Morphology and Molecular Phylogeny of a New Hypotrich Ciliate, Pseudourostyla guizhouensis sp. nov. from Southern China, with Notes on a Chinese Population of Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012 (Ciliophora, Hypotricha)

The morphology and molecular phylogeny of a soil hypotrich ciliate, Pseudourostyla guizhouensis sp. nov., collected from southern China, were investigated. Pseudourostyla guizhouensis sp. nov. has an elongate elliptical body measuring 180–310 × 65–85 μm in vivo; invariably two right and three or four left marginal rows; six or seven dorsal kineties; adoral zone consisting of 57–70 membranelles; 12–16 frontal cirri, one buccal cirrus, 13–20 midventral pairs, two frontoterminal cirri, two pretransverse cirri, and five to seven transverse cirri. Morphogenesis during physiological regeneration indicates that the marginal rows of each side originate from a common anlage that differentiates into several rows. Molecular phylogenetic analysis based on SSU rDNA sequence data reveals that P. guizhouensis sp. nov. clusters with the type species P. cristata (Jerka‐Dziadosz, 1964) Borror, 1972 and that the genus Pseudourostyla is monophyletic. The morphological characters of another soil hypotrich ciliate, Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012, are also described based on a Chinese (Guizhou) population.


MATERIALS AND METHODS Sampling and cultivation
Soil samples were collected on 19 July 2014 from the floodplain of a small river near the village of Zhonghuashan (27°34′48″N, 109°15′42″E, about 394 m above sea level), Aojiazhai Township, Tongren City, Guizhou Province, China. The annual average temperature of the sampling site is 15 °C and the annual average precipitation is 1,300 mm. About 1 kg of soil was air-dried in the laboratory. Ciliates were stimulated to excyst and emerge from the soil samples by employing the non-flooded Petri dish method described by Foissner (2016). Non-clonal cultures were established at room temperature (about 25 °C) in Petri dishes containing mineral water with squeezed rice grains to enrich the bacterial food.

Morphological investigations
Living cells were observed, using differential interference contrast microscopy and photographed using a digital camera (DP73, Olympus). The protargol silver staining method of Wilbert (1975) was used to reveal the nuclear apparatus and ciliature. Measurements of the stained specimens were carried out with an ocular micrometre. Drawings of stained specimens were performed at 1,250 × with the aid of a camera lucida. To illustrate the changes occurring during morphogenetic processes, the old (parental) ciliary structures are depicted by contour whereas new structures are shaded black. Terminology is mainly according to Berger (2006).

DNA extraction, PCR amplification, and gene sequencing
The genomic DNA of Pseudourostyla guizhouensis sp. nov. was extracted from single specimens, the procedure was conducted as previous studies (Chen et al. 2017;Luo et al. 2017;Shao et al. 2014). In short, each cell of the new species was isolated, washed repeatedly with sterile water to remove potential contamination, and transferred to a 2-ml microfuge tube with minimum volume of water. Genomic DNA was extracted, using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), following the manufacturer's instructions. The SSU rDNA was amplified with the eukaryotic universal primers Forward (5'-AAC CTG GTT GAT CCT GCC AGT-3') and Reverse (5'-TGA TCC  TTC TGC AGG TTC ACC TAC-3') (Huang et al. 2016;Medlin et al. 1988). High fidelity Taq polymerase (Takara Ex Taq, Takara Biotechnology, Dalian) was used to minimize the possibility of amplification errors. The amplification cycles were as follows: 5 min at 94 °C, followed by 30 cycles at 94 °C for 30 s, 56 °C for 1 min, 72 °C for 1 min 50 s; the final extension was 7 min at 72 °C (Gao et al. 2016).

