1 Pseudophyllomitus vesiculosus (Larsen and Patterson 1990) Lee, 2002, a poorly studied 2 phagotrophic biflagellate is the first characterized member of stramenopile environmental 3 clade MAST-6 4 5 Takashi Shiratoria,1, Rabindra Thakurb, and Ken-ichiro Ishidaa 6 7 aFaculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 8 305-8572, Japan 9 b Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 10 Ibaraki 305-8572, Japan 11 12 Running title: A first characterized member of MAST-6 13 1Corresponding Author: Fax: +81 298 53 4533 E-mail: [email protected] (T. Shiratori) 1 1 Abstract 2 There are many eukaryotic lineages that exclusively composed of environmental sequences 3 and lack information about which species are included. Regarding stramenopiles, at least 18 4 environmental lineages, known as marine stramenopiles (MAST), have been recognized. 5 Since each MAST lineage forms deep branches in the stramenopiles, the characterization of 6 MAST members is key to understanding the diversity and evolution of stramenopiles. In 7 this study, we established a culture of Pseudophyllomitus vesiculosus, which is a poorly 8 studied phagotrophic flagellate of uncertain taxonomic position. Our molecular 9 phylogenetic analyses based on small subunit ribosomal RNA gene sequences robustly 10 supported the inclusion of P. vesiculosus in the MAST-6 clade. Our microscopic 11 observations indicated that P. vesiculosus shared characteristics with stramenopiles, 12 including an anterior flagellum that exhibits sinusoidal waves and bears tubular 13 mastigonemes. The flagellar apparatus of P. vesiculosus was also similar to other 14 stramenopiles in having a transitional helix and five microtubular roots (R1–R4 and S 15 tubules) including R2 that split into two bands. On the other hand, P. vesiculosus was 16 distinguished from other deep-branching stramenopiles by the combination of flagellar 17 apparatus characteristics. Based on the phylogenetic analyses and microscopic observations, 18 we established Pseudophyllomitidae fam. nov in stramenopiles. 19 Key words: flagellar apparatus; MAST; phylogenetic analysis; stramenopiles; 20 ultrastructure 2 1 Introduction 2 Environmental DNA surveys focusing on small subunit ribosomal RNA (SSU rRNA) gene 3 sequences have uncovered the great diversity of protists (Bass and Cavalier-Smith 2004; 4 Lara et al. 2009; Lopez-Garcia et al. 2001). These environmental DNA surveys have 5 revealed not only the cryptic diversity of previously described taxa (Cavalier-Smith and 6 Von der Heyden, 2007; Holzmann et al. 2003; Lara et al. 2009), but also novel 7 environmental lineages that could not be assigned to any described groups (Choi et al. 8 2017; Kim et al. 2016). Culture-independent studies, such as fluorescence in situ 9 hybridization (FISH) and single cell based genome sequencing are effective approaches to 10 characterize these environmental lineages, and the methods have provided important 11 information, including cell shape and size, presence or absence of plastids, and mode of 12 nutrition (Jones et al. 2011; Yoon et al. 2011). However, traditional culture-based 13 taxonomic studies are also important for improved characterization of these environmental 14 lineages (del Campo et al. 2016; Moreira and López-García 2014). This approach provides 15 detailed morphological and ultrastructural information that complements 16 culture-independent studies and aids our understanding of the ecological role and 17 evolutionary history of targeted organisms. 18 Stramenopiles are a large eukaryotic assemblage characterized by the presence of 19 tripartite or bipartite tubular hairs (mastigonemes) on the anterior flagellum, and the group 20 includes various organisms ranging from multicellular seaweeds (Phaeophyceae) and 21 parasitic or saprotrophic fungus-like organisms (e.g., Hyphochytrea and Oomycetes) to 22 diverse photosynthetic and heterotrophic protists (e.g., Bikosea, Chrysophyceae, and 3 1 Bacillariophyceae). The stramenopiles also include several environmental lineages that are 2 not classified in any described groups, which have been described as MArine 3 STramenopiles (MAST) or Mystery Heterokont (MH) (Massana et al. 2004; Orsi et al. 4 2011; Richards and Bass 2005). Massana et al. (2014) rearranged newly and previously 5 described environmental lineages into 18 MAST groups (MAST-1, -2, -3, -4, -6, -7, -8, -9, 6 -10, -11, -12, -16, -20, -21, -22, -23, -24, and -25) by excluding synonyms and lineages that 7 are represented by chimeric sequences. 8 Molecular phylogenetic analyses using SSU rRNA gene sequences indicated that 9 most MAST lineages formed deep branches within the stramenopiles (Cavalier-Smith and 10 Scoble 2013; Massana et al. 2014). Therefore, the characterization of MAST species is key 11 to understanding the diversity and evolution of stramenopiles. Investigations of MAST 12 species to date have been mainly performed using culture independent approaches. 