Discovery of a new hypotrich ciliate from petroleum contaminated soil

Pollution after oil spill represents extreme habitat for survival and is a major concern for loss of species diversity in the affected area. In this study, we investigated soil samples collected from a petrochemical industry, Ulsan, South Korea. The soil was in the phase of recovery from the contamination of crude oil spill. Detailed investigation, based on morphology, ontogenesis, and molecular phylogenetic methods, resulted in discovery of a novel hypotrich ciliate, i.e., Metasterkiella koreana n. gen., n. sp., which is morphologically characterized by a semirigid body, undulating membranes in Oxytricha pattern, 18 frontal-ventral-transverse cirri with cirrus V/3 placed posteriorly, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kineties 1, 2, and 4. Interestingly, during ontogenesis, formation of three common anlagen for the proter and the opisthe and involvement of cirrus V/3 in anlagen formation was observed. The dorsal ontogenesis was typical of oxytrichids, i.e., simple fragmentation of dorsal kinety 3 and formation of dorsomarginal rows close to the right marginal row. The new species was found to be similar with Sterkiella subtropica, except for some minor differences in morphometry, and at gene level with only one base pair difference. In phylogenetic analyses, based on SSU rRNA gene sequence, M. koreana cluster in a clade away from Sterkiella species, which could be explained by the differences in the morphogenetic pattern between these two genera. It is proposed that S. subtropica probably belongs to Metasterkiella; however, we do not perform changes and wait for the reinvestigation of its morphogenetic pattern.


Introduction
It is not uncommon to find morphological variability within and between populations in ciliated protozoa [1][2][3][4][5]. Most of this variability is the result of laboratory culture conditions and stressful environmental conditions. Recent studies have shown that careful observations have resulted in recognition of cryptic ciliate species [3][4][5]. Indeed, crypticity is often predicted in molecular phylogenetic analyses and can be explained by insufficient acquisition and/or interpretation of the data. One example for this is represented by cyst species, a very recent concept in ciliates, but it can be estimated that it will significantly increase the number of ciliate species [3,6,7]. However, when ontogenesis within the genus is compared, most congeners have a rather similar mode with some minor variations [8]. In the present study, we investigated, in detail, the ontogenetic patterns of a novel hypotrich ciliate, i.e., Metasterkiella koreana n. gen., n. sp., which resembled morphologically very well with one Sterkiella species. Nonetheless, we observed a rather different mode of division which differs significantly when compared with the detailed ontogenetic data available for the genus Sterkiella [1,2]. Thus, we established a novel genus i.e., Metasterkiella for the novel species in accordance with the molecular analyses, which showed that the genus Sterkiella is a polyphyletic assemblage. We feel that such variable characters still exist within the genus and detailed investigation of which (i.e., morphology, ontogenesis, cyst structure) will help in differentiating the species often assigned to collective groups, e.g., Sterkiella histriomuscorum complex. Another example of morphological differentiation was presented in Kumar et al. [9] who described a novel Sterkiella species based on difference from other species by having constantly four (vs. five) transverse cirri. Similarly, Foissner [3] identified a novel species, i.e., Fragmospina depressa, which resembled with S. histriomuscorum but had undulating membranes close (vs. distant) to adoral zone of membranelles and resting cyst spinous (vs. wrinkled). All these examples suggest a fast evolution of the Sterkiella-like hypotrichs possibly originating from phylogenetically distant species.

Description of the sampling site and sample processing
Soil samples were collected from the spilled oil treatment facility, Onsan, Ulsan, South Korea (35˚24' 56"N; 129˚20' 38"E) on April 8 th , 2016, under control of the Safety Manager with prior permission from the company. Ciliates were reactivated from resting cysts from onemonth-dried soil samples (approximately 300 g) by employing the non-flooded Petri dish method [10]. A clonal culture of M. koreana was established as described in Kumar et al. [9], i.e., using Pringsheim's medium for culturing and the green alga Chlorogonium elongatum as food organism. Live observations were made using a microscope with bright-field and differential interference contrast illuminations at a magnification of 100-1000×. The protargol staining method described by Kamra and Sapra [11] was used with some modification to reveal the ciliature. Measurements of impregnated specimens were performed at a magnification of 1000× using an ocular micrometer. A Zeiss microscope camera was employed for photomicrography. The illustration of the live specimen was prepared using free-hand sketches, while those of impregnated specimens were made with a drawing device. Terminology is according to Berger [1] and Wallengren [12].

