A New Record of Oxytricha granulifera granulifera Foissner and Adam, 1983 (Protozoa, Ciliophora, Oxytrichidae) From a Hot Spring in Iceland, With Notes on Its Abnormal Form During Cultivation

We isolated a population of Oxytricha granulifera granulifera Foissner and Adam (1983) from a hot spring in Iceland. The pure culture of this isolate was established at room temperature in the laboratory. This allowed for a detailed investigation, informed by integrated approaches, of the isolate’s morphology and morphogenesis, as well as molecular phylogeny. Results showed that the morphological and morphogenetic characteristics of the Iceland population are consistent with those of other populations. During the 3-year long period of laboratory cultivation, some abnormal individuals appeared repeatedly in the culture system. Interestingly, the morphological characteristics of these abnormal cells were rather stable, and were as follows: 1) body slender and elliptical-shaped; 2) remarkably shortened adoral zone and significantly reduced number of adoral membranelles; and 3) loss of undulating membranes. Resting cysts, binary fission and conjugate reproduction were not found in abnormal specimens. Although the morphology of abnormal individuals changed significantly, the sequences of the SSU rDNA of the normal and abnormal morphotypes were the same. Phylogenetic analyses showed that the two morphotypes clustered in a clade with other populations of O. granulifera granulifera.

During a protistan fauna investigation, one encysted ciliate was isolated from a geothermal field (hot spring) in Iceland. This isolate was identified as O. granulifera granulifera Foissner and Adam (1983). Its pure culture was established at room temperature (20-25 • C) in Petri dishes, with rice grains to enrich the bacterial food. During the cultivation, some abnormal individuals appeared repeatedly in the system. Although the abnormal individuals appeared at different times, their morphological characteristics remained consistent, which made it easier to mistakenly deduct that a "new" species had been discovered. Oxytricha granulifera has not been recorded from hot springs, especially the abnormal forms in long-term indoor culture system have not been reported, therefore, the morphology and SSU rDNA sequences of the normal and abnormal forms are described in this article, and the morphogenesis of the normal form is also reported.

Sampling and Cultivation
The sludge sample was collected from a microbial mat in a geothermal field in Iceland near the Hellisheiðarvirkjun geothermal power station (64 • 01 11.5 N, 21 • 23 50.5 W), as described in Qu et al. (2018). The temperature was approximately 75 • C at the sampling site. The surface sediments were transferred to Petri dishes in water from the sample site and maintained as a raw culture in the laboratory for several days at room temperature (about 25 • C). The salinity (0 ) of the hot spring water was measured on site with a portable refractometer. The pH level (7.3) was measured using portable pH sensors. In the laboratory, the pure cultures of O. granulifera granulifera were established and maintained using volvic water and sterilized wheat grains. Wheat grains enriched bacteria as a food source for the ciliate. Clonal cultures were obtained by gradually transferring single ciliates from the enrichment culture to artificially distilled freshwater . One to two wheat grains per 20 mL cell culture were added to support the growth of bacteria. To maintain this population, approximately once every 2 weeks, several cells were picked up and transferred into a new Petri dish containing new water and rice grains to establish a new pure cultivation. The attempt to establish pure cultures using abnormal individuals solely failed, because the abnormal individuals gradually disappeared over the course of about one week. The pH level of the culture systems was 6.0-7.0.

Morphological and Ontogenetic Investigations
Live observations were carried out using a bright field microscope (Olympus BX53) equipped with differential interference contrast (DIC). The protargol silver staining method of Wilbert (1975) was used to reveal the ciliature and the nuclear apparatus. Protargol was synthesized mainly according to Pan et al. (2013). Drawings of stained specimens were conducted at a magnification of 1,000× with the aid of a camera lucida. Measurements were made with an ocular micrometer. To illustrate the changes that occur during ontogenetic processes, ciliary structures of parental cells were depicted by contours, whereas those of the daughter cells were shaded black. The terminology used herein follows Berger (1999) and Song and Shao (2017).

