Marine parasite Protaphelidium rhizoclonii gen. et sp. nov. designates the basal environmental cluster at the aphelid phylogenetic tree

Marine parasite Protaphelidium rhizoclonii gen. et sp. nov. designates the basal environmental cluster at the aphelid phylogenetic tree | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Marine parasite Protaphelidium rhizoclonii gen. et sp. nov. designates the basal environmental cluster at the aphelid phylogenetic tree Alexei Seliuk, Sergey Karpov This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4241557/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Mycological Progress → Version 1 posted 5 You are reading this latest preprint version Abstract Aphelids are a poorly known group of algal parasites that have raised considerable interest because of their pivotal phylogenetic position as a sister lineage to the kingdom Fungi. Recent breakthroughs in sampling, sequencing and bioinformatical analyses of environmental nucleic acids have revealed magnificent biodiversity of aphelids. Nevertheless, only 4 genera have been described ( Aphelidium , Paraphelidium , Amoeboaphelidium and Pseudaphelidium ); 18S rRNA gene sequences are published for all except for the marine genus Pseudaphelidium. Most of the environmental nucleic acid data is from analysis of freshwater samples. We isolated two new marine aphelid strains (X-138 and X-139), and herein describe the life cycle of Protaphelidium rhizoclonii gen. et sp. nov., a parasite of the green alga Rhizoclonium sp., and provide the first 18S rRNA gene sequences for cultivated marine aphelids. The new marine aphelid life cycle is mostly typical for aphelids, but also includes previously undescribed stages. Molecular phylogenetic analysis indicated that Protaphelidium rhizoclonii is a member of an environmental clade at the base of the aphelid tree. Aphelidiomycota marine fungi molecular phylogeny taxonomy Protaphelidium rhizoclonii Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Aphelids are parasitic protists with opisthokont zoospores. Knowledge of this group began about 150 years ago with the description of Aphelidium deformans , an unusual intracellular parasite of the alga Coleochaete solute (Zopf 1885 ). Aphelids evoked interest of naturalists due to their lifestyle, which superficially resembles that of chytrids. Based on their morphology, Scherffel ( 1925 ) considered their phylogenetic importance to be because of their intracellular development. Unlike members of the Chytridiomycota, aphelids have phagotrophic ameboid stages in their life cycle (Gromov 2000 ) and molecular analysis placed them in the Opisthosporidia sister to Fungi (Karpov et al. 2013 , 2014 ; Letcher et al. 2013 ). Electron-microscopy observations during the late 1960s and the 1970s contributed to our understanding of the biology of aphelids (Gromov and Mamkaeva 1968 , 1970a , b , 1975 , Schnepf et al. 1972). More recent studies have clarified the phylogenetic position of Aphelida as sister to Fungi, which makes them a pivotal group of protists (Torruella et al. 2018 ; Mikhailov et al. 2022 ; Wijayawardene et al. 2024 ). Although just a few aphelid species are officially described, the group is highly diverse, including many environmental sequences from different ecosystems (Seto et al. 2023 ; Yang et al. 2023 ). Currently, 20 species in four genera ( Aphelidium , Paraphelidium , Amoeboaphelidium and Pseudaphelidium ) have been described (Letcher and Powell 2019 ; Tcvetkova et al. 2019; Karpov et al. 2020 ; Seto et al. 2020 , 2022 ). All but one of these are freshwater species. The marine species, Pseudaphelidium drebesii , a parasite of the diatom Thallasiosira punctigera , was described without molecular information(Schweikert and Schnepf 1996 , 1997 ), hence its phylogenetic position is unknown. Here, we describe the morphology of the main life cycle stages of the marine aphelid strain X-138, which along with strain X-139, represents the new genus and species Protaphelidium rhizoclonii. Its novelty and position is supported by 18S rDNA based molecular phylogeny. MATERIAL AND METHODS Isolation and cultivation Strains were isolated from samples collected from the littoral zone of White Sea Biological Station named after A.N. Pertsov of Moscow State University in August 2023. Sample K-1 was collected using plankton net with mesh 23 µm and transferred into 50 ml falcon, sample Cl-4 was collected by hand squeezing out mass of algae from the littoral zone into 50 ml falcon. Sample K-1 was poured on Petri dishes with the addition of f/2 medium (Guillard and Ryther 1962 ; Guillard 1975 ) in 1:1 proportion; dishes were kept at 22°C under permanent light for 2 weeks. Sample Cl-4 was diluted with f/2 medium in proportion 1:1 in the 50 ml falcon centrifuge tubes, transferred to a laboratory and placed at 17°C under permanent light. For cultivation of the alga Rhizoclonium sp. we cut off single filaments and put them in Petri dishes with fresh f/2 medium at 17°C. Significant growth of algae occurred after 2–3 weeks, when the filaments of algae occupied the entire Petri dish area. An optimal temperature for parasite growth was 22°C. Each 2–3 weeks infected filaments of Rhizoclonium sp. were transferred into new medium and supplied with new host filaments. The strains X-138 and X-139 were established from isolates K-1 and Cl-4 correspondingly. Light microscopy Microscopic observations of living cultures were carried out on a Leica DM2500 upright microscope equipped with DIC and Phase contrast optics and a DS-Fi-3 camera (Nikon, USA) powered by NisElements AR software (Nikon). Several filaments of algae were carefully cut off from growth on a Petri dish and transferred to a droplet on a slide. DNA amplification and sequencing One or several infected Rhizoclonium sp. cells were cut off, manually isolated with a glass micropipette, transferred to 0.2-µl PCR tubes with 1–2 µl of medium and frozen at -21°C. The 18S rRNA gene of strain X-138 and X-139 was amplified with primer combination Q5antiDika (5’ - GCC ATG CAT GTC TAA GTA TAA A − 3’) and Q3Opisth (5’ - GGA AAC CTT GTT ACG ACT TTT A − 3’) using the Encyclo Plus PCR kit with polymerase mix (Evrogen, Russia). The PCR amplification program consisted of 2 min denaturation at 94°C; 35 cycles of a denaturation step at 94°C for 15 s, a 30 s annealing step at 62°C and an extension step at 72°C for 2 min; and a final elongation step of 7 min at 72°C. Sequencing was performed on the ABI Prism 3500 xl sequencer (Applied Biosystems, USA) with the primers, used for amplification, and a number of internal primers. Newly acquired 18S rRNA gene sequences of X-138 and X-139 have been deposited in GenBank with accession numbers: XXX Molecular phylogenetic analyses For molecular phylogenetic analyses of aphelids and rozellids we used a data set of 18S rDNA sequences from Seto et al. ( 2022 ). Sequences were automatically aligned with MAFFT 7.409 (Kuraku et al. 2013 ; Katoh et al. 2018 ). Ambiguously aligned regions were excluded manually. 1875 sites were retained in the final alignment. The maximum likelihood (ML) tree was inferred using RAxML 9.2.7 (Stamatakis 2014 ). A Bayesian analysis was run using MrBayes 3.2.7a (Ronquist et al. 2012 ). All parameters were retained as described in Seto et al. ( 2022 ). Execution of methods was performed using CIPRES (Miller et al. 2010 ). Resulting trees were visualized using iTOL (Letunic and Bork 2021 ). RESULTS Light microscopic observations The life cycle of the strain X-138 is similar to that of the marine aphelid Pseudaphelidium drebesii , however, with some differences. Invasive cysts Zoospores of X-138 attach to the host alga, encyst and penetrate the host cell wall with a penetration tube (Fig. 1A). The contents of the cyst migrate through the penetration tube into the host cell (Fig. 1B,C). Although most algal cells were infected with a single cyst, we found cells infected by numerous individual cysts or clusters containing up to 7 cysts (not shown) at the same spot on the algal surface (Fig. 1B–E). Generally, it is challenging to determine individual penetration tubes growing from a clump of cysts, but most cysts have their own penetration tube (Fig. 1C). However, we did find a pair of fused cysts with a common penetration tube (Fig. 1D,E). Occasionally, cysts group at the surface of previously infected and already empty algal cells. The contents of these cysts in the form of an amoeba are still capable of invading the algal cell (Fig. 1B,C). We also found simultaneous invasion of two amoebae from two cysts (Fig. 1C). Trophonts (amoeba - plasmodium) The intracellular amoeba is a trophic stage of the aphelids - it engulfs the host cytoplasm forming a central vacuole containing a residual body, which consists of red, undigested lipids (Fig. 1F,G). The parasite continues to grow and forms a multinuclear plasmodium, often with a loose and large residual body, while it totally consumes the cytoplasm of the host cell (Fig. 1H). The mature plasmodium then divides into a number of uninucleate cells (Fig. 1I), each of which produces a flagellum and thus, can be interpreted as zoospores (Fig. 1J, K). Intracellular zoospores Most zoospores inside host cells are spherical (~ 2.5 µm diam.) and can swim using a flagellum (~ 11 µm in length) (Fig. 1J, K). Flagellated zoospores, which move inside empty algal cells like amoeba using anterior lamellipodium then encyst. We often observed massive zoospore encystment inside the host cell (Fig. 1L–N). Newly formed internal cysts are able to form exit tubes through the host cell wall to release from the host (Fig. 1M-O). This may be the only way for the zoospores to exit because Rhizoclonium cell walls are rather thick and have no any holes. Meanwhile, tubes may connect two or even three adjacent, internal cysts and the contents of one cyst have been observed to move into the other, suggesting cell fusion (Fig. 1L, O). We base our opinion that flagellated cells encyst inside the host on the occurrence of zoospores together with cysts inside the algal cell (Fig. 1L). We also found algal cells containing numerous empty cysts (Fig. 1N). Extracellular zoospores We did not observe how aphelid X-138 releases zoospores from inside the host; therefore, we preliminarily propose that it might be the same as invasion. This can result in a number of rounded cells outside the host, which then produce a flagellum and become crawling amoeboflagellates with relatively short and stiff flagella (~ 11 µm) and cell bodies ~ 4 µm in length. These spores form anterior lamellipodia with subfilopodia, and filopodia (Fig. 1P–R). Rarely, we found swimming zoospores with a spherical body 3.5 µm in diameter and long flagellum ~ 17 µm (Fig. 1S). They often attach to the host surface for a very short time, or can reduce their movement, gradually settle down on the slide, and form filopodia convolving flagellum around the cell (Fig. 1T,U). Resting spores Resting spore formation is common in the X-138 strain (Fig. 1V–X). Spore walls are composed of thick inner and thin outer layer (Fig. 1X). The body contains lipid globules of different sizes located at the