Podomonas kaiyoae n. sp., a novel apusomonad growing axenically

Apusomonadida (apusomonads) is a group of heterotrophic biflagellates that feed on bacteria and small protists. Their diversity is not fully understood, and several major lineages remain to be identified in natural environments. Here, we report Podomonas kaiyoae n. sp., which was isolated from deep‐sea sediment and can be maintained as an axenic culture. While P. kaiyoae branched within one of the major unidentified lineages, the combination of the morphological characteristics is generally similar to that of Podomonas species, but can be distinguished from that of other Podomonas species based on the cell sizes.

APUSOMONADIDA (apusomonads) is a subgroup of Obazoa, and comprising heterotrophic flagellates that feed on bacteria and other small protists (Adl et al., 2019;Heiss et al., 2017). Because of their phylogenetic position among eukaryotes, their study is essential to understanding the origin and early evolution of metazoans. All established cultures of Apusomonadida thus far have been maintained along with prey bacteria, and their axenic culture has never been reported to date. Taxonomically, 22 species belonging to seven genera have been described, characterized by combinations of different morphological characteristics, such as the existence of a prominent pseudopodium, proboscis, which is the combined structure of the anterior flagellum and an associated sleeve-like extension of the cell body, and varied cell shape (Heiss et al., 2017). Nevertheless, their diversity is not fully understood, and many lineages represented only by environmental DNA (eDNA) sequences still remain to be identified in natural environments. A complete analysis of eDNA sequences performed by Torruella et al. (2017) revealed the presence of 30 independent clades (i.e. APU-01 to 30) in Apusomonadida.
Of these, only eight clades were characterized by previously described species, whereas the other 22 clades did not contain any species for which molecular sequences were available. Identification of members belonging to these 22 clades is awaited to understand the diversity of Apusomonadida more precisely.

Isolation and cultivation
A deep-sea sediment sample was collected off Sanriku (39.246067°N, 142.299683°E) at a depth of 762 m on May 4, 2015, during the KY15-08 cruise of the research vessel Kaiyo. The sample was subjected to initial incubation in Hemi medium (which contains 1% v/v horse serum and 0.1% v/v LB medium as organic carbon sources; Tashyreva et al., 2018), without any antibiotics at 19-20°C for ca. 24 h. A single cell of Podomonas kaiyoae n. sp. was carefully isolated from the incubated sample by micropipetting, washed in the artificial sea water twice, and then transferred into 200 μl Hemi medium. The established strain (YPF1504) was maintained under the same conditions as the initial incubation, and the absence of contaminating bacterial cells in the culture (i.e. axenic culture) was confirmed by careful microscopic observation and PCR using the extracted total DNA with universal bacterial primers ( Figure S1). The axenic culture was deposited to the National Institute for Environmental Studies (NIES, Tsukuba, Japan) as NIES-4540. The cells that grew under axenic conditions were transferred into KLB medium (Yabuki & Tame, 2015) in which unidentified bacterial contaminants from the culture of Microheliella maris deposited as CCAP 1945/1 (Yabuki et al., 2012) culture were preincubated. The cells that grew with the bacteria were kept in the KLB medium as the nonaxenic culture of P. kaiyoae.

Microscopy
Living cells of P. kaiyoyae growing under axenic conditions and with prey bacteria were observed under a BX43 microscope (Olympus) equipped with a digital camera (X9 super system; Micronet Inc.). For scanning electron microscopy, the axenic cells that grew on a glass slide were fixed with 2.5% glutaraldehyde in cultivation medium at 4°C. The fixed cells were washed with filtered artificial sea water (FASW) and then postfixed with 2.0% osmium tetroxide dissolved in FASW for 2 h. The postfixed cells were dehydrated using a graded ethanol series and dried with a JCPD-5 critical point drying device (JEOL), then coated with osmium using an OPC-80 osmium coater (Filgen). The specimens were imaged using a field-emission SEM (Quanta 450 FEG; FEI) operating at 5 kV.