DNA Extraction, PCR Amplification, and Sequencing
Total genomic DNA of the normal and abnormal forms of O. granulifera granulifera were extracted from single cells in pure cultures, using the DNeasy Blood and Tissue Kit (Qiagen, Gmbh, Germany) according to the manufacturer's instructions. Each cell was washed three times with distilled water and two times in ultra-pure water in order to remove contaminants and then was immediately transferred to 1.5-mL microfuge tubes with an ATL buffer (Jin et al., 2020). PCR amplifications of SSU rDNA were performed with universal eukaryotic primers 18S-F (5 -AAC CTG GTT GAT CCT GCC AGT-3 ) and 18S-R (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) (Medlin et al., 1988). The amplification conditions were as follows: a pre-run of 30 s at 98 • C followed by 35 cycles consisting of a denaturation at 98 • C for 30 s, an annealing at 60 • C for 20 s, and an extension at 72 • C for 1 min. After 35 cycles, the final extension step was run at 72 • C for 2 min. The PCR products were sequenced by Beijing Genomics Institute in Shenzhen, China. Three internal primers, 900S-F (5 -CGA TCA GAT ACC GTC CTA GT-3 ), 900S-R (5 -ACT AGG ACG GTA TCT GAT CG-3 ) and B (5 -AAY CTG GTT GAT YYT GCC AG-3 ) were used in sequencing.

Phylogenetic Analyses
Apart from the newly sequenced SSU rDNA sequences of the normal and abnormal forms of O. granulifera granulifera in this article, all of the sequences used in the phylogenetic analyses were obtained from the GenBank database (accession numbers see Figure 4). Novistrombidium orientale, Parastrobidinopsis minima, Strombidinopsis acuminata, and Strombidium apolatum were selected as outgroups since they were used to construct the phylogenies recently reported by Méndez-Sánchez et al. (2018) and Liao et al. (2020). Sequences were aligned using the MUSCLE package on the European Bioinformatics Institute web page 1 . Both ends of the alignment were then trimmed manually using BioEdit 7.0.9.1, resulting in a final matrix of 1,579 nucleotide positions. A maximum-likelihood (ML) tree was constructed using RAxML-HPC2 on XSEDE v.8.2.10 (Stamatakis, 2014) on the CIPRES Science Gateway (Miller et al., 2010). The model GTR + I + G was used. Support for the best ML tree came from 1,000 bootstrap replicates (Jin et al., 2020). Bayesian inference (BI) analysis was carried out on CIPRES Science Gateway using the MrBayes 3.2.6 on XSEDE (Ronquist and Huelsenbeck, 2003). TreeView v1.6.6 (Page, 1996) and MEGA 7.0 (Kumar et al., 2016) were used to visualize tree topologies.

