Study of a new strain of Sanchytrium tribonematis expands our knowledge on Sanchytriomycota (Fungi)

Recently, phylogenomic analyses of two parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a clade Sanchytriomycota sister to Blastocladiomycota. Sanchytrid species diversity is still very low and most of the isolates from different places belong to S. tribonematis. Here, we present a new strain X-137 CCPP ZIN RAS of fresh-water S. tribonematis having a peculiar morphology and an unusual stage in the life cycle. Its zoospores have a kinetosome composed of 9 microtubular singlets along its whole length (1.2 μm) with the pseudocilium containing an axoneme of 4 microtubules and enable to form an extremely long posterior filopodium (up to 10 body lengths). The kinetosome develops from a centriole of the centrosomal apparatus in sporangium, while another centriole becomes a non-flagellar kinetosome. Big zoospores with several pseudocilia and many (up to eleven) kinetosomes per cell have been found in S. tribonematis culture. They are probably a result of several zoospore or gamete fusions. These findings expand our still poor knowledge on the cell structure and biology of Sanchytriomycota.


Introduction
In recent years, the generalized use of molecular phylogeny tools to study microbial communities is unveiling an increasingly expanding diversity of microbial eukaryotes.In the particular case of fungi, the combination of molecular phylogeny and electron microscopy has revealed a wide cryptic diversity of zoosporic fungi.In the past decades, several new orders of chytridiomycetes, some of which had already been predicted based on molecular phylogeny analysis (James et al. 2006), have been formally described (Letcher et al. 2006;Mozley-Standridge et al. 2009;Simmons et al. 2009;Longcore and Simmons 2012;Karpov et al. 2014).At the same time, some organisms traditionally considered typical chytrids dramatically changed their taxonomic classification to match their position in molecular phylogenetic trees.An illustrative example is that of the former chytridiomycete genus Olpidium, which was reclassified first as a member of the zygomycete clade (James et al. 2006;Sekimoto et al. 2011), and recently on the base of phylogenomic study was confirmed as a phylum Olpidiomycota Doweld, forming a sister lineage to terrestrial fungi (Chang et al. 2021).
Some years ago, we presented the chytrid-like parasite of the yellow-green alga, Tribonema gayanum, as the new genus and species Amoeboradix gromovi (Mamkaeva et al. 2007).A. gromovi exhibited a typical chytrid morphology, similar to that of Rhizophydium anatropum (A.Br.) Sparrow (Couch 1935;Letcher and Powell 2012) but had amoeboid zoospores with highly reduced flagellum (pseudocilium) and an extremely long kinetosome composed of 9 microtubular singlets (Mamkaeva et al. 2007).It was formally described later as Amoeboradix gromovi Karpov, López-García, Mamkaeva & Moreira (Karpov et al. 2018).A year earlier, a new genus and species Sanchytrium tribonematis was described on the base of molecular phylogenetic and light microscopic studies and placed in Monoblepharidomycetes with unstable position among them (Karpov et al. 2017).Both species 69 Page 2 of 9 are closely related to each other and unexpectedly gave a long branch among higher fungi on the 18S phylogenetic tree (Karpov et al. 2018).Amoeboid zoospores also contained a microbody-lipid complex (MLC) but lacked fenestrated cisterna and ribosomal aggregation.Rounded cysts formed rhizoids inside the host; thick-walled sporangia were normally asymmetrical, rounded to pearshaped with one (Sanchytrium) or three (Amoeboradix) papillae for zoospore release.Ultrastructural studies of their zoospores revealed extreme peculiarities in both species: a pseudocilium with an unusually long and reduced kinetosome (Karpov et al. 2017(Karpov et al. , 2018(Karpov et al. , 2019)).The kinetosome of A. gromovi zoospores composed of 9 microtubular doublets/singlets 1.4 μm long, and S. tribonematis has a kinetosome composed of 9 singlets of microtubules 1 μm long.The kinetosome in both species produces a thin immobile pseudocilium supported by four microtubules instead of normal axoneme (Karpov et al. 2019).The centrioles in sporangia were also composed of nine singlets.Recently, a multigene phylogeny based on the genomes and transcriptomes of both species has shown that Amoeboradix and Sanchytrium form a separate lineage sister to Blastocladiomycota and were placed in the new phylum Sanchytriomycota (Galindo et al. 2021).
Here, we present small ribosomal subunit gene characterization and detailed light and electron microscopic study of Sanchytrium tribonematis strain X-137 CCPP ZIN RAS (Malysheva et al. 2016), which reveals new ultrastructural and biological peculiarities of sachytrids.