poles and separated with homogenous or granulated cytoplasm. Two residual bodies are always present between the two wall layers at the opposite poles. The shape of resting spores varies from elongated to spherical what can be interpreted as different stages of their maturation from elongated to spherical shape, because the cell wall is thickening and the contents constrain (Fig. 1V-X). Molecular phylogeny We amplified and sequenced full 18S rDNA from strains X-138 and X-139 maintained on Rhizoclonium sp. The sequences are nearly identical and the isolates certainly belong to the same species. We reconstructed a maximum likelihood (ML) phylogenetic tree including their 18S rDNA sequences with a set of published aphelid sequences using sequences of rozellids and nucleariids as an outgroup (Fig. 2). In our tree, all main aphelid clades are stable and our strains turned out to be in the basal clade (called here Clade I) containing environmental sequences only. X-138 and X-139 form a sister lineage with TAGIRI-24, which is an environmental sequence obtained from the East-China Sea (Takishita et al. 2005 ) and with Jp13Nb17E and Jp13Nb11E, which are from Japanese freshwater lakes (Ishida et al. 2015 ). DISCUSSION Morphology and life cycle Our newly described representative of the Aphelida has morphological peculiarities that are unknown for other aphelids. Its life cycle comprises two types of zoospores: swimming flagellates and crawling amoeboflagellates, with different lengths of flagella. One type of zoospore may transform to the other; we are not certain. We saw the beginning of the swimming form settling (Fig. 1T,U), but additional observations are needed to determine whether the swimming form settles and becomes an amoeboflagellate. Although the cell body of the swimming zoospore is similar in size as the amoeboflagellates, the length of the flagellum differs and is an important character distinguishing the two types of flagellates. Generally, change of locomotion from actively swimming to crawling amoeba occurs in Aphelidium and Paraphelidium before encystment, when zoospores settle down on algae filament, becoming amoeboflagellate with an immotile flagellum (Karpov et al. 2014 ; 2017 ). Transformation of a swimming zoospore to the amoeboid form, when not as a part of infection process, has been documented for Aph. insulamus (Karpov et al. 2020 , Fig. 2E,F). If no direct transformation between amoeboflagellate zoospores with short (11 µm) flagellum and spherical zoospores with long (17 µm) flagellum takes place, then we conclude that the two types of zoospores have different origins. Members of the Chytridiomycota and the Blastocladiomycota produce zoospores after sporangial cleavage and gametes after germination of a resting spore, where meiosis is proposed to occur (Sparrow 1960 ). Physoderma gerhardti zoospores from germinating resting spores bear flagella 5–10 µm longer than those on asexual zoospores (Sparrow 1977 , Table 1 ). Zoospores from asexual sporangia and zoospores from the resting spore of Synchytrium endobioticum differ in their outline and the length of flagellum, which can be seen on the author’s plates (Curtis 1921 , Figs. 72 and 171), but was not noted by the author. Table 1 Comparison of Pseudaphelidium drebesii and Protaphelidium rhizoclonii gen. et sp. nov. Pseudaphelidium drebesii (Schweikert and Schnepf, 1996 ) Protaphelidium rhizoclonii gen. et sp. nov. (present paper) Zoospore body length (µm) 5 4 Zoospore body width (µm) 3 2.6 Zoospore flagellum (µm) 15 11 Zoospore filopodia length (µm) - Up to 11 Infective cyst diam. (µm) 3 2.8 Multiple infection + + Cells after plasmodium cleavage (µm) Amoebae, 4–10 in length Amoeboflagellates, 4 in length Non-infective cysts (µm) 6.5 2.8 Penetration tube in non-infective cysts - + stack cysts - + non-infective encystment out of the host + - Host Thallasiosira punctigera Rhizoclonium sp Salinity (g/l), locality ~ 28, Wadden Sea ~ 25, White Sea Based on this comparison, we suggest that aphelid X-138 zoospores with long flagella may be produced by germination of resting spores whereas amoeboflagellates with shorter flagella could represent asexual zoospores formed after plasmodium cleavage. Some authors also suggest that resting spore can be a place where sexuality occurs (Seto et al. 2022 ). Unfortunately, germination of aphelid resting spores has not been described so far and we know little about its nature. The resting spore derives directly from the plasmodium, which contains several nuclei as proposed earlier for Amoeboaphelidium chlorellavorum by Gromov and Mamkaeva (1970). We interpreted several images of resting spores (Fig. 1U–W) as consecutive stages of their maturation, as was suggested earlier for other aphelids (Karpov et al. 2017 ). During this process, the cell changes shape from oval to spherical and produces thick inner and thin outer wall with a residual body between them. Strain X-138 has a residual body at each pole of the spore. A few TEM images of Aph. insulamus resting spores revealed one nucleus and one or several prominent lipid globules (Karpov et al. 2020 ). Based on our observations we propose a general scheme of the life cycle as follow (Fig. 3). Two generations of zoospores have been described in Pseudaphelidium drebesii , another marine aphelid (Schweikert and Schnepf 1996 , 1997 ): amoebae emanating from plasmodial division represent first generation; the second generation (the flagellates) appears after the first generation amoebae encyst; each cyst produces from 1 to 4 flagellates, depending on the amoeba’s size. The smallest amoebae (5 µm) yield one zoospore and the biggest amoebae (10 µm) yield up to four zoospores. We can explain this by incomplete plasmodium division resulting in amoeboid zoospores probably having 1–4 nuclei. That is not the case for the strain X-138, which produces a first generation of amoeboflagellates of equal dimensions and the second one (after excystment) of similar spreading amoeboflagellate zoospores. Cysts Stacked cysts on a surface of algae were also found in Amoeboaphelidium protococcorum (Gromov and Mamkaeva 1970b ) and Aph. parallelum (Seto et al. 2022 ). By analogy with some chytrids, this may be related to sexual reproduction that leads to formation of resting spores (Seto et al. 2022 ): one gamete encysts on the other encysted gamete followed by fusion of their contents (Canter 1969 ; Couch 1935 ; Koch 1951 ). One more explanation of stacked cysts on the algal surface suggests zoospore encystment on the empty cyst surface, forms penetration tube and injects its contents through empty cyst into the alga. Therefore, if the cysts appear one by one there should up to ten or more, like Gromov and Mamkaeva ( 1970b ) observed. Probably, the cysts just use the previous penetration tube of the first cyst. Moreover, zoospores can encyst on the algal surface nearby an empty cyst and form penetration tube straight in the tube of the neighbor as we observed (Fig. 1E). This phenomenon might be caused by thick cell wall of the host alga, i.e., it is easier for parasite to use common cyst tube or empty cysts for penetration. In all mentioned cases Amoeboaphelidium protococcorum (Gromov and Mamkaeva 1970b ), Aph. parallelum (Seto et al. 2022 ) and present paper the green algae with thick cell wall were infected, and we have never seen stacked cysts in the aphelids living on Tribonema gayanum having a gap in the cell wall. If the newly formed cyst just uses an empty tube of previous one, or the empty cyst, it makes nothing with resting spore formation. We really need to observe the cyst development and formation of resting spores to clarify the biological nature of the stacked cysts. The thick cell wall of Rhizoclonium can also cause difficulties for zoospores to release. Possibly, a capability of internal zoospores to encyst and penetrate through the cell wall is an adaptation of parasites to marine littoral algae. The intracellular encystment and formation of a tube through the host cell wall has been reported for Aph. insulamus (Karpov et al. 2020 ), what means that it is common for aphelids. Numerous intracellular encysted cells of X-138 strain produce tubes through host cell obviously to release from the host. Although we did not observe the moment of zoospore release, the presence of many emptied intracellular cysts support this idea. We were lucky to capture the moment of simultaneous invasion of two amoebae from two cysts independently (Fig. 1B,C). This leads to formation of two trophonts, what we could often observe (Fig. 1F). This way of penetration was described for all known aphelids and studied with TEM (Schnepf et al. 1971 ; Karpov and Paskerova 2020 ). Perhaps, further fusion of two trophonts occurs to produce a single plasmodium, as it was discussed recently for aphelids (Tcvetkova et al. 2023 ). That suggests a common phenomenon for aphelids. Identification, molecular phylogeny and taxonomy Isolates X-138 and X-139 have nearly identical full 18S rDNA sequences, suggesting that they belong to one species even though these strains were isolated from different samples collected ~ 700m from each other along the marine shore. Our phylogenetic reconstruction saved all main aphelid clades together with paraphyletic genus Aphelidium and shows, that Protaphelidium rhizoclonii gen. et sp. nov. is a single described species in the basal clade composed of environmental sequences only. This clade turns off the next after highly divergent Aph. collabens lineage (Seto et al. 2020 , 2022 ) and called here Clade I. The SSU rDNA of strain X-138 differs from that of sister marine clone TAGIRI-24 by 10.57% and of the closest described species Aphelidium parallelum (11.83%). These differences indicate at least genus level, though it is well known that genetic divergence for species level for aphelids is also rather high (Karpov et al. 2020 ). Despite a sister position to the marine clone, our strains have higher percentage of identity (90.22%) to an isolate under accession number OQ702871 from Seto et al. ( 2023 ) (not present on Fig. 2), obtained from fresh water samples in Michigan. Based on the position of X-138 in a clade of environmental sequences, and its unique life cycle features, we declare it as a new genus and new species Protaphelidium rhizoclonii Seliuk et Karpov. Pr. rhizoclonii gen. et sp. nov. resembles in some respects Pseudaphelidium drebesii , which parasitizes the marine diatom Thalassiosira punctigera , but essentially differs in others (Table 1 ). Taxonomy Among the studied aphelids, strain X-138 is morphologically closest to Pseudaphelidium drebesii ; both are marine and have two zoospore generations with encystment in between. Theoretically, X-138 could be a Pseudaphelidium species, but we choose not to place the new species in this genus because the phylogenetic position of Pseudaphelidium is unknown. Even if the genus Pseudaphelidium is represented by one of the published environmental sequences in Aphelida Clade I, the rDNA of Protaphelidium rhizoclonii differs from the nearest environmental sequence in Clade I by more than 10%, which corresponds a genus