Water samples were collected on April 8, 2016 from the outlet of an industrial company in Onsan, Ulsan, South Korea (35°25′55.9′′ N 129°21′07.2′′ E). Ciliates were isolated and fed with green algae Chlorogonium elongatum as a food organism (Ammermann et al., 1974). Live observations were made using a microscope with differential interference contrast illuminations at a magnification range of × 100–1,000. The protargol staining method described by Kamra and Sapra (1990) was used, with some modifications, to reveal the ciliature. Measurements of impregnated specimens were performed at a magnification of × 1,000 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. The terminology is according to Wallengren (1900) and Berger (1999).

Four clonal cultures were established, and the same were used for live observation and protargol impregnation to study morphology and morphogenesis. Three cells of H. tolerans were isolated and collected in 1.5-mL tubes for DNA extraction from a clonal culture with the help of glass micropipettes. Thus, the morphology, morphogenesis, and molecular phylogeny reported in the present study deal with the same species. The cells were washed at least three times with autoclaved distilled water for genomic DNA extraction using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), following the instruction of the manufacturer, except for the reduction of each reaction volume to one-tenth (Gong et al., 2007). Amplifications of the extracted DNA were carried out using the TaKaRa ExTaq DNA polymerase Kit (TaKaRa Bio-medicals, Otsu, Japan) using 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′) (Medlin et al., 1988). 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, the purified PCR products were directly sequenced on both strands on an ABI 3730 automatic sequencer at the Cosmo Genetech, Seoul, South Korea.

For phylogenetic analyses, the SSU rRNA gene sequence of H. tolerans n. sp. was aligned with 54 SSU rRNA gene sequences of hypotrich ciliates from GenBank using the MAFFT software v. 7¹ and choosing the iterative refinement methods Q-INS-I that consider the secondary structure of the SSU rRNA molecules (Katoh and Standley, 2013).

Ambiguously aligned regions were identified and excluded from the phylogenetic analyses with Gblocks v. 0.91b (Castresana, 2000) using parameters optimized for rRNA gene alignments, leaving 1,562 unambiguous positions. Maximum likelihood (ML) analyses were carried out using RAxML-HPC2 v. 8.0.24 (Stamatakis, 2014) on the CIPRES Science Gateway (Miller et al., 2010) with bootstrapping of 1,000 replicates. A Bayesian inference (BI) analysis was performed using Mr. Bayes v. 3.2.1 (Ronquist et al., 2012) and the TIM2 + I + G model, as selected by the jModel test v. 2.1.3 software (Posada, 2008) under the Akaike Information Criterion. Markov chain Monte Carlo 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 of all branches using the majority rule consensus approach. Phylogenetic trees were visualized using the free software package FigTree v. 1.4 by A. Rambaut.²

The present work is registered in ZooBank under urn:lsid:zoobank.org:pub:DC0F5E57-1A3A-42E0-B24F-7B36D325A001. The new species is registered in ZooBank under urn:lsid:zoobank.org:act:EB0BB241-1F83-416D-8B16-85A18304A34A.

Histriculus tolerans n. sp. (Figures 1–6 and Table 1).

Size in vivo 60–80 × 30–45 μm, usually about 70 × 40 μm, as calculated from some in vivo measurements (n = 7) and the morphometric data in Table 1, adding 15% for preparation shrinkage (Kumar et al., 2016), on average 60 × 40 μm in protargol preparations. Body rigid, outline elliptical with round anterior and posterior body end, dorsoventrally flattened about 2:1 (Figures 1A–E, 2A, 3A–D and Table 1). Nuclear apparatus in central quarters of cell in or slightly left of midline, composed of two macronuclear nodules and one or two micronuclei (Figures 1A,B,E, 2A–C, 3A–F and Table 1). Macronuclear nodules narrowly ellipsoidal to ellipsoidal, on average 15 × 6 μm in protargol preparations; contain many small nucleoli, 2–4 μm across. Micronuclei near or attached to macronuclear nodules, ellipsoidal, on average 5.7 × 3.5 μm in protargol preparations. Contractile vacuole in mid-body near left cell margin (Figures 1B,E, 2A). Cortical granules absent. Cytoplasm colorless, filled with cytoplasmic granules, fat droplets, and crystals of usual shapes (Figures 1B–D, 2A,B). Food vacuoles mainly in the posterior half of cell, in vivo up to 15 μm across, contains bacteria and flagellates (Chlorogonium elongatum) (Figures 1C, 2A). Movement with rapid crawling over and between soil particles.

