Redescription, Morphogenesis, and Molecular Phylogeny of Pseudosincirra longicirrata nov. comb., With Establishment of a New Genus Pseudosincirra nov. gen. (Ciliophora, Hypotrichia)

The morphology and morphogenesis of Pseudosincirra longicirrata nov. gen. and nov. comb., isolated from southern China, were investigated with living observation and protargol staining. Our population is similar to the original population in living characteristics and ciliary patterns. The main determinable morphogenetic features of P. longicirrata nov. comb. are the presence of five frontoventral-transverse cirral anlagen (FVT-anlagen) and a dorsomarginal kinety anlage. According to the origin of FVT-anlagen IV and V in proter, it can be determined that P. longicirrata nov. comb. possesses two frontoventral rows and one right marginal row. Hence, a new genus, Pseudosincirra nov. gen., is proposed, and the diagnosis of P. longicirrata nov. comb. is improved. The new genus is diagnosed as follows: adoral zone of membranelles and undulating membranes is in a Gonostomum pattern; there are three enlarged frontal cirri, one buccal cirrus, and one parabuccal cirrus; postperistomial cirrus and transverse cirri are lacking; there are two more or less long frontoventral rows and one right and two or more left marginal rows; cirri within all rows very widely spaced; dorsal kinety pattern is of Urosomoida type, that is, three dorsal kineties and one dorsomarginal kinety; and caudal cirri are present. Phylogenetic analyses based on the small subunit ribosomal (SSU rDNA) sequence data indicate that P. longicirrata nov. comb. clusters with Deviata and Perisincirra. It is considered that Pseudosincirra nov. gen. and Perisincirra paucicirrata should be assigned to the family Deviatidae; fine cirri, and cirri within all rows being relatively widely spaced, should be considered as plesiomorphies of Deviatidae; and Deviatidae is closely related to Dorsomarginalia or Strongylidium–Hemiamphisiella–Pseudouroleptus.

Stichotrichida is a group of hypotrichous ciliates that has always been difficult to classify. Its establishment is more due to the variable number and non-grouping of frontoventral-transverse cirral rows in its ciliary pattern (Berger, 2008;Lynn, 2008). Previous studies have shown that the dorsal infraciliature is at least as important as the ventral ciliary pattern for the estimation of the major phylogenetic relationships within the hypotrichs, and its application to the systematics of stichotrichids may prove to be particularly beneficial (Foissner et al., 2002;Berger, 2011).
The genus Perisincirra was erected by Jankowski (1978) with Perisincirra kahli (Grolière, 1975) Jankowski, 1978 as the type species. Unfortunately, some important morphological, morphogenetic, and molecular data are lacking for the type species P. kahli, so its diagnostic characters are insufficient for the systematics and classification of Perisincirra to be resolved. Thus, the inclusion of Perisincirra longicirrata Foissner et al., 2002 in this genus has been questioned. Here, the morphogenesis and phylogenetic position of Perisincirra longicirrata were investigated and discussed to determine whether it belongs to Perisincirra or if it represents a new genus.

Sample Site and Cultivation
On 12th September 2018, a mixture of water and silt was collected from a freshwater pond in Shenzhou Peninsula tourist resort (18 • 40 32.27 N; 110 • 20 49.88 E) in Wanning, China, when the water temperature was 25 • C and the pH was about 7.0. The cells of Pseudosincirra longicirrata nov. comb. were isolated from the sample and were cultured at room temperature (25 • C) in Petri dishes containing mineral water (Nongfu Spring) with rice grains added to promote the growth of bacteria as food for the ciliates. Although we failed to establish a clonal culture, there were no other hypotrichous morphospecies in the Petri dishes. Therefore, we are certain that the present morphological, morphogenetic, and molecular studies deal solely with Pseudosincirra longicirrata nov. comb.

Morphology
Cells from the cultures were studied in vivo using bright-field and differential interference contrast microscopy at magnifications of 40-1,000 (Bai et al., 2020). The protargol (Sigma-Aldrich) silver staining method was used to reveal the infraciliature and nuclear apparatus (Wilbert, 1975). The drawings of stained specimens were made with a drawing device (Wu et al., 2020). To illustrate the changes occurring during morphogenetical processes, old (parental) ciliary structures are depicted by contour, whereas new ones are shaded black. Terminology follows Berger (2011).