level difference. Thus, we establish a new genus and species for strains X-138 and X-139 of this marine parasite. Protaphelidium Seliuk et Karpov, present paper. Amoeboflagellate zoospores with a single opisthokont flagellum. Zoospore attaches to a host cell, encysts, penetrates into the cell interior, and develops into a phagocytotic plasmodium with central big digestion vacuole containing a prominent residual body composed of several lipid globules. Plasmodium cleaves to form uninucleate ameboflagellated cells, which encyst within the algal host. Cells release from algae through exit tubes and give rise to amoeboflagellate zoospores. Resting spore normally contains two opposite resting bodies between the inner and outer walls. Parasites of marine green algae predominantly. Etymology: πρώτα (greek) – the first, meaning the aphelid Clade I. Type species: Protaphelidium rhizoclonii Seliuk et Karpov, present paper. Protaphelidium rhizoclonii Seliuk et Karpov. Type: Fig. 1, present paper. Zoospores 4 µm long and 2.6 µm wide, flagellum 11 µm long. Plasmodium cleaves to form globular cells from which the amoeboflates arise. They form cysts measuring 2.8 µm in diameter, which penetrate host cell wall to release zoospores. Host: The marine green alga Rhizoclonium sp. Distribution: sea coastal waters. Type locality: White Sea, near village Poyakonda, N66.5549°, E33.09907°. Holotype: Fig. 1. Type strain: X-138 Etymology: after generic name of the host alga. Notes. Flagellated cells of Pr. rhizoclonii differ from Ps. drebesii by shorter flagellum (11 vs 15 µm), much smaller non-infective cysts (2.8 vs 6.5 µm), smaller cells after plasmodium cleavage (3 vs 5–10 µm). Plasmodium of Ps. drebesii cleaves to form non-flagellated amoebae, which are slowly (for some hours) released from diatom frustule via the girdle region, and then encyst. Meanwhile the plasmodium of Pr. rhizoclonii produces amoeboflagellates that encyst intracellularly and are released from the host through exit tubes without division as in Ps. drebesii . In Pr. rhizoclonii both zoospore generations have flagella, while in Ps. drebesii the first generation has non-flagellated, amoeboid zoospores. Penetration tube in non-infective cysts and stack cysts were not shown for Ps. drebesii. And lastly, they have different hosts. Declarations ACKNOWLEDGMENTS We thank WSBS named after by A.N. Pertsov for providing an opportunity to work. Authors express their indebtedness to A.I. Shestakov and to the staff of the department of microbiology of MSU for allowing us to use their equipment. We thank S.A. Poluzerov for help with sampling and A.E. Shipunova for modernization of our plankton mesh for a hand net. The authors are grateful to Joyce Longcore for manuscript discussion and correction. This work was supported by RSF grant 21-74-20089 (material collection, strain isolation, light microscopic studies, and writing), the Ministry of Science and Higher Education of the Russian Federation grant 075-15-2021-1069 (rDNA sequencing and molecular phylogeny), and made in the frame of lab topic 122031100260-0 ZIN RAS. Availability of data and material The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests. The authors have no relevant financial or non-financial interests to disclose. Funding This work supported by RSF grant 21-74-20089 and by grant of the Ministry of Science and Higher Education of the Russian Federation 075-15-2021-1069. Author Contributions Sample collection, isolation and cultivation, light microscopic observations and sequencing were performed by Alexei Seliuk. Sergey Karpov contributed to the study conception and design. Both authors commented on previous versions of the manuscript, read and approved the final manuscript. References Curtis KM (1921) IX.—The life-history and cytology of Synchytrium endobioticum (Schilb.), Perc., the cause of wart disease in potato. Philosophical Trans Royal Soc Lond Ser B Containing Papers Biol Character 210(372–381):409–478 Canter HM (1969) Studies on British chytrids. XXIX. A taxonomic revision of certain fungi found on the diatom Asterionella . Bot J Linn Soc 62(3):267–278 Couch JN (1935) New or little known Chytridiales . Mycologia 27:160–175 Gromov BV (2000) Algal parasites of the genera Aphelidium, Amoeboaphelidium , and Pseudaphelidium from the Cienkovski’s Monadea group as representatives of new class. Zool Zhurnal 79:517–525 (in Russian) Gromov BV, Mamkaeva KA (1968) Amoeboaphelidium protococcorum sp. n. and Amoeboaphelidium chlorellavorum sp. n. – endoparasites of protococcous algae. Acta Protozool 6:221–225 Gromov BV, Mamkaeva КA (1970a) Electron-microscopic investigations of development cycle and feeding behaviour of intracellular algal parasite of Chlorella , Amoeboaphelidium chlorellavorum . Tsitologiya 12:1191–1196 Gromov BV, Mamkaeva КA (1970b) The fine structure of Amoeboaphelidium protococcarum — an endoparasite of green alga Scenedesmus . Arch Hydrobiol 67:452–459 Gromov BV, Mamkaeva КA (1975) Zoospore ultrastructure of Aphelidium chlorococcarum Fott. Mikologiya i fitopatologiya 9:190–193 Guillard RRL (1975) Culture of Phytoplankton for Feeding Marine Invertebrates. In: Culture of Marine Invertebrate Animals. https://doi.org/10.1007/978-1-4615-8714-9_3 Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. Deep Sea Res Oceanogr Abstracts 9:7–10. https://doi.org/10.1016/0011-7471(62)90036-0 Ishida S, Nozaki D, Grossart HP, Kagami M (2015) Novel basal, fungal lineages from freshwater phytoplankton and lake samples. Environ Microbiol Rep 7(3):435–441. https://doi.org/10.1111/1758-2229.12268 Karpov SA, Mikhailov KV, Mirzaeva GS, Mirabdullaev IM, Mamkaeva KA, Titova NN, Aleoshin VV (2013) Obligately phagotrophic aphelids turned out to branch with the earliest-diverging fungi. Protist 164(2):195–205. https://doi.org/10.1016/j.protis.2012.08.001 Karpov SA, Mamkaeva MA, Aleoshin VV, Nassonova E, Lilje O, Gleason FH (2014) Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia. Front Microbiol 5:112. https://doi.org/10.3389/fmicb.2014.00112 Karpov SA, Mamkaeva MA, Moreira D, López-García P (2016) Molecular phylogeny of Aphelidium tribonemae reveals its sister relationship with A . aff. melosirae (Aphelida, Opisthosporidia). Protistology 10:97–103 Karpov SA, Tcvetkova VS, Mamkaeva MA, Torruella G, Timpano H, Moreira D, Mamanazarova KS, López-García P (2017) Morphological and Genetic Diversity of Opisthosporidia: New Aphelid Paraphelidium tribonemae gen. et sp. nov. J Eukaryot Microbiol 64(2):204–212 Karpov SA, Vishnyakov AE, López-García P, Zorina NA, Ciobanu M, Tcvetkova VS, Moreira D (2020) Morphology and molecular phylogeny of Aphelidium insulamus sp. nov. Opisthosporidia) Protistology 14:191–203Aphelida Karpov SA, Paskerova GG (2020) The aphelids, intracellular parasitoids of algae, consume the host cytoplasm from the inside. Protistology 14(4):258–263 Katoh K, Rozewicki J, Yamada KD (2018) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20(4). https://doi.org/10.1093/bib/bbx108 Koch WJ (1951) Studies in the genus Chytridium , with observations on a sexually reproducing species. J Elisha Mitchell Sci Soc 67(2):267–278 Kuraku S, Zmasek CM, Nishimura O, Katoh K (2013) aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Res 41 (Web Server issue). https://doi.org/10.1093/nar/gkt389 Letcher PM, Lopez S, Schmieder R, Lee PA, Behnke C, Powell MJ, McBride RC (2013) Characterization of Amoeboaphelidium protococcarum , an Algal Parasite New to the Cryptomycota Isolated from an Outdoor Algal Pond Used for the Production of Biofuel. PLoS ONE 8(2). https://doi.org/10.1371/journal.pone.0056232 Letcher PM, Powell MJ (2019) A taxonomic summary of Aphelidiaceae. IMA Fungus 10(1). https://doi.org/10.1186/s43008-019-0005-7 Letunic I, Bork P (2021) Interactive tree of life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49(W1). https://doi.org/10.1093/nar/gkab301 Mikhailov KV, Karpov SA, Letcher PM, Lee PA, Logacheva MD, Penin AA, Nesterenko MA, Pozdnyakov IR, Potapenko EV, Sherbakov DY et al (2022) Genomic analysis reveals cryptic diversity in aphelids and sheds light on the emergence of Fungi. Curr Biol 32(21). https://doi.org/10.1016/j.cub.2022.08.071 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop, GCE. https://doi.org/10.1109/GCE.2010.5676129 Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3). https://doi.org/10.1093/sysbio/sys029 Schnepf E, Hegewald E, Soeder C-J (1971) Electronenmikroskopische eobachtungen an parasite aus Scenedesmus -massenkulturen. Arch Mikrobiol 75:209–229 Schnepf E (1972) Strukturveränderungen am Plasmalemma Aphelidium -infizierter Scenedesmus -Zellen. Protoplasma 75(1):155–165 Scherffel A (1925) Endophytische Phycomyceten-Parasiten der bacilleriaceen und einige neue Monadinen. Arch Protistenkd 52:1–141 Schweikert M, Schnepf E (1996) Pseudaphelidium drebesii , gen. et spec. nov. (incerta sedis), a parasite of the marine centric diatom Thalassiosira punctigera . Archiv Fur Protistenkunde 147(1). https://doi.org/10.1016/S0003-9365(96)80004-2 Schweikert M, Schnepf E (1997) Electron microscopical observations on Pseudaphelidium drebesii Schweikert and Schnepf, a parasite of the centric diatom Thalassiosira punctigera . Protoplasma 199(3–4). https://doi.org/10.1007/BF01294500 Seto K, Matsuzawa T, Kuno H, Kagami M (2020) Morphology, ultrastructure, and molecular phylogeny of Aphelidium collabens sp. nov.(Aphelida), a parasitoid of a green alga Coccomyxa sp. Protist 171(3):125728 Seto K, Nakada T, Tanabe Y, Yoshida M, Kagami M (2022) Aphelidium parallelum , sp. nov., a new aphelid parasitic on selenastracean green algae. Mycologia 114(3). https://doi.org/10.1080/00275514.2022.2039487 Seto K, Simmons DR, Quandt CA, Frenken T, Dirks AC, Clemons RA, McKindles KM, McKay RML, James TY (2023) A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi. MBio 14(4):e0131323. https://doi.org/10.1128/mbio.01313-23 Sparrow FK (1960) Aquatic Phycomycetes. Ann Arbor Univ Michigan, p 1187 Sparrow FK (1977) Observations on chytridiaceous parasites of phanerogams. XXVI. Physoderma gerhardti Schroeter on Phalaris arundinacea L. Arch Microbiol 114:241–247 Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9). https://doi.org/10.1093/bioinformatics/btu033 Strittmatter M, Guerra T, Silva J, Gachon CMM (2016) A new flagellated dispersion stage in Paraphysoderma sedebokerense , a pathogen of Haematococcus pluvialis . J Appl Phycol 28(3). https://doi.org/10.1007/s10811-015-0700-8 Takishita K, Miyake H, Kawato M, Maruyama T (2005) Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny. Extremophiles 9(3). https://doi.org/10.1007/s00792-005-0432-9 Tcvetkova VS, Pozdnyakov IR, Seliuk AO, Zorina NA, Karpov SA (2023) Vegetative cell fusion and a new stage in the life cycle of the Aphelida (Opisthosporidia). J Eukaryot Microbiol 70(5):12977. https://doi.org/10.1111/jeu.12977 Torruella G, Grau-Bové X, Moreira D, Karpov SA, Burns JA, Sebé-Pedrós A, Völcker E, López-García P (2018) Global transcriptome analysis of the aphelid Paraphelidium tribonemae supports the phagotrophic origin of fungi. Commun Biology 1(1). https://doi.org/10.1038/s42003-018-0235-z Wijayawardene NN, Hyde KD, Mikhailov