Cirral pattern stylonychid (Berger, 1999). Invariably three, slightly enlarged, in vivo about 14-μm long frontal cirri, right cirrus posterior to distal end of adoral zone membranelles, left cirrus anterior of distal end of undulating membranes (Figures 2A,B, 3A–D). Buccal cirrus, about 10 μm distant from the anterior body end in protargol preparations, right of anterior end of undulating membranes (Figures 2A,B, 3A–D). Four frontoventral cirri, arranged in opposed tick mark shape (Figures 2A,B, 3A–D). Three postoral ventral cirri behind the buccal vertex and two slightly obliquely arranged pretransverse ventral cirri. Invariably, five distinctly enlarged transverse cirri, in vivo about 14 μm long, four leftmost reached up to the posterior body margin. Marginal rows confluent posteriorly, cirri about 12 μm long in protargol preparation. Gap between last cirri of rows about 2 μm in protargol preparation, left row composed of an average of 24 cirri; right row of 34 cirri (Figures 2A,B, 3A–D and Table 1).

Invariably six dorsal kineties, including two dorsomarginal rows, with bristles length at about 4 μm in vivo and about 2–3 μm in protargol preparations (Figures 1E, 2D, 3E,F). Kinety 1 slightly shortened anteriorly, consists of 23–28 bristles; kinety 2–4 bipolar, with 21–25, 16–23, and 15–18 bristles, respectively. Dorsomarginal row 1 shortened posteriorly, i.e., reaching two-third of body length, consists of 11–14 bristles; row 2 distinctly shortened posteriorly, with 6–11 bristles (Figures 1E, 2D, 3E,F, and Table 1).

Adoral zone about 51% of body length, composed of, on average, 32 membranelles, with cilia about 15 μm long in vivo. Paroral and endoral membranes of about equal length, slightly curved and nearly parallel (Figures 1C,D, 2A,C, 3A–D). Paroral commences about 10 μm posterior of anterior body end; endoral commences about 3 μm posterior of anterior end of paroral, at the level of buccal cirrus (Figures 1C,D, 2A,C, 3A–D and Table 1). Pharyngeal fibers of ordinary length and structure extend obliquely toward the right body margin (Figure 2A).

The genus Histriculus contains six species that should be compared with H. tolerans, namely, H. histrio, Histriculus sphagni (Stokes, 1891) Corliss, 1960; Histriculus complanatus (Stokes, 1887) Corliss, 1960; Histriculus minimus (Groličre, 1975) Berger and Foissner, 1997; H. vorax (Stokes, 1891) Corliss, 1960; and Histriculus similis (Quennerstedt, 1867) Corliss, 1960. Except for the type species, i.e., H. histrio, the description of the other species is mainly based on live observations, and thus detailed reinvestigations based on standard methods, especially live observations and protargol impregnation, were recommended by Berger (1999).

The type species can be easily separated from H. tolerans by having a larger body size in vivo (over 100 μm vs. about 70 μm), more adoral membranelles (40–58 vs. 29–33), and cirri in left (23–35 vs. 20–27) and right (33–51 vs. 31–37) marginal rows, bristles in dorsal kinety 1 (50–58 vs. 23–28), kinety 2 (38–46 vs. 21–25), kinety 3 (33–42 vs. 16–23), and dorsomarginal row 1 (25–35 vs. 11–14) (average values from Berger, 1999; Gupta et al., 2006; Kwon and Shin, 2011).

H. sphagni can be distinguished from H. tolerans in having rather straight (vs. slightly curved) undulating membranelles, additional cirrus (vs. no such cirrus) behind the frontal cirrus, and five (vs. six) dorsal kineties (Berger, 1999).

The new species can be separated from H. complanatus in the arrangement of transverse cirri, i.e., five transverse cirri arranged in a more or less tick mark shape (vs. arranged in rows) of which four leftmost (vs. three rightmost) reaching up to the cell margin (vs. protrudes beyond the posterior end) of the cell, buccal cirrus at the anterior right (vs. distinctly posterior) of undulating membranes, and marginal cirri of the same length [vs. increase in length posteriorly; for which Berger (1999) mentioned that inconspicuous caudal cirri could be present] throughout the body (data from illustrations of Stokes, 1887; Vuxanovici, 1961).

