNA sequence of isolate PCE was previously accessioned in GenBank as LC506390.

Zoobank LSID: urn:lsid:zoobank.org:act:6D80DFA9‐952E‐48A3‐8389‐E45F41D2141E.

Skoliomonadida ord. nov.

Diagnosis: Anaerobic flagellates with a conspicuous ventral feeding groove extending from the subapical flagellar insertion toward the posterior end of the cell. The right margin of the groove forms a distinct lip. The posterior flagellum bears a broad ventral‐facing vane. A large opening at the distal end of the groove leads into an elongate recurrent cytopharynx that curves dorsally and extends anteriorly within the cell. Includes Skoliomonadidae (fam. nov.).

Skoliomonadidae fam. nov.

Diagnosis: Same as order.

Type genus: Skoliomonas (Eglit and Simpson 2024).

Funding

This work was supported by the Japan Society for the Promotion of Science, 18J02091, Institute for Fermentation, Osaka, Y‐2024‐1‐001, and Natural Sciences and Engineering Research Council of Canada, 298366‐2019.

Supporting information

Figure S1: Overall topology corresponding to Figure1. Maximum‐likelihood (ML) tree of a small subunit (SSU) rDNA gene alignment. The ML tree was inferred from 126 OTUs, including 14 barthelonid sequences, with 1258 nucleotide positions. Support values (SH‐aLRT values and ML bootstrap values) ≤ 80% are omitted except within Notophryngea cl. nov. SH‐aLRT values and ML bootstrap values are presented for all other nodes. Nodes with SH‐aLRT values = 100% and ML bootstrap values = 100% are marked with open circles.

JEU-73-e70080-s004.pdf (376KB, pdf)

Figure S2: Transmission electron micrographs (TEM) of Parabarthelona vacuolata PAP020. (A) Secondary microtubules originate from R2. (B) Extended internal microtubules. Imt, internal microtubule; N, nucleus. Scale bars: 1 μm.

JEU-73-e70080-s002.png (3.7MB, png)

Figure S3: Transmission electron micrographs (TEM) of Parabarthelona vacuolata PAP020. (A–J) Serial sections of transversal view of B2. (K–M) Serial sections of longitudinal view of two basal bodies. AF, anterior flagellum; N, nucleus; PF, posterior flagellum, Arrows indicate axosomes. Scale bars: 500 nm.

JEU-73-e70080-s003.png (4.8MB, png)

Table S1: Geographic and environmental origins of barthelonid ASVs in EukBank.

JEU-73-e70080-s001.xlsx (10KB, xlsx)

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 18J02091 and the Institute for Fermentation, Osaka (IFO) grant number Y‐2024‐1‐001. Y.E. and A.G.B.S. gratefully acknowledge support by NSERC, through discovery grant 298366‐2019 to A.G.B.S.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Overall topology corresponding to Figure1. Maximum‐likelihood (ML) tree of a small subunit (SSU) rDNA gene alignment. The ML tree was inferred from 126 OTUs, including 14 barthelonid sequences, with 1258 nucleotide positions. Support values (SH‐aLRT values and ML bootstrap values) ≤ 80% are omitted except within Notophryngea cl. nov. SH‐aLRT values and ML bootstrap values are presented for all other nodes. Nodes with SH‐aLRT values = 100% and ML bootstrap values = 100% are marked with open circles.

JEU-73-e70080-s004.pdf (376KB, pdf)

Figure S2: Transmission electron micrographs (TEM) of Parabarthelona vacuolata PAP020. (A) Secondary microtubules originate from R2. (B) Extended internal microtubules. Imt, internal microtubule; N, nucleus. Scale bars: 1 μm.

JEU-73-e70080-s002.png (3.7MB, png)

Figure S3: Transmission electron micrographs (TEM) of Parabarthelona vacuolata PAP020. (A–J) Serial sections of transversal view of B2. (K–M) Serial sections of longitudinal view of two basal bodies. AF, anterior flagellum; N, nucleus; PF, posterior flagellum, Arrows indicate axosomes. Scale bars: 500 nm.

JEU-73-e70080-s003.png (4.8MB, png)

Table S1: Geographic and environmental origins of barthelonid ASVs in EukBank.

JEU-73-e70080-s001.xlsx (10KB, xlsx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from The Journal of Eukaryotic Microbiology are provided here courtesy of Wiley

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