Isolation and cultivation
The sample was isolated by V.S. Tcvetkova in April 2021 from the river Dymka near the village Dymy of the Leningrad region, Russia (N59°35.2298,E33°45.3070).Tribonema gayanum Pascher CALU-20 growing in No. 1 medium (Gromov and Titova 1991) was inoculated with a sample of water filtered through a piece of gauze.After several weeks of incubation, the culture lost its green color and threads of empty cell walls covered with characteristic sanchytrid sporangia were observed.Isolate X-137 was obtained by inoculation of monoxenic Tribonema cultures growing in liquid medium with a single infected filament collected from an infected culture via capillary micromanipulation.The strain was maintained in a culture on Tribonema gayanum (strain 20 CALU) as described by Karpov et al. (2016).

Light and transmission electron microscopy
Light microscopy observations of living cultures were carried out on a Zeiss Axioplan microscope equipped with colored MRm Axiocam (Zeiss, Germany).Photographs of forty zoospores were used for measuring.
For electron microscopy, the infected algal filaments were fixed with a mixture of 2% glutaraldehyde and 1% OsO4 on 0.1 M cacodylate buffer for 1 h on ice.Thereafter, the fixative mixture with algae was filtered through a 13-mmdiameter polycarbonate hydrophilic filter with 1-μm pore size (ipPore TM Track-Etched Membrane, Belgium) using a Swinny Filter Holder 13 mm (EMD MilliporeSigma, USA), and the filtered algae were embedded in 2% low melting agarose.Agarose plates with algae were washed in 0.1 M cacodylate buffer for 15 min, dehydrated in alcohol series and in propylene oxide and embedded in Agar LV resin (Agar Scientific Ltd., UK).Ultrathin sections were prepared using a Leica Ultracut ultramicrotome (Leica, Germany) with a diamond knife.After double staining by uranyl acetate and lead citrate, the sections were observed using a JEM 1400 (JEOL, Japan) microscope equipped with digital camera Olympus Veleta (Olympus, Japan).
The new 18S rRNA gene sequence of X-137 has been deposited in GenBank with the accession number: OR365268.

Light microscopic observations
Strain X-137 development on the yellow-green alga Tribonema gayanum is similar to Sanchytrium tribonematis in the life cycle and type of zoospores.Moving amoeboid zoospores are typically 4-7.2 μm long (medium 5.34) and 1.4-3.2μm (medium 2.32) wide with few lipid globules (Fig. 1A-C).They produce a broad and flat hyaline pseudopodium (lamellipodium) at the anterior end that can also form subfilopodia (Fig. 1A, B).Filopodia may rapidly appear and retract at any place of the cell when crawling on the substrate; some posterior filopodia can branch (Fig. 1A).Most zoospores have a tracking posterior pseudocilium (Fig. 1A, B) of different lengths (up to 8.8 μm), but this pseudocilium is rather labile and can totally retract.It differs from filopodia by its smooth stick-like appearance but sometimes has deforming swellings, which probably coursed by transporting vesicles (Fig. 2C).Most peculiar for zoospores of strain X-137 is the ability to form an extremely long posterior filopodium (Fig. 1C, D), which may be as long as 10 body lengths (Fig. 1D).
The zoospore encysts on the surface of the Tribonema filament and germinates penetrating the host cell wall and forms a branching rhizoid (Fig. 1E, F).The spherical sporangium grows gradually and forms normally one lateral papilla (Fig. 1G, H).Rarely, we noticed a second papilla in the mature sporangium, which is relatively small and does not exceed 10-12 μm.It produces appr.10 zoospores, which release through the papilla (Fig. 1I, J, Movie S1).

Electron microscopic observations
Using serial ultrathin sections, we studied released zoospores, and young and mature sporangia (Fig. 2).A nucleus occupies the center of the zoospore; lipid globules of medium size associate with loosely granulated microbodies, forming a microbody-lipid complex (MLC) (Figs. 2A and 3C).A Golgi body is present near the nucleus and a contractile vacuole at the cell periphery (Fig. 2A).Mitochondria with lamellar cristae locate more posteriorly and often circle a kinetosome (Fig. 2B).
The kinetosome of 1.1-1.4μm length is connected with a short centriole (Fig. 2D).They both are on the same longitudinal axis being turned at 180° to each other (Fig. 2D).Both structures have a cartwheel at their proximal end (Fig. 2D, E).The distal end of the kinetosome anchors to the plasma membrane and has a partition, the transverse plate with axosome, and short transitional fibers (Fig. 2D,  J).The kinetosome, which is 120 (at proximal end) to 140 (in the most thick middle part) nm wide, is composed of nine single microtubules that extend along the whole kinetosome length and connect to an intrakinetosomal cylinder (Fig. 2D, F-H).Only four of nine microtubules continue into a pseudocilium, gradually reducing on the way to its tip (Fig. 2B-D, K).
The cyst wall is composed of a thin electron-opaque layer (ca.20-30 nm) external to the plasma membrane (Fig. 2L,  M). Figure 2M shows the presence of full length kinetosome left from the zoospore at the cyst periphery.
The young sporangium has a much thicker (up to 220 nm) wall than the cyst.It penetrates a host wall forming a branching rhizoid (Fig. 2N).The nucleus divides along sporangium growth several times forming nuclei of future zoospores.Each nucleus has a couple of centrioles on its surface (centrosome) orthogonal to each other and composed of 9 microtubular singlets (not shown here).
In mature sporangium, we observed several uninucleate zoospores without long kinetosomes (Fig. 2O).On the serial sections of such sporangium (Fig. 2P-S), we found a growing kinetosome attached with its proximal end to the surface of nuclear projection (Fig. 2P, Q, R).A centriole attaches to the other side of the nuclear projection and lies orthogonal to the kinetosome (Fig. 2R, S).