KV, Gábor P, Aptroot A, Pires-Zottarelli CLA, Goto BT, Tokarev YS, Haelewaters D, Karunarathna SC et al (2024) Higher level classification of the Kingdom Fungi . Fungal Diversity (in press) Yang J, Yun J, Liu X, Du W, Xiang M (2023) Niche and ecosystem preference of earliest diverging fungi in soils. Mycology 14(3):239–255 Zopf W (1885) Zur Morphologie und Biologie der niederen Pilzthiere (Monadinen), zugleich ein Beitrag zur Phytopathologie. Verlag von Veit & Comp, Leipzig, Germany Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Mycological Progress → Version 1 posted Reviewers agreed at journal 20 Apr, 2024 Reviewers invited by journal 12 Apr, 2024 Editor invited by journal 11 Apr, 2024 Editor assigned by journal 10 Apr, 2024 First submitted to journal 09 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4241557","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290385940,"identity":"7490558f-8caa-4071-8c5d-85c18b9d8b1b","order_by":0,"name":"Alexei Seliuk","email":"","orcid":"","institution":"Saint Petersburg State University: Sankt-peterburgskij gosudarstvennyj universitet","correspondingAuthor":false,"prefix":"","firstName":"Alexei","middleName":"","lastName":"Seliuk","suffix":""},{"id":290385941,"identity":"efbc5d8f-68e9-41e3-9908-f63855544f91","order_by":1,"name":"Sergey Karpov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYFACNgjF2N4AJA0sSNDC3HMApEWCBC3sMxJAFBFa5NuPJX6u3GGT2Dvz+dUNPwokGPjbuxPwajE4k3ZY8uyZtMSZs3PKbvYAHSZx5uwG/FoY0hskG9sOGxvOzkm7wQPUYiCRi1+LfP/z5p8gLfY3z6Td/EOMFoYbacdAtsgxzmA/dpsoWwxuPEuzbDyTJsfYk8N2W8ZAgoegX+T704xvNu6w4WFsP/7s5ps/NnL87b0EHAYCjA0gkscATBJWjtDC/oA41aNgFIyCUTDiAABOz0pHTTgqRAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1509-1908","institution":"Zoological Institute RAS: FGBUN Zoologiceskij institut Rossijskoj akademii nauk","correspondingAuthor":true,"prefix":"","firstName":"Sergey","middleName":"","lastName":"Karpov","suffix":""}],"badges":[],"createdAt":"2024-04-09 11:35:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4241557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4241557/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11557-024-01998-6","type":"published","date":"2024-09-27T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54858155,"identity":"5514a450-17c7-4c70-b862-18c0ca1f695b","added_by":"auto","created_at":"2024-04-17 18:40:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2401772,"visible":true,"origin":"","legend":"\u003cp\u003eMain stages of the life cycle of \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e strain X-138 observed in living material with DIC and phase contrast (\u003cstrong\u003eO, P\u003c/strong\u003e). \u003cstrong\u003eA\u003c/strong\u003e: cyst (cy) with penetration tube (pt) on the surface of the host cell (hc); \u003cstrong\u003eB, C\u003c/strong\u003e: two optical sections of two empty cysts (arrows) with contents injected into the host cell; penetration tubes lie extremely close to each other (arrowheads) with penetrating amoeba from the left cyst (asterisk) and second amoeba not in focus (arrow) from the right cyst; \u003cstrong\u003eD\u003c/strong\u003e: three infective cysts (arrowheads), two with a common penetrating tube (arrow); \u003cstrong\u003eE\u003c/strong\u003e: two cysts with connection between them (arrowhead) and common penetration tube (arrow); \u003cstrong\u003eF\u003c/strong\u003e: two young trophonts (tr) in the host cell with corresponding residual bodies (rb) in the central vacuoles (cv); \u003cstrong\u003eG\u003c/strong\u003e: maturing trophont, which has engulfed almost half of the host cell; \u003cstrong\u003eH\u003c/strong\u003e: plasmodium with loose residual body; \u003cstrong\u003eI\u003c/strong\u003e: mature plasmodium with distinguishable spherical cells (arrow); \u003cstrong\u003eJ\u003c/strong\u003e, \u003cstrong\u003eK\u003c/strong\u003e: amoeboflagellate zoospores (zoo) with flagellum (f) in the algal cell; \u003cstrong\u003eL\u003c/strong\u003e: numerous cysts inside a host cell (cy), some of them are stack with connections (arrowheads), with remnant crawling amoebae (ac) which have not encysted yet and residual bodies lying on the opposite sides (rb); \u003cstrong\u003eM: \u003c/strong\u003ecysts with exit tubes through the cell wall of algae (arrowheads); \u003cstrong\u003eN\u003c/strong\u003e: numerous empty cysts (cy) with exit tubes (arrowheads); \u003cstrong\u003eO\u003c/strong\u003e: connections between three cysts (arrowheads) with cytoplasm migration into the middle cyst; exit tube of the cyst directed outside (arrow); \u003cstrong\u003eP–Q\u003c/strong\u003e: amoeboflagellate zoospores with flagellum (f), lamellipodia (la) and subfilopodia (arrowheads); \u003cstrong\u003eS\u003c/strong\u003e: zoospore with long flagellum (f) and \u003cstrong\u003eT,U:\u003c/strong\u003eits settlement down with convolving flagellum (arrow) and small filopodia (arrowheads); \u003cstrong\u003eV–X\u003c/strong\u003e resting spores with lipid globules (l), ejected residual bodies from opposite sides. Residual bodies lie in between of the spore wall (arrowhead) and outer thin wall (arrow).\u003c/p\u003e\n\u003cp\u003eScale bars: \u003cstrong\u003eA,F–S,V–X\u003c/strong\u003e – 10 µm; \u003cstrong\u003eB–E,T,U\u003c/strong\u003e – 5 µm.\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4241557/v1/00afb0c887bf1f235e20e05d.png"},{"id":54858153,"identity":"429ccd6a-3e4e-4b8f-b03d-1cc8375c1dff","added_by":"auto","created_at":"2024-04-17 18:40:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194131,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood consensus tree of aphelids and rozellids with some of nucleariids based on 18S rDNA sequences, showing position of strains X-138 and X-139 (in bold). The tree was constructed using 1875 nucleotide characters. Node support values are as follows: bootstrap values (RAxML; above 50% shown) followed by Bayesian posterior probabilities (MrBayes; above 95% shown).\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4241557/v1/1ea33277c08c82164e2a6183.png"},{"id":54858145,"identity":"d04f2ab1-9456-41c7-b8a3-e7e28faf09a3","added_by":"auto","created_at":"2024-04-17 18:40:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233764,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral scheme of the \u003cem\u003eProtaphelidium\u003c/em\u003e \u003cem\u003erhizoclonii \u003c/em\u003egen. et sp. nov. life cycle.\u003c/p\u003e\n\u003cp\u003eDash arrow shows a proposed way of the origin of zoospores with long flagellum.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4241557/v1/2c6788eee6c3654594713fbe.png"},{"id":65627134,"identity":"9c6b1bb7-8826-436f-bd5d-bd62a957b8b8","added_by":"auto","created_at":"2024-09-30 16:12:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4178389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4241557/v1/52d8ac40-b898-4cd8-9788-90760787c8b7.pdf"}],"financialInterests":"","formattedTitle":"Marine parasite Protaphelidium rhizoclonii gen. et sp. nov. designates the basal environmental cluster at the aphelid phylogenetic tree","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAphelids are parasitic protists with opisthokont zoospores. Knowledge of this group began about 150 years ago with the description of \u003cem\u003eAphelidium deformans\u003c/em\u003e, an unusual intracellular parasite of the alga \u003cem\u003eColeochaete solute\u003c/em\u003e (Zopf \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1885\u003c/span\u003e). Aphelids evoked interest of naturalists due to their lifestyle, which superficially resembles that of chytrids. Based on their morphology, Scherffel (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1925\u003c/span\u003e) considered their phylogenetic importance to be because of their intracellular development. Unlike members of the Chytridiomycota, aphelids have phagotrophic ameboid stages in their life cycle (Gromov \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and molecular analysis placed them in the Opisthosporidia sister to Fungi (Karpov et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Letcher et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eElectron-microscopy observations during the late 1960s and the 1970s contributed to our understanding of the biology of aphelids (Gromov and Mamkaeva \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1968\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1970a\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1975\u003c/span\u003e, Schnepf et al. 1972). More recent studies have clarified the phylogenetic position of Aphelida as sister to Fungi, which makes them a pivotal group of protists (Torruella et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mikhailov et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wijayawardene et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough just a few aphelid species are officially described, the group is highly diverse, including many environmental sequences from different ecosystems (Seto et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Currently, 20 species in four genera (\u003cem\u003eAphelidium\u003c/em\u003e, \u003cem\u003eParaphelidium\u003c/em\u003e, \u003cem\u003eAmoeboaphelidium\u003c/em\u003e and \u003cem\u003ePseudaphelidium\u003c/em\u003e) have been described (Letcher and Powell \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tcvetkova et al. 2019; Karpov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Seto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All but one of these are freshwater species. The marine species, \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e, a parasite of the diatom \u003cem\u003eThallasiosira punctigera\u003c/em\u003e, was described without molecular information(Schweikert and Schnepf \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), hence its phylogenetic position is unknown.\u003c/p\u003e \u003cp\u003eHere, we describe the morphology of the main life cycle stages of the marine aphelid strain X-138, which along with strain X-139, represents the new genus and species \u003cem\u003eProtaphelidium rhizoclonii.\u003c/em\u003e Its novelty and position is supported by 18S rDNA based molecular phylogeny.