DNA extraction, PCR amplification, and sequencing
Five cells were collected from a clonal culture with the help of glass micropipettes and washed three times with autoclaved distilled water (same culture was used for live observation and protargol staining to study morphology and ontogenesis). Genomic DNA was extracted using the RED Extract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), following the manufacturer's instruction except for the reduction of each reaction volume to one-tenth [13]. Extracted DNA (1 μl) was dispensed into a PCR tube containing 22 μl of autoclaved distilled water, and amplifications were carried out using the TaKaRa ExTaq DNA polymerase Kit (TaKaRa Bio-medicals, Otsu, Japan) in a total volume of 30 μl with the universal eukaryotic primers Euk A (FW 5'-AAC CTG GTT GAT CCT GCC AG-3') and Euk B (RV 5'-CAC TTG GAC GTC TTC CTA GT-3') [14]. The PCR program for SSU rRNA gene amplification included an initial denaturation at 94˚C for 3 min, followed by 35 cycles of 94˚C for 1 min, 56˚C for 45 s and 72˚C for 80 s, with a final extension step at 72˚C for 10 min. After confirmation of the appropriate size in 1.2% agarose gel, the purified PCR products were directly sequenced on both strands on an ABI 3730 automatic sequencer (Cosmo genetech., Seoul, Korea).

Phylogenetic analyses
For phylogenetic analyses, the SSU rRNA gene sequence of M. koreana was aligned with 54 SSU rRNA gene sequences of hypotrich ciliates from GenBank using the MAFFT software v. 7.047 (choosing the iterative refinement methods Q-INS-I that considers the secondary structure of the SSU rRNA molecules) [15].
Ambiguously aligned regions were identified and excluded from the phylogenetic analyses with GBlocks v.0.91b [16] using parameters optimized for rRNA alignments, leaving 1,514 unambiguous positions. A Bayesian inference (BI) analysis was performed using MrBayes v.3.2.1 [17] and the TIM2+I+G model, as selected by the jModel test v.2.1.3 software [18] under the Akaike Information Criterion corrected (AICc). Markov chain Monte Carlo (MCMC) simulations were run, with two sets of four chains using the default settings, for 1,000,000 generations with trees sampled every 100 generations and discarding the first 25% of the sampled trees as burn-in. The remaining trees were used to generate a consensus tree and to calculate the posterior probabilities (PP) of all branches using the majority-rule consensus approach. Maximum likelihood (ML) analyses were carried out using RAxML-HPC2 v. 8.0.24 [19] on the CIPRES Science Gateway [20] with bootstrapping of 1000 replicates. Phylogenetic trees were visualized using the free software package FigTree v. 1.4 by A. Rambaut at http://tree.bio.ed.ac.uk/software/figtree/. To assess the probability of the monophyletic relationships between Metasterkiella koreana and the genus Sterkiella, the approximately unbiased (AU) test was performed in CONSEL v. 0.1j [21,22]. Steps were as described in Shazib et al. [23]. Two constrained ML analyses were carried out, i.e., (i) monophyly of M. koreana and Sterkiella cavicola, and (ii) monophyly of M. koreana and all other Sterkiella species.

Data availability
The newly obtained SSU rRNA gene sequence of M. koreana is available from the GenBank/ EMBL databases (accession number: KY448243). Slides with fixed specimens are available from the National Institute of Biological Resources (NIBR), Incheon, Korea with registration numbers NIBRPR0000107886, NIBRPR0000107887, and NIBRPR0000107888. The slides contain many specimens, with relevant cells marked by a black ink circle on the cover glass.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:ADCE754A-CD51-4D72-AF73-B31813D36C5D. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Results
Description of Metasterkiella koreana n. gen. n. sp.