DNA Extraction, PCR Amplification, and Sequencing
Single cell of Pseudosincirra longicirrata nov. comb. was washed three times with sterilized water to remove contaminants and then transferred to a 1.5-ml microfuge tube with a minimum volume of water. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer's instructions. Small subunit ribosomal (SSU rDNA) amplification from the extracted DNA was carried out using primers 82S-F (5 -GAA ACT GCG AAT GGC TC-3 ), 900F (5 -CGA TCA GAT ACC GTC CTA GT-3 ), Pro B (5 -GGT TAA AAA GCT CGT AGT-3 ), 900R (5 -ACT AGG ACG GTA TCT GAT CG-3 ), and 18S-R (5 -GAT CCT TCT GCA GGT TCA CCT AC -3 ). The conditions for PCR were as follows: denaturation at 98 • C for 2 min; followed by 30 cycles of denaturation at 98 • C for 10 s, annealing at 56 • C for 15 s, extension at 72 • C for 1 min 50 s, and a final extension step at 72 • C for 7 min. Sequencing was performed bidirectionally by the Tsingke Biotechnology Co., Ltd. Xi'an Branch.

Diagnosis
The new genus is diagnosed as follows: adoral zone of membranelles and undulating membranes is in Gonostomum pattern. There are three enlarged frontal cirri, one buccal cirrus, and one parabuccal cirrus. Postperistomial cirrus and transverse cirri are lacking. There are two long frontoventral rows and one right and two or more left marginal rows, and cirri within all rows are widely spaced. Dorsal kineties are in Urosomoida pattern, that is, three dorsal kineties and one dorsomarginal kinety. Caudal cirri are present.

Etymology
The name is a composite of pseudo-(false, i.e., resembling but not equaling) and suffix (-sincirra) of the genus name Perisincirra Jankowski, 1978. This indicates that Pseudosincirra has a cirral pattern similar to that of Perisincirra and has a feminine gender.

Type of Species
The type of species is Perisincirra longicirrata Foissner et al., 2002. Remarks Pseudosincirra longicirrata nov. comb. was previously assigned in Perisincirra as Perisincirra longicirrata. However, according to the origin of frontoventral-transverse cirral anlagen (FVTanlagen) IV and V (for details, see Divisional Morphogenesis), it can be deduced that right marginal rows 1 and 2 of Pseudosincirra longicirrata nov. comb. described in Foissner et al. (2002) are actually frontoventral rows. It differs from the type species of Perisincirra, P. kahli (Grolière, 1975) Jankowski, 1978, in having two (vs. one) long ventral rows. Hence, a new genus, Pseudosincirra nov. gen., was erected.

Voucher Slides
Seven voucher slides (no. GQ2018091202A-G) with protargolstained specimens were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.

Morphological Redescription
The body size is 90-130 × 30-50 µm in vivo (n = 16), usually about 120 × 40 µm; the length-to-width ratio is about 3:1 in live and 2-2.8:1, on average 2.5:1, in protargol preparations due to cell expansion caused by the fixative. The body is usually elongated ellipsoidal in shape, occasionally fusiform with the anterior region slightly more narrowed and the posterior portion indistinctly pointed (Figures 1A,D,E). The body is flexible but not contractile, only slightly flattened dorsoventrally. Invariably, two ellipsoidal macronuclear nodules, about 17 × 9 µm in size (after protargol staining), are located behind the buccal vertex near midline. Usually there are two, sometimes three, globular micronuclei about 2 µm in diameter, two of which are closely associated with macronuclear nodules (Figures 1C,J). A single contractile vacuole without distinct collecting canals, about 13 µm in diameter, is contracting at intervals of about 12 s, located about 33% down the length of the body near the left cell margin. Cortical granules are lacking. The cytoplasm is colorless, usually packed with highly refractive fat globules 1-6 µm across, making cells appear dark under low magnification. Crystals are sparse or lacking. Many individuals have food vacuole with orange contents on the right side of their bodies, about 20 µm across. Locomotion by slowly crawling on substrate was observed; when suspended, swimming while rotating about the longitudinal axis was observed.
The adoral zone of membranelles is about 27% of the body length in vivo and 30% on average in protargol preparations due to strong cell expansion caused by the fixative. Oral apparatus is in Gonostomum pattern. The adoral zone is composed of 22-26 membranelles (n = 16) with cilia up to 17-µm long. The endoral is located behind and parallel to the paroral and are almost equal in length (Figures 1B,F,G; Table 1).
The ciliary pattern on the ventral side is rather constant although the number of cirri within rows is variable. All cirri are rather long and conspicuous, with cilia of the frontal cirri and the marginal cirri about 20-µm long. Constantly, there are three frontal cirri with the rightmost one in front of cirrus III/2. Buccal cirrus is ahead of the endoral and slightly behind the anterior end of the paroral. There are two long frontoventral rows; the inner row (FVR1) is composed of 13-16 cirri and commences slightly behind the level of cirrus III/2, and the outer row (FVR2) consists of 14-18 cirri and commences at the approximately same level as the rightmost frontal cirrus; both rows terminate at the posterior end of the cell. There are three widely spaced left marginal rows, commencing near the proximal end of the adoral zone, composed of 10-13, 10-12, and 7-9 cirri, from inner to outer row, respectively. Invariably, there is one right marginal row, with 12-16 cirri, which is almost bipolar (Figures 1B,F,G). All cirri are fine, and most frontoventral and marginal cirri are composed of four basal bodies and widely spaced. Dorsal bristles are about 5-µm long; constantly arranged in four kineties; and composed of 12-14, 12-16, 11-13, and 5-8 dikinetids, respectively. Dorsal kineties 2 and 3 are almost bipolar; kinety 1 commences about 20% down the length of the body; kinety 4 (dorsomarginal kinety) terminates about 35% down the length of body. Usually, there are three caudal cirri, one at the posterior end of each of dorsal kineties 1-3; sometimes, there are four caudal cirri (in about 25% of the individuals), two at the posterior end of dorsal kinety 1 and one at the posterior end of each of dorsal kineties 2 and 3 ( Figures 1C,H,I).