Morphology of Normal Forms
Size 90-130 × 30-45 µm (average 110 × 35 µm, n = 10) in vivo, and 79-135 × 28-61 µm (average 102 × 42 µm) in protargol preparations. Body flexible and slightly contractile, elliptical in shape with a ratio of length: width approximately 3.5:1, right margin usually straight and left margin more or less convex. Cell dorsoventrally flattened with a width: thickness ratio of approximately 2:1, ventral side flat and dorsal slightly convex (degree depending of nutrition status). Constantly two globular to ellipsoid macronuclear nodules, on average 16.1 × 9.2 µm in protargol preparations, located in anterior and posterior third of cell. One to four globular micronuclei, on average 2.2 µm across, attached to macronuclear nodules ( Figures 1C,  3A,B). Contractile vacuole (about 8 µm across) in mid-body, close to left cell margin; pulsing at intervals of about 8 s ( Figures 1A, 2B,D). Cytoplasm colorless, usually containing food vacuoles (about 5 µm), lipid droplets (about 3 µm) and minute crystals ( Figures 1A, 2A,B). Colorless cortical granules (about 1 µm across), arranged in longitudinal rows on dorsal side and sparsely arranged in irregular short rows on ventral side (Figures 2C,D). Locomotion by swimming in water or crawling on substrate rapidly.
Adoral zone 33-43 µm long after protargol staining, occupying ca. 36% of body length, composed of 26-32 (on average 30) membranelles, with cilia about 12 µm long (Table 1 and Figures 1A,B, 3A,B). Undulating membranes in Oxytrichapattern (Figures 1B, 3A). Three slightly enlarged frontal cirri arranged at the anterior end of the cell, one buccal cirrus 1 http://www.ebi.ac.uk located near anterior end of paroral membrane, invariably four frontoventral cirri positioned between the anterior portion of the right marginal row and the paroral, cirrus III/2 arranged anterior to the level of cirrus VI/3 (Figure 1B), three postoral ventral cirri behind the proximal end of the adoral zone, cirrus IV/2 located more anteriorly than V/4 ( Figure 1B), two pretransverse cirri arranged in front of the transverse cirri, five transverse cirri in a hook-shaped row, inconspicuously projecting above posterior body margin. One right and one left marginal row comprised of 24-36 and 21-34 cirri (cilia about 10 µm long), respectively. Marginal rows not confluent posteriorly (Figures 1B, 3A). All of the cirri about 12 µm long in vivo.
Five rows of dorsal bristles in typical Oxytricha pattern. Two bipolar rows left of midline of body, each associated with a caudal cirrus at its rear end; row 3 ends at posterior third; row 4 starts below mid-body and also produces a caudal cirrus at its posterior end; row 5 terminates ahead of mid-body (Figures 1C, 3B). Dorsal bristles about 2 µm long in vivo.

Ontogenesis
The ontogenetic process of O. granulifera granulifera is very similar to that of the genus. For further details see Figures 3F-L. The main morphogenetic features are: 1) the oral primordium of the opisthe originates de novo and the parental adoral zone of membranelles remains unchanged and inherited by the proter; 2) six streaks of frontal-ventral-transverse cirral anlagen are formed; 3) the marginal rows and the nuclear apparatus develop in the usual manner; 4) dorsal kineties 1-3 develop within the parental kineties, dorsal kinety 4 originates from kinety 3 to kinety 5 originates dorsomarginally, that is, from an anlage near the right marginal row (Figures 3K,L). Ontogenesis confirms there is only one dorsomarginal kinety.

Detection of Abnormal Forms in Culture System
The present population of O. granulifera granulifera was isolated in the summer of 2017 and has been cultured at room temperature in the laboratory since then. However, since November 2018 many abnormal individuals have been found in some cultures. Usually, the course of these abnormal individuals can be described as follows: to begin with, a large number of normal populations bloomed in Petri dishes, and then a lot of abnormal resting cysts (Figures 2F-H) were quickly formed, and a certain proportion (about 10-15%) of these cysts then excysted to form abnormal individuals (Figures 2I-M). This phenomenon occurred repeatedly in the culture system.
Abnormal individuals have never been found to be able to form resting cysts, or carry on dividing and conjugate reproduction. The attempt to cultivate these abnormal individuals failed and the population gradually disappeared within one week.

Sequence Similarity and Phylogenetic Analyses
The partial SSU rDNA sequences of the normal and abnormal forms of O. granulifera granulifera were deposited into the GenBank database with the accession numbers MW143562 and MW143561, with a length of 1,677 and 1,631 bp, respectively. The similarity of the common 1,631 bp covered by both forms is 100%.
The topology of ML and BI trees are similar, and, therefore, only the ML tree is shown (Figure 4). Subfamily Oxytrichinae was polyphyletic, and five subgroups were recognized (Oxytrichinae-I-V). All strains of Oxytricha granulifera except X53486 were exclusively located within Oxytrichinae-IV, while O. granulifera X53486 clustered   All data were measured from protargol-stained specimens. CV, coefficient of variation in %; DK, dorsal kinety; Max, maximum; Mean, arithmetic mean; Med, median value; Min, minimum; n, number of cells measured; SD, standard deviation; SE, standard error of arithmetic mean.
Frontiers in Marine Science | www.frontiersin.org in Oxytrichinae-III. The normal and abnormal forms of O. granulifera granulifera from the present research clustered together without full support (ML/BI, 89, 1.00). These two sequences were close to four other strains of O. granulifera granulifera (AM412768, AM412769, AM412771, and AM412772) and a strain of O. granulifera (AF164122). This branch then grouped with other O. granulifera strains.