13 Environmental DNA surveys showed that the abundance of MAST sequences reached 14 around 20% of total picoeukaryotes in the surface water of open oceans (Massana et al. 15 2004), while some MAST lineages were specific to anaerobic water column or sediment 16 (Massana et al. 2014). FISH experiments using lineage-specific oligonucleotide probes and 17 incubation experiments revealed approximate cell size, habitat, and the prey of some 18 MAST members (Kolodziej and Stoeck 2007; Massana et al. 2009; Piwosz and Pernthaler 19 2010). On the other hand, culture-dependent approaches for the identification of MAST 20 members have not been frequently performed and only a few MAST species have been 21 identified so far (MAST-3: Incisomonas marina and Solenicola setigera) (Cavalier-smith 22 and Scoble 2013; Gómez et al. 2011). 4 1 In this study, we established a culture of Pseudophyllomitus vesiculosus (strain 2 SRT537) from marine detritus on the tidal flat sediment of Amami Island, Okinawa, Japan. 3 Pseudophyllomitus is a genus of heterotrophic free-living flagellates established by Lee 4 (2002), and it includes four species that were reassigned from Phyllomitus based on 5 morphological differences. Pseudophyllomitus has a sac-shaped cell body and two 6 heterodynamic flagella that emerge from a subapical gullet or pocket (Lee 2002). Although 7 Pseudophyllomitus spp. were reported in field surveys of marine sediment samples (Aydin 8 and Lee 2012; Larsen and Patterson 1990; Lee 2015; Lee and Patterson 2000), molecular 9 and ultrastructural data of this genus are still unavailable, thus the taxonomic position is 10 uncertain. Our molecular phylogenetic analysis using SSU rRNA gene sequences showed 11 that P. vesiculosus is a member of the MAST-6. We also performed microscopic 12 observations of P. vesiculosus, and compared the morphology and ultrastructure with other 13 deep-branching stramenopiles for MAST-6 characterization. 14 5 1 Results 2 Light microscopic observation 3 The cells of P. vesiculosus strain SRT537 were flexible, sac-shaped, and slightly 4 dorsoventrally flattened (Fig. 1A, B). Cell length was 9.3–18.3 µm (13.58 ± 1.95 µm, n = 5 62), and the width was 6.3–12.4 µm (8.89 ± 1.31 µm, n = 62). Two flagella were inserted 6 subapically, and were arranged at a right angle (Fig. 1A–D). The anterior flagellum was 7 14.4–24.9 µm (19.31 ± 2.10 µm, n = 58), curved over the right side of the cell, and it 8 exhibited sinusoidal waves during swimming. The posterior flagellum was as long as or 9 slightly longer than the cell, and it trailed behind the cell body. Cells had many large 10 vesicles (approximately 1.1–2.4 µm in diameter) in the peripheral region (Fig. 1A–E). Cells 11 occasionally included lipid globules that were probably derived from prey diatoms (Fig. 1A, 12 B, D). A nucleus with a nucleolus was positioned at the center or slightly anterior region of 13 the cell (Fig. 1A, B). A rod or bar laid against anterior side of the nucleus (Fig. 1A). Cells 14 swam immediately above the substrate with attaching the tip of the posterior flagellum to 15 the substrate or in the water column with rotating movements. 16 Transmission electron microscopic observation 17 Whole-mount observation using a transmission electron microscope showed that P. 18 vesiculosus strain SRT537 had bipartite tubular mastigonemes on the anterior flagellum 19 (Fig. 1F). The tubular mastigoneme consisted of a shaft and a terminal filament (Fig. 1G, 20 H). The posterior flagellum was acronematic (Fig. 1F). 21 In ultrathin section observation, thin extensions were observed on the cell surface 22 (Fig, 2A). The extensions considered to be fixation artifacts because they were not 23 observed in light microscopy (Fig. 1A-E) and whole-mount observation (Fig. 1F) as well as 6 1 ultrathin section observation using chemical fixation specimen (not shown). Except for 2 the extensions, no appendages (e.g., cell wall, cell coat, and scales) were observed on cell 3 surface (Fig. 2A). A large nucleus with a conspicuous nucleolus was observed at the middle 4 to anterior region of the cell (Fig. 2A). A Golgi apparatus was located at the anterior region 5 of the cell, along the anterior side of the nucleus (Fig. 2A). Large vesicles that occasionally 6 contained amorphous inclusions were arranged along the peripheral region of the cell (Fig. 7 2A). Several round or oval mitochondrial profiles with tubular cristae were scattered 8 throughout the cell (Fig. 2A, B). Amorphous microbodies, including dense dots were 9 closely associated with the nucleus (Fig. 2C). The endoplasmic reticulum (ER) around the 10 nucleus included bunches of mastigonemes (Fig. 2D). Small vesicles containing electron 11 dense inclusions were observed just beneath the plasma membrane (Fig. 2E). 12 The flagellar transitional region had a dense transitional plate and a single 13 transitional helix with 10–12 gyros that were positioned at the distal side of the plate (Fig. 14 3A, B). Two basal bodies were arranged at approximately 70–90° (Fig. 3C, D). The basal 15 bodies were not in the same plane, and the right side of the posterior basal body was closely 16 associated with the left side of the anterior basal body (Fig.