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and cultivation\u003c/h2\u003e \u003cp\u003eStrains were isolated from samples collected from the littoral zone of White Sea Biological Station named after A.N. Pertsov of Moscow State University in August 2023. Sample K-1 was collected using plankton net with mesh 23 \u0026micro;m and transferred into 50 ml falcon, sample Cl-4 was collected by hand squeezing out mass of algae from the littoral zone into 50 ml falcon. Sample K-1 was poured on Petri dishes with the addition of f/2 medium (Guillard and Ryther \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1962\u003c/span\u003e; Guillard \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) in 1:1 proportion; dishes were kept at 22\u0026deg;C under permanent light for 2 weeks. Sample Cl-4 was diluted with f/2 medium in proportion 1:1 in the 50 ml falcon centrifuge tubes, transferred to a laboratory and placed at 17\u0026deg;C under permanent light. For cultivation of the alga \u003cem\u003eRhizoclonium\u003c/em\u003e sp. we cut off single filaments and put them in Petri dishes with fresh f/2 medium at 17\u0026deg;C. Significant growth of algae occurred after 2\u0026ndash;3 weeks, when the filaments of algae occupied the entire Petri dish area. An optimal temperature for parasite growth was 22\u0026deg;C. Each 2\u0026ndash;3 weeks infected filaments of \u003cem\u003eRhizoclonium\u003c/em\u003e sp. were transferred into new medium and supplied with new host filaments. The strains X-138 and X-139 were established from isolates K-1 and Cl-4 correspondingly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLight microscopy\u003c/h2\u003e \u003cp\u003eMicroscopic observations of living cultures were carried out on a Leica DM2500 upright microscope equipped with DIC and Phase contrast optics and a DS-Fi-3 camera (Nikon, USA) powered by NisElements AR software (Nikon). Several filaments of algae were carefully cut off from growth on a Petri dish and transferred to a droplet on a slide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA amplification and sequencing\u003c/h2\u003e \u003cp\u003eOne or several infected \u003cem\u003eRhizoclonium\u003c/em\u003e sp. cells were cut off, manually isolated with a glass micropipette, transferred to 0.2-\u0026micro;l PCR tubes with 1\u0026ndash;2 \u0026micro;l of medium and frozen at -21\u0026deg;C. The 18S rRNA gene of strain X-138 and X-139 was amplified with primer combination Q5antiDika (5\u0026rsquo; - GCC ATG CAT GTC TAA GTA TAA A \u0026minus;\u0026thinsp;3\u0026rsquo;) and Q3Opisth (5\u0026rsquo; - GGA AAC CTT GTT ACG ACT TTT A \u0026minus;\u0026thinsp;3\u0026rsquo;) using the Encyclo Plus PCR kit with polymerase mix (Evrogen, Russia). The PCR amplification program consisted of 2 min denaturation at 94\u0026deg;C; 35 cycles of a denaturation step at 94\u0026deg;C for 15 s, a 30 s annealing step at 62\u0026deg;C and an extension step at 72\u0026deg;C for 2 min; and a final elongation step of 7 min at 72\u0026deg;C. Sequencing was performed on the ABI Prism 3500 xl sequencer (Applied Biosystems, USA) with the primers, used for amplification, and a number of internal primers. Newly acquired 18S rRNA gene sequences of X-138 and X-139 have been deposited in GenBank with accession numbers: XXX\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular phylogenetic analyses\u003c/h2\u003e \u003cp\u003eFor molecular phylogenetic analyses of aphelids and rozellids we used a data set of 18S rDNA sequences from Seto et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sequences were automatically aligned with MAFFT 7.409 (Kuraku et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Katoh et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Ambiguously aligned regions were excluded manually. 1875 sites were retained in the final alignment. The maximum likelihood (ML) tree was inferred using RAxML 9.2.7 (Stamatakis \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A Bayesian analysis was run using MrBayes 3.2.7a (Ronquist et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). All parameters were retained as described in Seto et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Execution of methods was performed using CIPRES (Miller et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Resulting trees were visualized using iTOL (Letunic and Bork \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLight microscopic observations\u003c/h2\u003e \u003cp\u003eThe life cycle of the strain X-138 is similar to that of the marine aphelid \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e, however, with some differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eInvasive cysts\u003c/h2\u003e \u003cp\u003eZoospores of X-138 attach to the host alga, encyst and penetrate the host cell wall with a penetration tube (Fig.\u0026nbsp;1A). The contents of the cyst migrate through the penetration tube into the host cell (Fig.\u0026nbsp;1B,C). Although most algal cells were infected with a single cyst, we found cells infected by numerous individual cysts or clusters containing up to 7 cysts (not shown) at the same spot on the algal surface (Fig.\u0026nbsp;1B\u0026ndash;E). Generally, it is challenging to determine individual penetration tubes growing from a clump of cysts, but most cysts have their own penetration tube (Fig.\u0026nbsp;1C). However, we did find a pair of fused cysts with a common penetration tube (Fig.\u0026nbsp;1D,E). Occasionally, cysts group at the surface of previously infected and already empty algal cells. The contents of these cysts in the form of an amoeba are still capable of invading the algal cell (Fig.\u0026nbsp;1B,C). We also found simultaneous invasion of two amoebae from two cysts (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTrophonts (amoeba - plasmodium)\u003c/h2\u003e \u003cp\u003eThe intracellular amoeba is a trophic stage of the aphelids - it engulfs the host cytoplasm forming a central vacuole containing a residual body, which consists of red, undigested lipids (Fig.\u0026nbsp;1F,G). The parasite continues to grow and forms a multinuclear plasmodium, often with a loose and large residual body, while it totally consumes the cytoplasm of the host cell (Fig.\u0026nbsp;1H). The mature plasmodium then divides into a number of uninucleate cells (Fig.\u0026nbsp;1I), each of which produces a flagellum and thus, can be interpreted as zoospores (Fig.\u0026nbsp;1J, K).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular zoospores\u003c/h2\u003e \u003cp\u003eMost zoospores inside host cells are spherical (~\u0026thinsp;2.5 \u0026micro;m diam.) and can swim using a flagellum (~\u0026thinsp;11 \u0026micro;m in length) (Fig.\u0026nbsp;1J, K). Flagellated zoospores, which move inside empty algal cells like amoeba using anterior lamellipodium then encyst. We often observed massive zoospore encystment inside the host cell (Fig.\u0026nbsp;1L\u0026ndash;N). Newly formed internal cysts are able to form exit tubes through the host cell wall to release from the host (Fig.\u0026nbsp;1M-O). This may be the only way for the zoospores to exit because \u003cem\u003eRhizoclonium\u003c/em\u003e cell walls are rather thick and have no any holes. Meanwhile, tubes may connect two or even three adjacent, internal cysts and the contents of one cyst have been observed to move into the other, suggesting cell fusion (Fig.\u0026nbsp;1L, O). We base our opinion that flagellated cells encyst inside the host on the occurrence of zoospores together with cysts inside the algal cell (Fig.\u0026nbsp;1L). We also found algal cells containing numerous empty cysts (Fig.\u0026nbsp;1N).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExtracellular zoospores\u003c/h2\u003e \u003cp\u003eWe did not observe how aphelid X-138 releases zoospores from inside the host; therefore, we preliminarily propose that it might be the same as invasion. This can result in a number of rounded cells outside the host, which then produce a flagellum and become crawling amoeboflagellates with relatively short and stiff flagella (~\u0026thinsp;11 \u0026micro;m) and cell bodies\u0026thinsp;~\u0026thinsp;4 \u0026micro;m in length. These spores form anterior lamellipodia with subfilopodia, and filopodia (Fig.\u0026nbsp;1P\u0026ndash;R). Rarely, we found swimming zoospores with a spherical body 3.5 \u0026micro;m in diameter and long flagellum\u0026thinsp;~\u0026thinsp;17 \u0026micro;m (Fig.\u0026nbsp;1S). They often attach to the host surface for a very short time, or can reduce their movement, gradually settle down on the slide, and form filopodia convolving flagellum around the cell (Fig.\u0026nbsp;1T,U).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eResting spores\u003c/h2\u003e \u003cp\u003eResting spore formation is common in the X-138 strain (Fig.\u0026nbsp;1V\u0026ndash;X). Spore walls are composed of thick inner and thin outer layer (Fig.\u0026nbsp;1X). The body contains lipid globules of different sizes located at the poles and separated with homogenous or granulated cytoplasm. Two residual bodies are always present between the two wall layers at the opposite poles. The shape of resting spores varies from elongated to spherical what can be interpreted as different stages of their maturation from elongated to spherical shape, because the cell wall is thickening and the contents constrain (Fig.\u0026nbsp;1V-X).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular phylogeny\u003c/h2\u003e \u003cp\u003eWe amplified and sequenced full 18S rDNA from strains X-138 and X-139 maintained on \u003cem\u003eRhizoclonium\u003c/em\u003e sp. The sequences are nearly identical and the isolates certainly belong to the same species. We reconstructed a maximum likelihood (ML) phylogenetic tree including their 18S rDNA sequences with a set of published aphelid sequences using sequences of rozellids and nucleariids as an outgroup (Fig.\u0026nbsp;2). In our tree, all main aphelid clades are stable and our strains turned out to be in the basal clade (called here Clade I) containing environmental sequences only. X-138 and X-139 form a sister lineage with TAGIRI-24, which is an environmental sequence obtained from the East-China Sea (Takishita et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and with Jp13Nb17E and Jp13Nb11E, which are from Japanese freshwater lakes (Ishida et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorphology and life cycle\u003c/h2\u003e \u003cp\u003eOur newly described representative of the Aphelida has morphological peculiarities that are unknown for other aphelids. Its life cycle comprises two types of zoospores: swimming flagellates and crawling amoeboflagellates, with different lengths of flagella. One type of zoospore may transform to the other; we are not certain. We saw the beginning of the swimming form settling (Fig.\u0026nbsp;1T,U), but additional observations are needed to determine whether the swimming form settles and becomes an amoeboflagellate. Although the cell body of the swimming zoospore is similar in size as the amoeboflagellates, the length of the flagellum differs and is an important character distinguishing the two types of flagellates.\u003c/p\u003e \u003cp\u003eGenerally, change of locomotion from actively swimming to crawling amoeba occurs in \u003cem\u003eAphelidium\u003c/em\u003e and \u003cem\u003eParaphelidium\u003c/em\u003e before encystment, when zoospores settle down on algae filament, becoming amoeboflagellate with an immotile flagellum (Karpov et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Transformation of a swimming zoospore to the amoeboid form, when not as a part of infection process, has been documented for \u003cem\u003eAph. insulamus\u003c/em\u003e (Karpov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Fig.\u0026nbsp;2E,F). If no direct transformation between amoeboflagellate zoospores with short (11 \u0026micro;m) flagellum and spherical zoospores with long (17 \u0026micro;m) flagellum takes place, then we conclude that the two types of zoospores have different origins.