SSU rRNA sequence of X-137
We amplified and sequenced a near-full SSU rRNA gene from the strain X-137, which has introns of group I with five insertion positions.Sequences of strain X-137, excluding introns, were 98.96% identical to that of the type strain X-128 of S. tribonematis (Karpov et al. 2017), but it differs in the unique pattern of intron localization.

Discussion
Deeper study of sanchytrid strains revealed new data on their biology, zoospore polymorphism, and ultrastructure.We have shown for the first time the mode of zoospore release from sporangium and unambiguously demonstrated a relatively small number of matured zoospores, which release one by one through a narrow discharge pore using active amoeboid movement.

Ultrastructure
Sanchytrid X-137 definitely differs from Amoeboradix gromovi by sporangial shape and dimensions, and kinetosome structure.As in Sanchytrium tribonematis, the sporangium of strain X-137 is spherical (8-10 μm diam) with one papilla, while A. gromovi has elongated asymmetrical sporangia (up to 18 μm in length) with 2-3 papillae (Table 1).The kinetosome of X-137 contains 9 microtubular singlets along the whole length, as in S. tribonematis, while the kinetosome of A. gromovi has microtubular doublets in the proximal part and singlets in the distal half (Karpov et al. 2018(Karpov et al. , 2019)).Hence, strain X-137 is morphologically similar to S. tribonematis having the same sporangial structure and dimensions and zoospore organization including structure of pseudocilium and its kinetosome and definitely belongs to the genus Sanchytrium.At the same time, the kinetosome X-137 is 0.1-0.4μm longer than in S. tribonematis (strain X-127), and the zoospore produces an extralong posterior filopodium that is a prominent character of this strain.

Molecular phylogeny
We analyzed the full SSU gene in comparison with known sequences of previously described strains (Karpov et al. 2017) and did not find significant reasons to consider that strain X-137 belongs to a new species of genus Sanchytrium.Since all Sanchytrium strains have highly intronised rDNA, we used two different ways trying to reconstruct their phylogeny.Sanchytrium's introns relate to group I introns, which means that they catalyze their own splicing from the pre-mRNA.Consequently, it is not justified to count them constructing phylogeny.In this scenario, there are no synapomorphies between strain X-137 and others and only autapomorphies in the form of difference in individual nucleotides can be found.Otherwise, if we take into account introns, there occurs intriguing difference between all known sanchytrid's SSU.Each strain has a unique pattern of intron localization.At present, we do not have enough data to consider that phenomenon as interspecific character.Further investigations on broader material have to be done to clarify this situation.

Development of kinetosomal apparatus in sanchytrids
New data on the intrasporangial zoospores of Sanchytrium tribonematis X-137 suggest a way of centriole transformation into kinetid (centriole + kinetosome with pseudocilium) of released zoospore.The nuclear centrosome in the sporangium consists of two short orthogonal centrioles composed of 9 microtubular singlets, which take part in the nuclear division during multinuclear plasmodium formation as it was shown earlier for S. tribonematis (Karpov et al. 2019).After the plasmodium division into zoospores, each pair of centrioles transforms in the kinetosomal apparatus: one, probably, the mother centriole, grows and becomes a kinetosome; a daughter centriole detaches the nucleus and turns at an angle 90° from its orthogonal position becoming antiparallel to the kinetosome later producing a pseudocilium in the released zoospore.Such antiparallel location of centriole and kinetosome is common for mature zoospores of both genera Amoeboradix and Sanchytrium (Karpov et al. 2018(Karpov et al. , 2019)).When the zoospore attaches to the host surface and becomes a cyst the pseudocilium is retracted, but the long kinetosome and centriole stay intact for some time (Fig. 2L, M).We do not know, either the kinetid in sporangium totally degrades and cell produces two new centrioles taking part in nuclear division, or the kinetosome only disassembles and the centriole produces a daughter centriole.