Phylogenetic analyses
The SSU rDNA sequence of Pseudourostyla guizhouensis sp. nov. was aligned to the sequences of 71 other spirotrich ciliates from the GenBank database, using the online program Muscle 3.7 (Edgar 2004). Apodiophrys ovalis, Diophrys scutum, Paradiophrys zhangi, and Uronychia multicirrus were selected as the outgroup taxa. Regions that could not be aligned unambiguously were removed and the ends were trimmed manually, resulting in a matrix of 1,632 characters. The program MrModeltest v.2.2 (Nylander 2004) selected the GTR + I (= 0.5618) + G (= 0.4527) as the best model with Akaike Information Criterion (AIC). The Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) which was run with two sets of four chains for 1,000,000 generations and a sampling frequency of 100 generations. The first 25% of sampled trees were discarded as burn-in prior to constructing 50% majority rule consensus trees. The Maximum-likelihood (ML) analysis was carried out online, using RAxML-HPC2 on XSEDE (8.0.24) on the CIPRES Science Gateway (Stamatakis et al. 2008). TreeView v1.6.6 (Page 1996) and MEGA 4.0 (Tamura et al. 2007) were used to visualize tree topologies.
In order to determine the statistical probability of the hypothesis that the family Pseudourostylidae is monophyletic, constrained ML analyses were generated by PAUP v.4.0. Internal relationships within the constrained groups and relationships among the remaining taxa were unspecified. The reliability of the constrained tree was compared to the unconstrained ML topologies, using the approximately unbiased (AU) test (Shimodaira 2002) implemented in the CONSEL v 0.1 (Shimodaira and Hasegawa 2001).

Nomenclatural Acts
This article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN 2012), and hence the new name contained herein is available under that Code. The 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 https://zoobank.org/. This work was published in a journal with an ISSN.

Pseudourostyla guizhouensis sp. nov.
Description. Body 180-310 × 65-85 µm in vivo and 175-300 × 50-88 µm after protargol staining, ratio of length to width 3-4:1, elongate elliptical in outline, with lateral margins more or less parallel, both ends broadly rounded (Fig. 1A, 2A), dorsoventrally flattened about 2:1, very flexible but not contractile. Cytoplasm colorless and packed with granular inclusions. Posterior cell portion usually filled with food vacuoles rendering cell dark at low magnification ( This article is protected by copyright. All rights reserved. Adoral zone of membranelles about 38% of body length in fixed specimens, composed of 57-70 membranelles (Table 1) with cilia 12-15 µm long. Undulating membranes relatively short, slightly curved and optically intersecting. Pharyngeal fibers distinct after protargol staining, extending posteriorly. Frontal cirri forming a bicorona comprising six to eight pairs of cirri, with cilia 12-15 µm long. Single buccal cirrus, two frontoterminal cirri, and 13-20 midventral pairs in a zig-zag row extending almost to the two pre-transverse ventral cirri. Five to seven transverse cirri. Three or four left and invariably two right marginal rows. Six or seven longitudinal dorsal kineties of almost cell length and dorsal cilia about 5 µm long.
Physiological regeneration. Two reorganizers were found ( Fig. 1H-J, 2I, J); 2). In these specimens: 1) the proximal portion of the parental adoral zone is resorbed and replaced by new membranelles (Fig. 1H, 2I); 2) the frontal-midventral-transverse (FVT) cirral streak I forms the leftmost cirrus in the anterior corona and the undulating membranes, streak II forms the second cirrus in the anterior corona and the buccal cirrus, streaks III to n-1 forms other cirri in bicorona and midventral pairs, the last five to seven streaks generate one transverse cirrus each, the last two streaks contribute one pretransverse cirrus each, and the last streak forms two frontoterminal cirri ( SSU rDNA sequences and phylogenetic analyses. The SSU rDNA sequence of Pseudourostyla guizhouensis sp. nov. (GenBank accession number KX139470) is 1,727 bp long and has a G+C content of 45.2%. The phylogenetic trees inferred from the SSU rDNA sequences using ML and BI, have similar topologies; therefore, a single topology from the ML tree is presented with support values from both algorithms indicated on branches (Fig. 4). Pseudourostyla spp. form a fully supported clade (ML 100%, BI 1.00), and P. guizhouensis sp. nov. is sister to the type P. cristata (DQ019318). The genera Hemicycliostyla, Pseudourostyla, and Trichototaxis are distinctly separated in the genealogies, and the hypothesis that these three genera form a monophyletic group is rejected by AU tests (P = 0.012).

Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012
Description of the Chinese (Guizhou) population. Body about 260 × 80 μm in vivo and 250 × 94 μm after protargol staining, elongate elliptical in outline, slightly narrowed at both ends and very flexible (Fig. 3A, F). One contractile vacuole left of buccal vertex (Fig. 3F). Posterior cell portion in some specimens full of captured prey, possibly the testate amoeba Difflugia (Fig. 3A, F). Cortical granules colorless, about 0.6 μm in diameter, scattering irregularly. 166-195 ellipsoidal macronuclear nodules, approximately 10 × 5 μm in size after protargol staining ( Fig. 3C; Table 1). The number of micronuclei is uncertain (three micronuclei were observed in only one out of eight specimens). Locomotion usually by crawling moderately fast on debris and rotating about main body axis when swimming.
Adoral zone of membranelles occupying about 27% of cell length (in protargol preparations), question mark-shaped, with 55-66 membranelles. Undulating membranes in Oxytricha-pattern, i.e., paroral and endoral slightly curved and optically intersecting (Fig. 3D, K). Pharyngeal fibers distinct after protargol impregnation, extending posteriorly (Fig. 3I). Eleven to 15 frontal cirri arranged in a bicorona. About 12 midventral pairs in a zig-zag row extending to mid-body (Fig. 3D). Two or three buccal cirri, usually one parabuccal cirrus, and two or three frontoterminal cirri. Seven or eight left and six or seven right marginal rows with anterior portion of outermost marginal rows extending onto dorsal surface and terminating with dorsal bristles (Fig. 3E); marginal rows occupy large

Accepted Article
This article is protected by copyright. All rights reserved. portion of cell surface. Five to eight transverse cirri (Fig. 3D, J). Three or four dorsal kineties extending almost entire cell length (Fig. 3E, L).

Pseudourostyla guizhouensis sp. nov.
Morphological comparison with related taxa. The major morphological features of the new species and its congeners are presented in Table 2. The key distinguishing feature of Pseudourostyla guizhouensis sp. nov. is that it invariably has two right marginal rows and thus it can be easily separated from those species with four or more right marginal rows, i.e., P. cristata (both the neotype and the Lake Biwa populations), P. cristatoides, P. dimorpha, P. levis, P. pelotensis, and P. subtropica. The two remaining Pseudourostyla species, namely P. muscorum and P. nova, have two right marginal rows, similarly as P. guizhouensis sp. nov.. The main differences between P. guizhouensis sp. nov. and P. nova are the numbers of frontal cirri (12-16 vs. 4-6), midventral pairs (13-20 vs. 22-36), left marginal rows (three or four vs. invariably two), and macronuclear nodules (26-40 vs. 9-25). Furthermore, P. guizhouensis sp. nov. has a bicorona that is typical in the genus Pseudourostyla, whereas the frontal cirri in P. nova are arranged in a single row or monocorona (Chen et al. 2014;Wiackowski 1988). P. guizhouensis sp. nov. can be distinguished from P. muscroum by the numbers of buccal cirri (one vs. nine) and transverse cirri (5-7 vs. 8-15) (Berger 2006; Kahl 1932).
Morphogenetic comparison. Very early reorganizers of Pseudourostyla guizhouensis sp. nov. were not available. From the longitudinal splitting of the marginal cirral anlagen of a middle stage reorganizer (Fig. 1H, J), we speculate that the new marginal cirral rows develop from one anlage on each side, each of which probably originated from the dedifferentiation of the rightmost parental marginal rows; these anlagen then split and form three left and two right new marginal rows (Fig. 2I). This process is similar to that in P. cristata and P. cristatoides, this is, the marginal rows of each side are formed from a single anlage which appears within the rightmost parental row (Chen et al. 2010;Jung et al. 2012). Further similarities of the cortical development in P. guizhouensis sp. nov. with that in P. cristata and P. cristatoides are that the dorsal kinety anlagen originate in parental rows and generate new dorsal kineties extending anteriorly and posteriorly (Chen et al. 2010;Jung et al. 2012).

Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012
The Chinese population of Hemicycliostyla franzi was firstly found in subtropical soils from the Hunan province (Shen et al. 1992), but only a brief characterization the present species was provided. A more detailed description and illustrations of Chinese population are provided based on Guizhou specimens in present paper.

Comparison of Chinese (Guizhou) population with African and Indian population. The Chinese
(Guizhou) population of Hemicycliostyla franzi matches well the African (Foissner 1987;Berger 2006) and Indian populations (Kumar et al. 2010) in the ciliary pattern and other unique characteristics, i.e., the numbers of marginal rows and macronuclear nodules, an anterior portion of the outermost marginal rows composed of dorsal bristles, marginal rows occupying a large portion of the cell surface, and the absence of caudal cirri. Furthermore, all these three populations were found in terrestrial habitats. There are, however, minor differences among these three populations: 1) the adoral zone of the Chinese (Guizhou) specimens of Hemicycliostyla franzi occupies 27% of body length, which matches its length in the African specimens (30%), whereas it is much longer in the Indian specimens (39%); 2) the numbers of buccal cirri, transverse cirri, and marginal rows in the Guizhou specimens are similar to those in the African specimens, but differ from those in the Indian specimens (Berger 2006;Foissner 1987;Kumar et al. 2010); 3) both the Chinese (Guizhou) and the Indian specimens possess 0.6 µm-sized, spherical cortical granules, whereas the African specimens

Accepted Article
This article is protected by copyright. All rights reserved. possesses 1.0-2.0 × 1.5 µm-sized, ellipsoidal cortical granules which can be extruded (Berger 2006;Paiva et al. 2012); 4) the pharyngeal wall is without peculiarities in the Chinese (Guizhou) specimens, whereas the African specimens have many about 1 μm long rods (Berger 2006;Foissner 1987).

Phylogeny of the family Pseudourostylidae and Pseudourostyla guizhouensis sp. nov.
Members of the family Pseudourostylidae are characterized by having a bicorona, a midventral complex composed of cirral pairs only, and more than one left marginal cirral row (Berger 2006). Consequently, three genera, namely Hemicycliostyla, Pseudourostyla, and Trichototaxis, were assigned to the Pseudourostylidae by Berger (2006) who noted, however, that details of the cirral pattern of the latter two genera were lacking at that time; so, their placement in this family was tentative. Two new species of Trichototaxis have since been described, including details of their cirral pattern, i.e., T. marina Lu et al., 2014 andT. songi Chen et al., 2007. Although both species possess a bicorona, a midventral complex composed of cirral pairs only, and more than one left marginal row, morphogenetic and molecular data did not support the familial placement of Trichototaxis (Chen et al. 2007;Lu et al. 2014). In addition, the type species of Hemicycliostyla, H. sphagni Stokes, 1886, has since been redescribed, and an Indian population of H. franzi has been reported (Kumar et al. 2010;Paiva et al. 2012).
In the SSU rDNA tree presented here, Pseudourostyla, Hemicycliostyla, and Trichototaxis fall into three separate clades, which is consistent with previous findings (Lu et al. 2014). Furthermore, the AU test rejects the possibility that the family Pseudourostylidae is monophyletic. Differences in morphogenetic processes also suggest a more distant relationship among the three pseudourostylid genera. These include: 1) marginal cirral rows originate from individual anlagen in Hemicycliostyla, but from a common anlage in Pseudourostyla; 2) the macronuclear nodules fuse into a single mass during morphogenesis in Pseudourostyla, whereas they fuse into more than one mass in Trichototaxis marina (Lu et al. 2014). However, since morphogenetic and molecular data for the type species of Trichototaxis, T. stagnatilis Stokes, 1891, are not available, it would be premature to revise the family Pseudourostylidae.
In the present study, Pseudourostyla guizhouensis sp. nov. clustered with other Pseudourostyla species with moderate to high support, which corresponds well with the morphological data, i.e., a large body, a bicorona, midventral pairs, two or more marginal rows on each side, and the absence of caudal cirri. Furthermore, the morphogenetic pattern in P. guizhouensis sp. nov. resembles its congeners. The monophyly of Pseudourostyla is thus well supported by our analyses.