18S rRNA gene sequencing and phylogenetic analyses
Total DNA was extracted from axenic cells using a DNeasy Plant Mini Kit (Qiagen K. K.). The nearcomplete 18S rRNA gene sequence was amplified from the DNA samples using PCR with the eukaryotespecific primers Euk1A (Sogin & Gunderson, 1987) and EukB (Medlin et al., 1988). PCR consisted of 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. Amplified DNA fragments were cloned using a StrataClone PCR Cloning Kit (Agilent Technologies), and the clones were sequenced on both the strands. The 18S rRNA gene sequence of P. kaiyoae determined in this study has been deposited in GenBank (accession number: LC662742).
The 18S rRNA gene sequence of P. kaiyoae was added to the sequence alignment analyzed by Torruella et al. (2017) and manually aligned using Mesquite (Maddison & Maddison, 2011). The alignment was trimmed in accordance with the sites analyzed by Torruella et al. (2017), and several gappy sites were additionally removed to reconstruct the dataset. A maximum likelihood tree and bootstrap values from 1000 replicates were obtained using RaxML-NG v. 1.1.0 (Stamatakis, 2006) with the GTR + Γ + I model. Bayesian analysis was run using MrBayes v3.2.7a (Ronquist et al., 2012) with the GTR + Γ + I model. Markov chain Monte Carlo (one cold and three heated) with default chain temperatures was run for 1 × 10 6 generations, with sampling trees at 100-generation intervals. The first 2.5 × 10 5 generations were discarded as "burn-in" because it was confirmed that the scores of the sampled log-likelihood plateaued before 2.5 × 10 5 generations.

R E SU LT S A N D DI SC US SION
The cells of P. kaiyoae glided on the dish bottom trailing the posterior flagellum, while they occasionally settled and attached to the bottom using developed pseudopodia ( Figure 1A) and showed contractingextending movements (Movie S1). The cell size excluding the pseudopodium was 13.49 ± 1.01 μm in length and 5.56 ± 0.67 μm in width (N = 13). The short anterior flagellum emerged from the subapical area and its base was covered by a sleeve ( Figure 1A). The anterior flagellum measured approximately half the cell length and always showed a nodding motion during gliding. The trailing posterior flagellum occasionally overlapped with the pseudopodium. The length of the posterior flagellum, including the part overlapping with the cell, was approximately 1.5-2× times the length of the cell (Movie S1). The pseudopodium that emerged from the posterior end of the settled cells extended into a few thin branches ( Figure 1A). Podomonas kaiyoae grew axenically in the Hemi medium, whereas they grew feeding on prey bacteria in the KLB medium. The feeding cells were 12.77 ± 0.94 μm long and 4.83 ± 0.91 μm wide (N = 13), which was not distinguishable from the size of the axenic cells. However, vacuoles and granules were more abundant in feeding cells than in axenic cells, and a line of refractile granules was also observed ( Figure 1B). As the pseudopodium was rarely observed in the feeding cells, these cells did not attach and only glided on the bottom. In addition, the frequency of contracting-extending movements was lower than that in axenic cells (Movie S2). Although the acroneme was not clearly recognizable under light microscope ( Figure 1A,B, Movies S1 and S2), it was recognized at the end of both flagella under scanning electron microscope (SEM) ( Figure 1C). There was a variation in the length of acronemes ( Figure S2), but it may have been caused by a fixation artifact. An anterior projection of the cell body, which is called "tusk" was also recognized