Notes on ontogenesis
The ontogenetic stages show some variability, however, two distinguishing features were recorded, i.e., involvement of cirrus V/3 in anlagen formation and a common origin of anlagen II, V, and VI for the proter and the opisthe (Figs 4A-4F, 5A-5G, 6A-6F and 7A-7F).
The oral primordium develops close to transverse cirri and extends towards the buccal vertex (Figs 4A and 6A). The scattered basal bodies at the anterior end of the oral primordium develop into the opisthe's anlagen I and II (Figs 4B and 6B). Cirrus IV/2 disaggregates and forms the opisthe's anlage III (Figs 4C and 6C). A group of basal bodies originates de novo on the right side of cirrus V/4 and splits transversely. The anterior portion of this group proliferates anteriorly, forming the proter's anlagen V and VI; the posterior portion elongates posteriorly by incorporating cirrus V/3, which further splits longitudinally to form the opisthe's anlagen V and VI (Figs 4C-4F, 5A, 5B, 6C-6F and 7A and 7B). Anlage II of the opisthe extends with anterior portion crossing the buccal vertex and joining the disaggregating buccal cirrus in early dividers (Figs 4C-4F and 6C-6F). The disaggregation of cirrus V/4 forms the opisthe anlage IV (Figs 4F and 6F). Anlage I of the proter, i.e., the partially reorganized paroral and endoral, generates first frontal cirrus I/1 as well as the paroral and the endoral for the proter (Figs 5B-5D and 7C and 7D). Cirri III/2 and IV/3 disaggregate and give rise to the anlagen III and IV of the proter (Figs 4A, 4B and 7A and 7B). In the opisthe, anlage I separates from the posterior ends of anlagen II to VI and forms the paroral, endoral and cirrus I/1 (Figs 5C and 7A). Thus, six parental cirri and parental undulating membranes are involved in anlagen formation. The 18 frontal-ventral-transverse cirri arise from these anlagen, splitting in a 1, 3, 3, 3, 4, 4 pattern (Figs 5D, 5E and 7D and 7E). A new adoral zone of membranelles for the opisthe develops from the oral primordium, while the parental adoral zone of membranelles is retained unchanged for the proter.
The marginal anlagen arise at each of two levels by "within-row" anlagen formation utilizing one or two of the parental cirri at each level. The marginal anlagen elongate deploying four or five parental cirri and differentiate into new marginal rows. The remaining parental marginal cirri are resorbed (Figs 5C-5E and 7C-7E).
On the dorsal surface, three anlagen are formed within row from dorsal kineties 1, 2 and 3 at two levels (one set for the proter and one for the opisthe) (Figs 5F, 5G and 7F). The third dorsal primordium fragments at the middle giving rise to the third and fourth kineties. The two dorso-marginal rows arise near the right marginal row (Figs 5D, 5E and 7E). Each caudal cirrus originates at the posterior end of the new dorsal kineties 1, 2, and 4 (Figs 5G and 7F).
Nuclear division proceeds in the usual manner for oxytrichids. In middle dividers the macronuclear nodules fuse to form a single mass which divides twice to produce the typical four

SSU rRNA gene sequence and phylogeny
The SSU rRNA gene sequence of Metasterkiella koreana is 1,652 bp in length and has a GC content of 45.03%. It has been deposited in the NCBI database under the accession number KY448243. Phylogenetic trees inferred from the SSU rRNA gene sequences using ML and BI present similar topologies; therefore, only the BI tree is shown here (Fig 8). Phylogenetic

Justification of the genus Metasterkiella
Berger and Foissner [24] and Berger [1] divided the 18-cirri oxytrichids into the Stylonychinae (rigid body, lack of cortical granules, cirrus V/3 not involved in anlagen formation) and Oxytrichinae (flexible body, cirrus V/3 involved in anlagen formation). However, a recent redescription of Rigidohymena candens showed that its body is flexible although being a stylonychiid ciliate [25]. Nevertheless, its stylonychiid home is supported by the ontogenetic inactivity of cirrus V/3 and by the 18S rRNA gene. This indicates that cell rigidity might not be a reliable diagnostic feature of the genus Rigidohymena and the subfamily Stylonychinae. The genus Sterkiella possesses a semirigid body and cirrus V/3 does not participate in anlagen formation [2]. Furthermore, anlagen form separately for the proter and the opisthe [2]; except for a population of Sterkiella histriomuscorum described by Berger et al. [26] showing a common origin of anlagen for proter and the opisthe with intact V/3. The morphology of the present species resembles that of Sterkiella species, however its ontogenesis differs significantly, i.e., at least three common anlagen are formed and cirrus V/3 is involved in anlagen formation.    We consider this as an important difference, especially when this feature (V/3 participation) has been used to separate species at the genus level (e.g., Rigidohymena, Australocirrus). Therefore, we propose a new genus, Metasterkiella, for our new species. Classification in a separate genus might help to reduce the non-monophyly problem of the genus Sterkiella. The  https://doi.org/10.1371/journal.pone.0178657.g008 population described by Berger et al. [26] shows another morphogenetic pattern, i.e., cirrus V/ 3 remains intact and some cirral anlagen have a common origin. Possibly, a new genus will be needed for this population. A further support for establishment of Metasterkiella comes from a discovery of the genus Fragmospina Foissner, 2016. Its type species, F. depressa Foissner, 2016, resembles Sterkiella species, especially S. histriomuscorum but differs in the course of the paroral membrane which almost touches frontal adoral membranelles.  [27]) of dorsal kinety 3. Considering these differences probably a subspecies rank would be more appropriate for the new species, as also indicated by SSU rRNA gene sequences which differ only by a single nucleotide position. However, it has been reported that SSU rRNA gene sequences are not informative for separating different species [28,29], i.e., morphologically distinct species are shown genetically closely related by SSU rRNA gene sequences; even some species have identical SSU rRNA gene sequence but differ at other rRNA regions [29].
Fragmospina depressa can be distinguished from this new species by having a paroral membrane close (vs. distant) to the adoral membranelles and the structure of resting cyst, i.e., spinous (vs. wrinkled) surface.