Abnormal Form Could Easily Be Deduced as a "New" Species
The general infraciliature and arrangement of cortical granules of the normal and abnormal individuals are consistent. However, some morphological characteristics of abnormal individuals are quite different from those of normal individuals, and are mainly as follows: 1) the body size: in life 90-128 × 31-43 µm in normal forms vs 115-140 × 21-24 µm in abnormal forms; 2) the body shape: very narrowly elliptical in abnormal forma vs elliptical in normal forms; 3) the length of the adoral zone (14% of the body length in abnormal forms vs 36% of the body length in normal forms) and the number of membranelles (on average in abnormal forms 17 vs on average 30 membranes in normal forms); 4) the undulating membranes (absence in abnormal forms vs presence in normal forms); 5) number of macronuclei (1 to 4 in abnormal forms vs invariably 2 in normal forms); 6) mortality (abnormal individuals slow vs fast in normal individuals). However, because the abnormal individuals appear spontaneously, repeatedly and have stable morphological characteristics in the culture system, it could be easy to regard them as a "new" species without continuous and comprehensive investigation.

Occurrence of Abnormal Resting Cysts and Individuals
The population of O. granulifera granulifera Foissner and Adam (1983) was isolated from terrestrial environment, and no abnormal cysts or form were reported. While our population was isolated from sludge in a hot spring, and abnormal cysts and individuals occurred spontaneously in the culture system. Some researchers have reported abnormal cysts from artificial inducing in some ciliate species. Hashimoto (1962Hashimoto ( , 1964 induced abnormal cysts of Oxytricha fallax by embedding cells in agar, exposed to high temperatures, starved, or changing the composition of the culture medium. Another research article reported that abnormal cysts were formed in Euplotes taylori due to pressure of the coverslip (Garnjobst, 1928). As for our case, we speculate that the lack of food (necessary nutrients) and space after the outbreak of the high population density from single source of food (wheat grains) might trigger the potential polymorphism, then the abnormal cysts and forms appeared. Transverse cirri, number
Frontiers in Marine Science | www.frontiersin.org The simpler characteristics enabled the abnormal forms to consume less energy than the formal forms which served as a strategy for the ciliate to endure the deteriorative environment. This speculation should be tested by transcriptomics in future work.

CONCLUSION
O. granulifera granulifera was detected in a hot spring in Iceland for the first time. So far, this species has been recorded in all of the main biogeographic regions, indicating its worldwide distribution. Currently, laboratory cultivation is necessary for the study of morphology, ontogenesis and molecular biology of ciliates (Foissner, 1996;Lian et al., 2020;Wang et al., 2020;Zhang et al., 2020). In this study, owing to stable morphological features of abnormal individuals of this species, the possibility that it may be misidentified as a "new" species has been discussed above. Attention to this possibility must be paid in future research. Likewise, the causes of deformities also need to be investigated further.

Protargol Stained Specimens
The protargol slides containing the normal (ZR20181112A-G) and the abnormal specimens (ZR20181129A-F) have been deposited in the Laboratory of Hydrobiology, Hebei University, China.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

AUTHOR CONTRIBUTIONS
SF and TS sampled the material. RZ and QZ conducted indoor culture of ciliate population and silver staining, generated the molecular data, and wrote the manuscript. XH, FL, ZQ, SF, and TS revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Natural Science Foundation of China (Project Numbers: 31872206 and 41976086) and the Scientific Research Foundation of Hebei Province for returned scholars (C201801).