\u003c/p\u003e \u003cp\u003eMembers of the Chytridiomycota and the Blastocladiomycota produce zoospores after sporangial cleavage and gametes after germination of a resting spore, where meiosis is proposed to occur (Sparrow \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). \u003cem\u003ePhysoderma gerhardti\u003c/em\u003e zoospores from germinating resting spores bear flagella 5\u0026ndash;10 \u0026micro;m longer than those on asexual zoospores (Sparrow \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Zoospores from asexual sporangia and zoospores from the resting spore of \u003cem\u003eSynchytrium endobioticum\u003c/em\u003e differ in their outline and the length of flagellum, which can be seen on the author\u0026rsquo;s plates (Curtis \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1921\u003c/span\u003e, Figs.\u0026nbsp;72 and 171), but was not noted by the author.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e and \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e gen. et sp. nov.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e (Schweikert and Schnepf, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e gen. et sp. nov. (present paper)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZoospore body length (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZoospore body width (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZoospore flagellum (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZoospore filopodia length (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUp to 11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfective cyst diam. (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple infection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCells after plasmodium \u003c/p\u003e \u003cp\u003ecleavage (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmoebae, 4\u0026ndash;10 in length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmoeboflagellates, 4 in length\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-infective cysts (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePenetration tube in\u003c/p\u003e \u003cp\u003enon-infective cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003estack cysts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enon-infective encystment out of the host\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eThallasiosira punctigera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRhizoclonium sp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalinity (g/l), locality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e~\u0026thinsp;28, Wadden Sea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e~\u0026thinsp;25, White Sea\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on this comparison, we suggest that aphelid X-138 zoospores with long flagella may be produced by germination of resting spores whereas amoeboflagellates with shorter flagella could represent asexual zoospores formed after plasmodium cleavage. Some authors also suggest that resting spore can be a place where sexuality occurs (Seto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Unfortunately, germination of aphelid resting spores has not been described so far and we know little about its nature. The resting spore derives directly from the plasmodium, which contains several nuclei as proposed earlier for \u003cem\u003eAmoeboaphelidium chlorellavorum\u003c/em\u003e by Gromov and Mamkaeva (1970). We interpreted several images of resting spores (Fig.\u0026nbsp;1U\u0026ndash;W) as consecutive stages of their maturation, as was suggested earlier for other aphelids (Karpov et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). During this process, the cell changes shape from oval to spherical and produces thick inner and thin outer wall with a residual body between them. Strain X-138 has a residual body at each pole of the spore. A few TEM images of \u003cem\u003eAph. insulamus\u003c/em\u003e resting spores revealed one nucleus and one or several prominent lipid globules (Karpov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on our observations we propose a general scheme of the life cycle as follow (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eTwo generations of zoospores have been described in \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e, another marine aphelid (Schweikert and Schnepf \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e): amoebae emanating from plasmodial division represent first generation; the second generation (the flagellates) appears after the first generation amoebae encyst; each cyst produces from 1 to 4 flagellates, depending on the amoeba\u0026rsquo;s size. The smallest amoebae (5 \u0026micro;m) yield one zoospore and the biggest amoebae (10 \u0026micro;m) yield up to four zoospores. We can explain this by incomplete plasmodium division resulting in amoeboid zoospores probably having 1\u0026ndash;4 nuclei.\u003c/p\u003e \u003cp\u003eThat is not the case for the strain X-138, which produces a first generation of amoeboflagellates of equal dimensions and the second one (after excystment) of similar spreading amoeboflagellate zoospores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCysts\u003c/h2\u003e \u003cp\u003eStacked cysts on a surface of algae were also found in \u003cem\u003eAmoeboaphelidium protococcorum\u003c/em\u003e (Gromov and Mamkaeva \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1970b\u003c/span\u003e) and \u003cem\u003eAph. parallelum\u003c/em\u003e (Seto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By analogy with some chytrids, this may be related to sexual reproduction that leads to formation of resting spores (Seto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e): one gamete encysts on the other encysted gamete followed by fusion of their contents (Canter \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Couch \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1935\u003c/span\u003e; Koch \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1951\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne more explanation of stacked cysts on the algal surface suggests zoospore encystment on the empty cyst surface, forms penetration tube and injects its contents through empty cyst into the alga. Therefore, if the cysts appear one by one there should up to ten or more, like Gromov and Mamkaeva (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1970b\u003c/span\u003e) observed. Probably, the cysts just use the previous penetration tube of the first cyst. Moreover, zoospores can encyst on the algal surface nearby an empty cyst and form penetration tube straight in the tube of the neighbor as we observed (Fig.\u0026nbsp;1E).\u003c/p\u003e \u003cp\u003eThis phenomenon might be caused by thick cell wall of the host alga, i.e., it is easier for parasite to use common cyst tube or empty cysts for penetration. In all mentioned cases \u003cem\u003eAmoeboaphelidium protococcorum\u003c/em\u003e (Gromov and Mamkaeva \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1970b\u003c/span\u003e), \u003cem\u003eAph. parallelum\u003c/em\u003e (Seto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and present paper the green algae with thick cell wall were infected, and we have never seen stacked cysts in the aphelids living on \u003cem\u003eTribonema gayanum\u003c/em\u003e having a gap in the cell wall.\u003c/p\u003e \u003cp\u003eIf the newly formed cyst just uses an empty tube of previous one, or the empty cyst, it makes nothing with resting spore formation. We really need to observe the cyst development and formation of resting spores to clarify the biological nature of the stacked cysts.\u003c/p\u003e \u003cp\u003eThe thick cell wall of \u003cem\u003eRhizoclonium\u003c/em\u003e can also cause difficulties for zoospores to release. Possibly, a capability of internal zoospores to encyst and penetrate through the cell wall is an adaptation of parasites to marine littoral algae. The intracellular encystment and formation of a tube through the host cell wall has been reported for \u003cem\u003eAph. insulamus\u003c/em\u003e (Karpov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), what means that it is common for aphelids. Numerous intracellular encysted cells of X-138 strain produce tubes through host cell obviously to release from the host. Although we did not observe the moment of zoospore release, the presence of many emptied intracellular cysts support this idea.\u003c/p\u003e \u003cp\u003eWe were lucky to capture the moment of simultaneous invasion of two amoebae from two cysts independently (Fig.\u0026nbsp;1B,C). This leads to formation of two trophonts, what we could often observe (Fig.\u0026nbsp;1F). This way of penetration was described for all known aphelids and studied with TEM (Schnepf et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Karpov and Paskerova \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Perhaps, further fusion of two trophonts occurs to produce a single plasmodium, as it was discussed recently for aphelids (Tcvetkova et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). That suggests a common phenomenon for aphelids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIdentification, molecular phylogeny and taxonomy\u003c/h2\u003e \u003cp\u003eIsolates X-138 and X-139 have nearly identical full 18S rDNA sequences, suggesting that they belong to one species even though these strains were isolated from different samples collected\u0026thinsp;~\u0026thinsp;700m from each other along the marine shore.\u003c/p\u003e \u003cp\u003eOur phylogenetic reconstruction saved all main aphelid clades together with paraphyletic genus \u003cem\u003eAphelidium\u003c/em\u003e and shows, that \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e gen. et sp. nov. is a single described species in the basal clade composed of environmental sequences only. This clade turns off the next after highly divergent \u003cem\u003eAph. collabens\u003c/em\u003e lineage (Seto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and called here Clade I. The SSU rDNA of strain X-138 differs from that of sister marine clone TAGIRI-24 by 10.57% and of the closest described species \u003cem\u003eAphelidium parallelum\u003c/em\u003e (11.83%). These differences indicate at least genus level, though it is well known that genetic divergence for species level for aphelids is also rather high (Karpov et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite a sister position to the marine clone, our strains have higher percentage of identity (90.22%) to an isolate under accession number OQ702871 from Seto et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (not present on Fig.\u0026nbsp;2), obtained from fresh water samples in Michigan. Based on the position of X-138 in a clade of environmental sequences, and its unique life cycle features, we declare it as a new genus and new species \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e Seliuk et Karpov.