Big zoospores
Taking into account centriole-kinetosome transformation, we can try to explain a phenomenon of multiple kinetosomes in big zoospores of S. tribonematis, which were found for the first time and not studied yet in details.
Two types of zoospores were also shown for the aphelids (Tcvetkova et al. 2023).Big amoeboid zoospores with 2-4 nuclei have been found in the culture of Amoeboaphelidium protococcorum (Gromov and Mamkaeva 1968) and big zoospores with 3-12 stiff flagella described recently in Aphelidium insulamus (Tcvetkova et al. 2023).The authors supposed that such big cells can be a result of incomplete plasmodium division and probably do not survive.Theoretically, if big cells of S. tribonematis have several small nuclei and kinetosomes, they can represent non divided plasmodial cells releasing a sporangium by some unknown reasons.However, its multinuclear sporangium has no long kinetosomes, which appear later after the division into uninuclear cells.Therefore, the big cells cannot be a portion of the sporangium.Another version that big cells in strain X-137 appear as a result of zoospore or gamete fusion appears to be more realistic.The fusion of amoeboid zoospores supposed to occur in Rhizophydium anatropum (Couch 1935;Letcher and Powell 2012).Rh. anatropum has amoeboid zoospores of about 3.3 μm in diameter with a long immotile posterior flagellum of ca. 10 μm and the hyaline anterior zone producing filopodia.J.N. Couch observed a copulation of 2 or 3 zoospores/gametes and suggested that they can fuse producing a resting spore, although he did not observe a complete cell fusion (Couch 1935).
The fusion of flagellated cells (gametes) with temporal formation of a biflagellate planozygote is well known for the blastoclad Allomyces (James et al. 2014 for references) forming a sister lineage to sanchytrids.The amoeboid gamete fusion has also been described for other blastoclad genera, such as Paraphysoderma, Physoderma, Urophlyctis, and Catenaria (Sparrow 1977;Strittmatter et al. 2016).This amoeba fusion results in a bigger immotile amoeboid cell with short radial pseudopodia, which corresponds to the zygote in blastoclads (Strittmatter et al. 2016).The big amoeboid cells with short radial pseudopodia of S. tribonematis strain X-137 also could represent the zygotic stage.Unfortunately, we did not yet observe subsequent fate of the big cells and decided to continue our study of this very interesting phenomenon.

Fig. 1
Fig. 1 Light microscopy of the life cycle stages of Sanchytrium tribonematis strain X-137 on the filaments of Tribonema gayanum.A-D amoeboid zoospores with pseudocilium and long posterior filopodium (C and D), E young sporangium with penetration stalk (arrow), F, G immature sporangia with branched rhizoid (F, arrows)

Fig. 2
Fig.2Ultrastructure of life cycle stages of Sanchytrium tribonematis strain X-137.A longitudinal section (LS) of zoospore, B LS of kinetosome and pseudocilium at zoospore posterior end, C cross section (CS) of pseudocilium at proximal part with 4 microtubules and transporting vesicle, and distal end with 3 microtubules, D LS of nonflagellar kinetosome (c) and kinetosome with a base of pseudocilium, E-K CSs of kinetosome at different levels from proximal end with cartwheel structure (E), to the middle (F-I), and distal end with transversal plate and axosome (J) and base of pseudocilium with reduced number of tubules (K).L newly formed cyst on the algal surface (to the left), M CS of kinetosome still left in the cyst.N Young sporangium with rhizoids (arrows) in the Tribonema cell.O Section of mature sporangium with zoospores.P-S Consecutive serial sections of growing kinetosome and a centriole in the intrasporangial zoospore.Scale bars: A, L, N 1 μm, B 300 nm, C 150 nm, D-K, M 100 nm, O 1.5 μm, P-S 200 nm ◂

Fig. 3
Fig.3LM and TEM illustrations of big zoospores with several pseudocilia and kinetosomes of Sanchytrium tribonematis strain X-137.A, B LM view of large zoospores with more than one pseudocilium, C peripheral cytoplasm containing at least 4 obliquely sectioned kinetosomes, insert: CS at the very base of 3 pseudocilia with different number of microtubules, D, E sections of the same zoospore with 11 cross sectioned kinetosomes, F CS of two full length kinetosomes in zoospore, G CS of kinetosomes in the center of big cell.Scale bars: A, B 10 μm, C, G 250 nm, D, E 1.5 μm, F 100 nm ◂