Development of the Ventral Ciliature
The oral primordium for the opisthe appears in the postoral area between the inner left marginal row and frontoventral row 1. With the proliferation of basal bodies, the oral primordium lengthens posteriorly and differentiates posteriad (Figures 2A-D, 4A,B). Three streaks are formed anteriorly to the right of the oral primordium (Figures 2C,D, 4C,E).
In the proter, the parental adoral zone remains intact. Anlage I forms from the dedifferentiated parental paroral and endoral, anlage II develops from the dedifferentiated parental buccal cirrus, and anlage III is generated from the dedifferentiated parental cirrus III/2 (as shown in Figures 2D,E,  4D). Up to this point, three FVT-anlagen are formed in each filial product.
Soon after, in the proter, anlage IV is generated within the frontoventral row 2, and anlage V develops de novo to the right of frontoventral row 2. In the opisthe, anlagen IV and V are generated within frontoventral rows 1 and 2, respectively (Figures 2E,G, 4F). Then, in the proter, anlagen IV and V migrate to the mid-region of the body (Figures 2G, 3A, 4H,I). Finally, anlage I produces the left frontal cirrus and undulating membranes, anlage II generates the middle frontal cirrus and buccal cirrus, anlage III develops into the right frontal cirrus and cirrus III/2, while anlagen IV and V generate frontoventral rows 1 and 2, respectively (Figures 3A,C, 4K).

Development of Marginal Rows and Dorsal Kineties
The marginal row anlagen and dorsal kinety anlagen develop intrakinetally within the parental marginal rows and dorsal kineties 1-3 in each daughter cell, respectively. These anlagen enlarge by proliferation of basal bodies and stretch in both directions to eventually replace the parental rows (Figures 2E-H, 3A,B, 4G-I). In addition, a short streak of basal bodies, i.e., the dorsal kinety 4 anlage, develops ahead of the anteriormost portion of the right marginal anlage in both the proter and the opisthe (Figures 3A, 4I). One or two caudal cirri are formed at the posterior end of dorsal kinety anlage 1, and constantly, one caudal cirrus is formed at the posterior end of each dorsal kineties anlagen 2 and 3 (Figures 3D, 4L).

Division of Nuclear Apparatus
In this process, the two macronuclear nodules fuse into a single mass (Figures 3B, 4J). At later stages of morphogenesis, the mass splits and is distributed between the proter and the opisthe (Figures 3D, 4L). The micronuclei divide mitotically (Figures 2H, 3B,D, 4J,L).