Thecamonas oxonienis EU542598
Podomonas kaiyoae n. sp. LC662742 0.09 under SEM ( Figure 1D). Small nodules were irregularly arranged on the cell surface ( Figure 1C-E) and they were possibly undischarged extrusomes. The possible discharged extrusomes were also recognized ( Figure 1E). Other structures decorating and/or supporting the cell membrane, such as distinct thecal plates, were not observed. Phylogenetic analysis showed that P. kaiyoae branched within the clade that was labeled "APU-11" by Torruella et al. (2017) with high statistical support (90% ML bootstrap value and 1.00 Bayesian posterior probability, Figure 1F). The sequences showing the closest affinity to P. kaiyoae with 91% ML bootstrap value and 1.00 Bayesian posterior probability also came from deep-sea sediments, which is consistent with the fact that P. kaiyoae was found in deep-sea sediments ( Figure 1F). Although APU-11 branched relatively close to APU-10, 12, 13, and 14, including Podomonas species, neither their monophyly nor their close relationship was well supported ( Figure 1F).
In contrast, such a pseudopodium was not prominent in the feeding cells of P. kaiyoae, whereas a prominent pseudopodium has been observed in the cells of the vegetative stage (i.e. feeding stage) in the other apusomonads. The pseudopodium of P. kaiyoae might have extended to increase the cell surface for efficient acquisition of nutrients from the medium. The line of the refractile granules and the reduced sleeve, which are recognized as key characteristics of the genus Podomonas (Cavalier-Smith & Chao, 2010;Heiss et al., 2017), were also shared with P. kaiyoae. The acroneme of the anterior flagellum, which is also the diagnostic characteristics of Podomonas (Cavalier-Smith & Chao, 2010), was hardly detected under the light microscope; however, SEM observation showed that P. kaiyoae also possessed the acroneme at the tip of both flagella. The tusk was a shared characteristic with the members of Thecamonas, Podomonas, and Chelonemonas (Heiss et al., 2015). Extrusomes were reported from Multimonas only, and the distribution pattern and the discharged form of the extrusomes of P. kaiyoae is very similar to those of Multimonas. Considering the combination of morphological characteristics, P. kaiyoae is unique in Apusomonadida, but relatively similar to previously described members of Podomonas. A possible close relationship between Podomonas and P. kaiyoae was also suggested by the phylogenetic analysis, although there was no statistical support. Hence, we concluded that P. kaiyoae should be treated as a member of genus Podomonas in this study, while the existence of the extrusomes is notable and this taxonomic assignment may need to be updated in future studies. As five species have been described in genus Podomonas to date (Cavalier-Smith & Chao, 2010;Myľnikov, 1999) and all of them can be distinguished from P. kaiyoae based on their cell sizes (Table S1), P. kaiyoae should be described as a new species of the genus Podomonas. In addition, P. kaiyoae was shown to grow on Hemi medium alone. Thus, to the best of our knowledge, this is the first report of an axenic culture of Apusomonadida. Such an axenic culture may be useful in various future studies on the physiology of apusomonads, and it is possible that other unidentified apusomonads could be discovered and established as cultures on Hemi medium.
Hapantotype: ATNS-AL-58990, a stub mounting preserved cells suitable for viewing by scanning electron microscopy, is deposited in the herbarium of the National Museum of Nature and Science, Tsukuba, Japan.
Etymology: The species name was derived from the R/V Kaiyo, which collected the sample to find this species, and has now been retired.

AC K NOW L E DGM E N T S
We thank Dr. Shinji Tsuchida (JAMSTEC) for managing the KY15-08 cruise as the chief scientist and providing the initial material (deep-sea sediment sample), Dr. Katsunori Fujikura (JAMSTEC) for managing MEXT Tohoku Ecosystem-Associated Marine Sciences Project (Grant Number JPMXD1111105260) in which the KY15-08 cruise was conducted, and the Captain and crew of the R/V "Kaiyo" and the ROV "Hyper-Dolphin" team. We appreciate Dr. Guifré Torruella (IRB Barcelona) for sharing the alignment file of Apusomonadida. This study was partially supported by grants from the Japan Society for the Promotion of Science (20K06792) awarded to AY and the Institute of Fermentation, Osaka (G-2020-1-014) awarded to AY. ORC I D Akinori Yabuki https://orcid. org/0000-0002-4070-0226