H. minimus differs from H. tolerans by a smaller body size (30–40 vs. 60–75 μm), arrangement of micronuclei, i.e., one in between two macronuclear nodules (vs. one or two attached at various positions or near to the nodules), and 18–21 (vs. 31–37) cirri in the right marginal row (Berger, 1999).

H. vorax has been reported with rather variable shapes, i.e., about 170 μm (Stokes, 1891), small adult cells 70–140 × 50–60 μm, and large adult cells 190–250 × 100–120 μm (Curds, 1966). The body margin is often indented, with the left margin concave and the right convex, which was not observed in the present species. The new species can be distinguished from H. vorax in having a smaller body length, i.e., 60–75 × 40–55 μm. Further differences include widely spaced and fewer marginal cirri (vs. narrowly spaced with 31–37 cirri in the right marginal row) in H. vorax. Berger (1999) mentioned the possibility that H. vorax may possess caudal cirri due to the longer and prominently projecting cirri beyond the posterior end of the cell.

H. similis can be distinguished from H. tolerans in having a long body in vivo, i.e., 110–130 μm (vs. 60–75 μm), body about three (vs. two) times as long as broad, and the adoral zone of membranelles was about 38% (vs. 51%) of body length.

The divisional morphogenesis of H. tolerans resembles that of H. histrio as described by Gupta et al. (2006). The only significant difference observed was that the oral primordium in H. tolerans does not contribute to the formation of anlage II of the proter (for a review, see Berger, 1999; Gupta et al., 2006). For the remaining Histriculus species, no morphogenetic data are known. However, Curds (1965, 1966) reported morphogenesis in H. vorax by the formation of an endogenous bud that also needs to be reinvestigated since Pang and Zhang (1981) assumed it to be the cannibalism induced by overfeeding. No endogenous bud formation was observed in H. tolerans.

The SSU rRNA gene sequence of H. tolerans matches well with H. histrio (99% similarity and four base pair difference), the only species within the genus for which gene sequence is available. In the phylogenetic analyses, the new species clustered with the type species with the full support of 100/1.00 (ML/BI). The detailed analyses are restricted due to the limited sequences available within the genus; however, Histriculus is well fitted in the subfamily Stylonychinae clustering in a clade with Tetmemena, Stylonychia, and Sterkiella species (Figure 7).

In our analyses, the genus Sterkiella appeared to be a polyphyletic group, possibly due to the differences in the morphogenetic pattern (Kumar et al., 2015, 2017; Bharti et al., 2018). Similarly, a slight variation in morphogenesis was observed in the present study, i.e., no contribution of the oral primordium toward the formation of anlage II of the proter in contrast to the type species where such contribution was reported by Gupta et al. (2006). Furthermore, recent observations on the resting cyst structures have resulted in the identification of the cryptic ciliate species (Foissner, 2016; Kumar et al., 2016; Bharti et al., 2019). We believe that the detailed observations, i.e., future addition of cyst data along with morphology and morphogenesis, as well as molecular sequences, for the remaining Histriculus species will clarify whether the genus Histriculus stays monophyletic or polyphyletic.

Ciliates have been reported to play a significant role in the bioaccumulation or bio-concentration since they come directly in contact with chemicals in contrast to other single-cell microbes, such as bacteria, yeast, and algae, that possess a cell wall and may affect the uptake of chemicals (Cooley et al., 1972; Carter and Cameron, 1973). Furthermore, several studies have highlighted the potential of protozoan ciliates as a model organism for various in vitro metal toxicological assessments as well as their role in the self-purification of natural aquatic ecosystems (Cooley et al., 1972; Carter and Cameron, 1973; Larsen et al., 1997; Pauli and Berger, 1997; Sauvant et al., 1999; Bogaerts et al., 2001; Schultz et al., 2005; Vilas-Boas et al., 2020).

The heavy metal concentration during the year (2015–2018) from the sampling site ranges significantly and on many occasions rose higher than the permissible levels, e.g., Cd concentration reached up to 0.252 mg/L, Pb up to 0.44 mg/L, and As up to 0.456 mg/L (data retrieved from the water quality monitoring system of the Korean Ministry of Environment, see text footnote 3). Thus, it is believed that the species present in this habitat must possess some defense mechanism to overcome the stress of the heavy metal. The present study reports a novel species that might have the potential to survive heavy metal stress and that it can be used as a model organism for evaluating heavy metal toxicity. However, further studies are required for generating reference data that can be used in future ecotoxicological analyses using H. tolerans.