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePr. rhizoclonii\u003c/em\u003e gen. et sp. nov. resembles in some respects \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e, which parasitizes the marine diatom \u003cem\u003eThalassiosira punctigera\u003c/em\u003e, but essentially differs in others (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomy\u003c/h2\u003e \u003cp\u003eAmong the studied aphelids, strain X-138 is morphologically closest to \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e; both are marine and have two zoospore generations with encystment in between. Theoretically, X-138 could be a \u003cem\u003ePseudaphelidium\u003c/em\u003e species, but we choose not to place the new species in this genus because the phylogenetic position of \u003cem\u003ePseudaphelidium\u003c/em\u003e is unknown. Even if the genus \u003cem\u003ePseudaphelidium\u003c/em\u003e is represented by one of the published environmental sequences in Aphelida Clade I, the rDNA of \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e differs from the nearest environmental sequence in Clade I by more than 10%, which corresponds a genus level difference. Thus, we establish a new genus and species for strains X-138 and X-139 of this marine parasite.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtaphelidium\u003c/b\u003e Seliuk et Karpov, present paper.\u003c/p\u003e \u003cp\u003eAmoeboflagellate zoospores with a single opisthokont flagellum. Zoospore attaches to a host cell, encysts, penetrates into the cell interior, and develops into a phagocytotic plasmodium with central big digestion vacuole containing a prominent residual body composed of several lipid globules. Plasmodium cleaves to form uninucleate ameboflagellated cells, which encyst within the algal host. Cells release from algae through exit tubes and give rise to amoeboflagellate zoospores. Resting spore normally contains two opposite resting bodies between the inner and outer walls. Parasites of marine green algae predominantly.\u003c/p\u003e \u003cp\u003eEtymology: πρώτα (greek) \u0026ndash; the first, meaning the aphelid Clade I.\u003c/p\u003e \u003cp\u003eType species: \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e Seliuk et Karpov, present paper.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtaphelidium rhizoclonii\u003c/b\u003e Seliuk et Karpov. Type: Fig.\u0026nbsp;1, present paper.\u003c/p\u003e \u003cp\u003eZoospores 4 \u0026micro;m long and 2.6 \u0026micro;m wide, flagellum 11 \u0026micro;m long. Plasmodium cleaves to form globular cells from which the amoeboflates arise. They form cysts measuring 2.8 \u0026micro;m in\u003c/p\u003e \u003cp\u003ediameter, which penetrate host cell wall to release zoospores.\u003c/p\u003e \u003cp\u003eHost: The marine green alga \u003cem\u003eRhizoclonium\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eDistribution: sea coastal waters.\u003c/p\u003e \u003cp\u003eType locality: White Sea, near village Poyakonda, N66.5549\u0026deg;, E33.09907\u0026deg;.\u003c/p\u003e \u003cp\u003eHolotype: Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eType strain: X-138\u003c/p\u003e \u003cp\u003eEtymology: after generic name of the host alga.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNotes.\u003c/b\u003e Flagellated cells of \u003cem\u003ePr. rhizoclonii\u003c/em\u003e differ from \u003cem\u003ePs. drebesii\u003c/em\u003e by shorter flagellum (11 vs 15 \u0026micro;m), much smaller non-infective cysts (2.8 vs 6.5 \u0026micro;m), smaller cells after plasmodium cleavage (3 vs 5\u0026ndash;10 \u0026micro;m). Plasmodium of \u003cem\u003ePs. drebesii\u003c/em\u003e cleaves to form non-flagellated amoebae, which are slowly (for some hours) released from diatom frustule via the girdle region, and then encyst. Meanwhile the plasmodium of \u003cem\u003ePr. rhizoclonii\u003c/em\u003e produces amoeboflagellates that encyst intracellularly and are released from the host through exit tubes without division as in \u003cem\u003ePs. drebesii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003ePr. rhizoclonii\u003c/em\u003e both zoospore generations have flagella, while in \u003cem\u003ePs. drebesii\u003c/em\u003e the first generation has non-flagellated, amoeboid zoospores. Penetration tube in non-infective cysts and stack cysts were not shown for \u003cem\u003ePs. drebesii.\u003c/em\u003e And lastly, they have different hosts.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank WSBS named after by A.N. Pertsov for providing an opportunity to work. Authors express their indebtedness to A.I. Shestakov and to the staff of the department of microbiology of MSU for allowing us to use their equipment. We thank S.A. Poluzerov for help with sampling and A.E. Shipunova for modernization of our plankton mesh for a hand net. The authors are grateful to Joyce Longcore for manuscript discussion and correction. This work was supported by RSF grant 21-74-20089 (material collection, strain isolation, light microscopic studies, and writing), the Ministry of Science and Higher Education of the Russian Federation grant 075-15-2021-1069 (rDNA sequencing and molecular phylogeny), and made in the frame of lab topic 122031100260-0 ZIN RAS.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work supported by RSF grant 21-74-20089 and by grant of the Ministry of Science and Higher Education of the Russian Federation 075-15-2021-1069.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample collection, isolation and cultivation, light microscopic observations and sequencing were performed by Alexei Seliuk. Sergey Karpov contributed to the study conception and design. Both authors commented on previous versions of the manuscript, read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCurtis KM (1921) IX.\u0026mdash;The life-history and cytology of \u003cem\u003eSynchytrium endobioticum\u003c/em\u003e (Schilb.), Perc., the cause of wart disease in potato. Philosophical Trans Royal Soc Lond Ser B Containing Papers Biol Character 210(372\u0026ndash;381):409\u0026ndash;478\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanter HM (1969) Studies on British chytrids. XXIX. A taxonomic revision of certain fungi found on the diatom \u003cem\u003eAsterionella\u003c/em\u003e. Bot J Linn Soc 62(3):267\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCouch JN (1935) New or little known \u003cem\u003eChytridiales\u003c/em\u003e. Mycologia 27:160\u0026ndash;175\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGromov BV (2000) Algal parasites of the genera \u003cem\u003eAphelidium, Amoeboaphelidium\u003c/em\u003e, and \u003cem\u003ePseudaphelidium\u003c/em\u003e from the Cienkovski\u0026rsquo;s Monadea group as representatives of new class. Zool Zhurnal 79:517\u0026ndash;525 (in Russian)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGromov BV, Mamkaeva KA (1968) \u003cem\u003eAmoeboaphelidium protococcorum\u003c/em\u003e sp. n. and \u003cem\u003eAmoeboaphelidium chlorellavorum\u003c/em\u003e sp. n. \u0026ndash; endoparasites of protococcous algae. Acta Protozool 6:221\u0026ndash;225\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGromov BV, Mamkaeva КA (1970a) Electron-microscopic investigations of development cycle and feeding behaviour of intracellular algal parasite of \u003cem\u003eChlorella\u003c/em\u003e, \u003cem\u003eAmoeboaphelidium chlorellavorum\u003c/em\u003e. Tsitologiya 12:1191\u0026ndash;1196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGromov BV, Mamkaeva КA (1970b) The fine structure of \u003cem\u003eAmoeboaphelidium protococcarum\u003c/em\u003e \u0026mdash; an endoparasite of green alga \u003cem\u003eScenedesmus\u003c/em\u003e. Arch Hydrobiol 67:452\u0026ndash;459\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGromov BV, Mamkaeva КA (1975) Zoospore ultrastructure of \u003cem\u003eAphelidium chlorococcarum\u003c/em\u003e Fott. Mikologiya i fitopatologiya 9:190\u0026ndash;193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuillard RRL (1975) Culture of Phytoplankton for Feeding Marine Invertebrates. In: Culture of Marine Invertebrate Animals. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4615-8714-9_3\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4615-8714-9_3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. Deep Sea Res Oceanogr Abstracts 9:7\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0011-7471(62)90036-0\u003c/span\u003e\u003cspan address=\"10.1016/0011-7471(62)90036-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshida S, Nozaki D, Grossart HP, Kagami M (2015) Novel basal, fungal lineages from freshwater phytoplankton and lake samples. Environ Microbiol Rep 7(3):435\u0026ndash;441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1758-2229.12268\u003c/span\u003e\u003cspan address=\"10.1111/1758-2229.12268\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Mikhailov KV, Mirzaeva GS, Mirabdullaev IM, Mamkaeva KA, Titova NN, Aleoshin VV (2013) Obligately phagotrophic aphelids turned out to branch with the earliest-diverging fungi. Protist 164(2):195\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.protis.2012.08.001\u003c/span\u003e\u003cspan address=\"10.1016/j.protis.2012.08.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Mamkaeva MA, Aleoshin VV, Nassonova E, Lilje O, Gleason FH (2014) Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia. Front Microbiol 5:112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2014.00112\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2014.00112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Mamkaeva MA, Moreira D, L\u0026oacute;pez-Garc\u0026iacute;a P (2016) Molecular phylogeny of \u003cem\u003eAphelidium tribonemae\u003c/em\u003e reveals its sister relationship with \u003cem\u003eA\u003c/em\u003e. aff. \u003cem\u003emelosirae\u003c/em\u003e (Aphelida, Opisthosporidia). Protistology 10:97\u0026ndash;103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Tcvetkova VS, Mamkaeva MA, Torruella G, Timpano H, Moreira D, Mamanazarova KS, L\u0026oacute;pez-Garc\u0026iacute;a P (2017) Morphological and Genetic Diversity of Opisthosporidia: New Aphelid \u003cem\u003eParaphelidium tribonemae\u003c/em\u003e gen. et sp. nov. J Eukaryot Microbiol 64(2):204\u0026ndash;212\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Vishnyakov AE, L\u0026oacute;pez-Garc\u0026iacute;a P, Zorina NA, Ciobanu M, Tcvetkova VS, Moreira D (2020) Morphology and molecular phylogeny of \u003cem\u003eAphelidium insulamus\u003c/em\u003e sp. nov. Opisthosporidia) Protistology 14:191\u0026ndash;203Aphelida\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarpov SA, Paskerova GG (2020) The aphelids, intracellular parasitoids of algae, consume the host cytoplasm from the inside. Protistology 14(4):258\u0026ndash;263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Rozewicki J, Yamada KD (2018) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bib/bbx108\u003c/span\u003e\u003cspan address=\"10.1093/bib/bbx108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoch WJ (1951) Studies in the genus \u003cem\u003eChytridium\u003c/em\u003e, with observations on a sexually reproducing species. J Elisha Mitchell Sci Soc 67(2):267\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuraku S, Zmasek CM, Nishimura O, Katoh K (2013) aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Res 41 (Web Server issue). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkt389\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkt389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetcher PM, Lopez S, Schmieder R, Lee PA, Behnke C, Powell MJ, McBride RC (2013) Characterization of \u003cem\u003eAmoeboaphelidium protococcarum\u003c/em\u003e, an Algal Parasite New to the Cryptomycota Isolated from an Outdoor Algal Pond Used for the Production of Biofuel. PLoS ONE 8(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0056232\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0056232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetcher PM, Powell MJ (2019) A taxonomic summary of Aphelidiaceae. IMA Fungus 10(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43008-019-0005-7\u003c/span\u003e\u003cspan address=\"10.1186/s43008-019-0005-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Bork P (2021) Interactive tree of life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49(W1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkab301\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkab301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikhailov KV, Karpov SA, Letcher PM, Lee PA, Logacheva MD, Penin AA, Nesterenko MA, Pozdnyakov IR, Potapenko EV, Sherbakov DY et al (2022) Genomic analysis reveals cryptic diversity in aphelids and sheds light on the emergence of Fungi. Curr Biol 32(21). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2022.08.071\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2022.08.071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop, GCE. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1109/GCE.2010.5676129\u003c/span\u003e\u003cspan address=\"10.1109/GCE.2010.5676129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, H\u0026ouml;hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/sysbio/sys029\u003c/span\u003e\u003cspan address=\"10.1093/sysbio/sys029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchnepf E, Hegewald E, Soeder C-J (1971) Electronenmikroskopische eobachtungen an parasite aus \u003cem\u003eScenedesmus\u003c/em\u003e-massenkulturen. Arch Mikrobiol 75:209\u0026ndash;229\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchnepf E (1972) Strukturver\u0026auml;nderungen am Plasmalemma \u003cem\u003eAphelidium\u003c/em\u003e-infizierter \u003cem\u003eScenedesmus\u003c/em\u003e-Zellen. Protoplasma 75(1):155\u0026ndash;165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScherffel A (1925) Endophytische Phycomyceten-Parasiten der bacilleriaceen und einige neue Monadinen. Arch Protistenkd 52:1\u0026ndash;141\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweikert M, Schnepf E (1996) \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e, gen. et spec. nov. (incerta sedis), a parasite of the marine centric diatom \u003cem\u003eThalassiosira punctigera\u003c/em\u003e. Archiv Fur Protistenkunde 147(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0003-9365(96)80004-2\u003c/span\u003e\u003cspan address=\"10.1016/S0003-9365(96)80004-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweikert M, Schnepf E (1997) Electron microscopical observations on \u003cem\u003ePseudaphelidium drebesii\u003c/em\u003e Schweikert and Schnepf, a parasite of the centric diatom \u003cem\u003eThalassiosira punctigera\u003c/em\u003e. Protoplasma 199(3\u0026ndash;4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01294500\u003c/span\u003e\u003cspan address=\"10.1007/BF01294500\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeto K, Matsuzawa T, Kuno H, Kagami M (2020) Morphology, ultrastructure, and molecular phylogeny of \u003cem\u003eAphelidium collabens\u003c/em\u003e sp. nov.(Aphelida), a parasitoid of a green alga \u003cem\u003eCoccomyxa\u003c/em\u003e sp. Protist 171(3):125728\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeto K, Nakada T, Tanabe Y, Yoshida M, Kagami M (2022) \u003cem\u003eAphelidium parallelum\u003c/em\u003e, sp. nov., a new aphelid parasitic on selenastracean green algae. Mycologia 114(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/00275514.2022.2039487\u003c/span\u003e\u003cspan address=\"10.1080/00275514.2022.2039487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeto K, Simmons DR, Quandt CA, Frenken T, Dirks AC, Clemons RA, McKindles KM, McKay RML, James TY (2023) A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi. MBio 14(4):e0131323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.01313-23\u003c/span\u003e\u003cspan address=\"10.1128/mbio.01313-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparrow FK (1960) Aquatic Phycomycetes. Ann Arbor Univ Michigan, p 1187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparrow FK (1977) Observations on chytridiaceous parasites of phanerogams. XXVI. \u003cem\u003ePhysoderma gerhardti\u003c/em\u003e Schroeter on \u003cem\u003ePhalaris arundinacea\u003c/em\u003e L. Arch Microbiol 114:241\u0026ndash;247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btu033\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btu033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrittmatter M, Guerra T, Silva J, Gachon CMM (2016) A new flagellated dispersion stage in \u003cem\u003eParaphysoderma sedebokerense\u003c/em\u003e, a pathogen of \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e. J Appl Phycol 28(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10811-015-0700-8\u003c/span\u003e\u003cspan address=\"10.1007/s10811-015-0700-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakishita K, Miyake H, Kawato M, Maruyama T (2005) Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny. Extremophiles 9(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00792-005-0432-9\u003c/span\u003e\u003cspan address=\"10.1007/s00792-005-0432-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTcvetkova VS, Pozdnyakov IR, Seliuk AO, Zorina NA, Karpov SA (2023) Vegetative cell fusion and a new stage in the life cycle of the Aphelida (Opisthosporidia). J Eukaryot Microbiol 70(5):12977. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jeu.12977\u003c/span\u003e\u003cspan address=\"10.1111/jeu.12977\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorruella G, Grau-Bov\u0026eacute; X, Moreira D, Karpov SA, Burns JA, Seb\u0026eacute;-Pedr\u0026oacute;s A, V\u0026ouml;lcker E, L\u0026oacute;pez-Garc\u0026iacute;a P (2018) Global transcriptome analysis of the aphelid \u003cem\u003eParaphelidium tribonemae\u003c/em\u003e supports the phagotrophic origin of fungi. Commun Biology 1(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-018-0235-z\u003c/span\u003e\u003cspan address=\"10.1038/s42003-018-0235-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWijayawardene NN, Hyde KD, Mikhailov KV, G\u0026aacute;bor P, Aptroot A, Pires-Zottarelli CLA, Goto BT, Tokarev YS, Haelewaters D, Karunarathna SC et al (2024) Higher level classification of the Kingdom \u003cem\u003eFungi\u003c/em\u003e. Fungal Diversity (in press)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Yun J, Liu X, Du W, Xiang M (2023) Niche and ecosystem preference of earliest diverging fungi in soils. Mycology 14(3):239\u0026ndash;255\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZopf W (1885) Zur Morphologie und Biologie der niederen Pilzthiere (Monadinen), zugleich ein Beitrag zur Phytopathologie. Verlag von Veit \u0026amp; Comp, Leipzig, Germany\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycological-progress","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mypr","sideBox":"Learn more about [Mycological Progress](https://www.springer.com/journal/11557)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mypr/default.aspx","title":"Mycological Progress","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aphelidiomycota, marine fungi, molecular phylogeny, taxonomy, Protaphelidium rhizoclonii","lastPublishedDoi":"10.21203/rs.3.rs-4241557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4241557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAphelids are a poorly known group of algal parasites that have raised considerable interest because of their pivotal phylogenetic position as a sister lineage to the kingdom Fungi. Recent breakthroughs in sampling, sequencing and bioinformatical analyses of environmental nucleic acids have revealed magnificent biodiversity of aphelids. Nevertheless, only 4 genera have been described (\u003cem\u003eAphelidium\u003c/em\u003e, \u003cem\u003eParaphelidium\u003c/em\u003e, \u003cem\u003eAmoeboaphelidium\u003c/em\u003e and \u003cem\u003ePseudaphelidium\u003c/em\u003e); 18S rRNA gene sequences are published for all except for the marine genus \u003cem\u003ePseudaphelidium.\u003c/em\u003e Most of the environmental nucleic acid data is from analysis of freshwater samples. We isolated two new marine aphelid strains (X-138 and X-139), and herein describe the life cycle of \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e gen. et sp. nov., a parasite of the green alga \u003cem\u003eRhizoclonium\u003c/em\u003e sp., and provide the first 18S rRNA gene sequences for cultivated marine aphelids. The new marine aphelid life cycle is mostly typical for aphelids, but also includes previously undescribed stages. Molecular phylogenetic analysis indicated that \u003cem\u003eProtaphelidium rhizoclonii\u003c/em\u003e is a member of an environmental clade at the base of the aphelid tree.\u003c/p\u003e","manuscriptTitle":"Marine parasite Protaphelidium rhizoclonii gen. et sp. nov. designates the basal environmental cluster at the aphelid phylogenetic tree","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 18:40:19","doi":"10.21203/rs.3.rs-4241557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-20T10:24:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-12T06:49:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycological Progress","date":"2024-04-11T21:07:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-11T03:55:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycological Progress","date":"2024-04-10T01:52:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycological-progress","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mypr","sideBox":"Learn more about [Mycological Progress](https://www.springer.com/journal/11557)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mypr/default.aspx","title":"Mycological Progress","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"591fc7ae-1f20-44b1-a78c-caae157a94e4","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:00:56+00:00","versionOfRecord":{"articleIdentity":"rs-4241557","link":"https://doi.org/10.1007/s11557-024-01998-6","journal":{"identity":"mycological-progress","isVorOnly":false,"title":"Mycological Progress"},"publishedOn":"2024-09-27 15:57:10","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-04-17 18:40:19","video":"","vorDoi":"10.1007/s11557-024-01998-6","vorDoiUrl":"https://doi.org/10.1007/s11557-024-01998-6","workflowStages":[]},"version":"v1","identity":"rs-4241557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4241557","identity":"rs-4241557","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}