Phylogenetic position of Metasterkiella koreana
Phylogenetic analyses placed Metasterkiella koreana close to Sterkiella subtropica, with full support (1.00 BI and 98% ML; Fig 8). Although these two species differ in their habitat, ciliature (minor difference), and interestingly in the ontogenetic pattern; it has been reported in Chen et al. [27] that the ontogenesis of S. subtropica is very similar to that of the type species, Sterkiella cavicola, in which separate anlagen are formed for the proter and the opisthe [2,27], whereas three common anlagen are formed in the new species. The single nucleotide difference in the SSU rRNA gene of these two species indicate that S. subtropica probably belongs to Metasterkiella; since, both species group away from other Sterkiella species. A detailed reinvestigation on the ontogenesis of Sterkiella subtropica will confirm its final assignment; however, considering the significant difference in the ontogenesis we are confident in raising the new species up to the genus level. In support of our classification, several examples exists like, Parasterkiella thompsoni (Foissner, 1996) Küppers et al., 2011, Fragmospina depressa, which would have been easily identified as Sterkiella species but separated based on detailed investigations on morphology and cyst structure. At the end, it is clear that addition of related molecular sequences, e.g., Fragmospina, S. histriomuscorum populations, and gene sequences from other loci will further clarify whether the genus Sterkiella is monophyletic or not. Furthermore, it would also not be surprising to find different morphogenetic patterns in the species of the genus Sterkiella, e.g., S. histriomuscorum complex, S. tricirrata, which are often considered as synonyms.

Ciliates from petroleum contaminated soils
To the best of our knowledge, this is the first report on the description of a novel ciliate species from petroleum contaminated soil. However, some reports exist on the response of certain ciliates species to crude oil contamination [31,32]. The contamination was a result of an accidental leakage from the industrial plant in Onsan, Ulsan, South Korea. Thereby, causing spread of crude oil in the surrounding area. When the samples were collected the soil was under treatment for recovery, however, the presence of small oil droplets were still seen in the Petri dish. Interestingly, after rewetting the soil samples very few ciliates excysted, the new species was found to be freely moving around the petroleum oil droplets in the Petri dish, indicating its probable tolerance for petroleum contamination. Whether the new species have some role in purification of petroleum oil needs further investigation at the molecular as well as biochemical level. Genus Metasterkiella n. gen.

Diagnosis
Body Semirigid. Frontal-ventral-transverse cirri arranged in typical oxytrichid pattern. One right and one left row of marginal cirri. Six dorsal kineties including two dorsomarginal rows, kinety 3 with simple fragmentation; caudal cirri present. Undulating membranes in Oxytricha pattern. Common origin of anlagen II, V and VI for proter and opisthe, cirrus V/3 involved in anlagen formation.

Etymology
Composite of the Greek prefix meta (next to, among after) and the genus-group name Sterkiella, referring to the similarity in ciliature with Sterkiella.

Metasterkiella koreana n. sp. Diagnosis
Size about 85 × 50 μm in vivo; body elongate to broadly ellipsoidal. Nuclear apparatus composed of two ellipsoidal macronuclear nodules and two micronuclei on average. Invariably, 18 fronto-ventral-transverse cirri. Right and left marginal rows composed of an average of 21 and 20 cirri, respectively. Adoral zone 39% of body length and composed of an average of 24 membranelles. Three narrowly spaced, inconspicuous caudal cirri in body's midline. Resting cyst with wrinkled surface. Soil habitat.

Type material
The slide containing the holotype specimen (Figs 2F and 3A) and two paratype slides with protargol-stained morphostatic specimens have been deposited at the National Institute of Biological Resources (NIBR), Incheon, Korea with registration numbers NIBRPR0000107886, NIBRPR0000107887, and NIBRPR0000107888. The slides contain many specimens, with relevant cells marked by a black ink circle on the cover glass. The SSU rRNA gene sequence is deposited in GenBank (accession number: KY448243).

Etymology
The species-group name koreana refers to the country where the species was discovered, i.e., Korea.

Occurrence and ecology
As yet found only at the type location. At the time of sampling, soil was under treatment for spilled crude oil.

S1 Fig. Sampling images with the Safety Manager.
(PDF) S1 File.