Phylogenetic Analyses Based on SSU rDNA Gene Sequences
The SSU rDNA sequence of Pseudosincirra longicirrata nov. comb. was deposited in GenBank with the accession number OK173050. The length and GC content of the new sequence are 1,613 bp and 45.38%, respectively. Since the topologies of ML tree and BI tree are basically the same, only the ML tree is shown with nodal support for both algorithms (Figure 5).
Compared with Fragmocirrus, the new genus has a Gonostomum-patterned (vs. Oxytricha-patterned) adoral zone of membranelles and undulating membranes, a single buccal cirrus and a single parabuccal cirrus (vs. a buccal row FIGURE 5 | Maximum likelihood (ML) tree based on the SSU rDNA sequence data. The newly sequenced Pseudosincirra longicirrata nov. gen. and nov. comb. are indicated in red. Numbers at the nodes represent the bootstrap values of ML and the posterior probabilities of Bayesian analysis (BI), respectively. A hyphen "-" indicates the disagreement between the BI tree and the reference ML tree. All branches are drawn to scale. The scale bar corresponds to 0.01 expected substitutions per site. and a parabuccal row), transverse cirri absent (vs. present), fine cirri (vs. moderately thick), cirri within all rows very widely (vs. narrowly) spaced, and caudal cirri at the end of dorsal kineties 1-3 (vs. at the end of dorsal kineties 1 and 2 only) (Berger, 2011).
Kahliella resembles Pseudosincirra nov. gen. in terms of the structure of its adoral zone of membranelles and undulating membranes, but can be separated from the latter by having three (vs. two) frontoventral rows, moderately thick (vs. fine) cirri and cirri within all rows narrowly (vs. widely) spaced, parental left marginal rows retained (vs. resorbed) in postdividers, and caudal cirri absent (vs. present) (Berger, 2011).
Pseudosincirra nov. gen. differs from Parakahliella in having a Gonostomum-patterned (vs. Oxytricha-patterned) adoral zone of membranelles and undulating membranes, fine (vs. moderately thick) cirri, all rows with very widely (vs. narrowly) spaced cirri, a single buccal and a single parabuccal cirrus (vs. a buccal row and a parabuccal row), parental dorsal kineties absorbed (vs. retained) in postdividers, and caudal cirri at the end of dorsal kineties 1-3 (vs. at the end of dorsal kineties 1 and 2 only) (Berger, 2011). Pseudosincirra nov. gen. resembles Perisincirra in having widely spaced cirri in all rows, but can be separated from the latter by having two (vs. one) long ventral rows (Berger, 2011).
Identification of the Chinese Population of Pseudosincirra longicirrata nov. comb. and Comparison With Two African Populations Pseudosincirra longicirrata nov. comb. was first reported by Foissner et al. (2002) as Perisincirra longicirrata based on populations discovered in Benin and Namibia. The present population differs from the Benin and Namibian populations in having the following: more adoral membranelles (22-26 vs. 18-22); a smaller ratio of adoral zone length to body length (24-37% and average 30 vs. 29-44% and average 37%); more cirri in the frontoventral rows 1 and 2 (13-16 and 14-18 vs. 7-12 and 9-14) and in the middle left marginal row (10-12 vs. 4-10); usually three, sometimes four, caudal cirri (vs. invariably three caudal cirri); two or three micronuclei (vs. one or two micronuclei); and shorter cilia on the ventral side (20 vs. 30-µm long). However, we consider these differences to be population-dependent and therefore not significant for species-level separation. The identity of the present population is therefore not in doubt.

Morphogenesis
Previous studies on Pseudosincirra longicirrata nov. comb. were limited to morphology, so this is the first report of its morphogenesis (Foissner et al., 2002). According to the origin of FVT-anlagen IV and V in the proter, it can be deduced that right marginal rows 1 and 2 described in Foissner et al. (2002) are actually frontoventral rows. Hence, only one right marginal row is present. Considering the formative mode of FVT-anlagen IV and V, we compare P. longicirrata nov. comb. with some similar species that possess three clearly differentiated frontal cirri and at least two more or less long frontoventral rows and whose FVT-anlagen development is well known, i.e., Afrokahliella paramacrostoma Li et al., 2021a;Deviata abbrevescens Eigner, 1995;D. baciliformis;D. brasiliensis;D. parabaciliformis;Fragmocirrus espeletiae Foissner, 2000;Khaliella simplex (Horváth, 1934) Berger, 2011Neogeneia hortualis Eigner, 1995; and Parakahliella macrostoma (Foissner, 1982) Berger et al., 1985 (for details, see Table 2). In all these genera, not all the FVT-anlagen IV-VI in both daughter cells develop within the corresponding parental frontoventral rows IV-VI, as in Parastrongylidium oswaldi (Aescht and Foissner, 1992). We speculated that this point may be phylogenetically informative pending greater taxon sampling and the availability of further information on molecular phylogeny of these species.

Molecular Phylogeny
Pseudosincirra longicirrata nov. comb. falls in the Deviata baciliformis + D. brasiliensis + D. parabaciliformis + D. rositae + Perisincirra paucicirrata clade, the close relationship between these six species being supported by having fine cirri, i.e., cirri in the ventral and marginal rows are mostly composed of two or four cilia and cirri within all rows are relatively widely spaced. The presence or absence of dorsomarginal kineties and the number of dorsal kineties vary in these species and other species in the family Deviatidae Foissner, 2016. We agree with Foissner (2016) that "the feature of presence/absence of dorsomarginal kineties evolved several times independently" and posit that Pseudosincirra and Perisincirra paucicirrata should be assigned to Deviatidae. Furthermore, we suggest that the possession of fine cirri and the relatively widely spaced cirri within all rows should be considered as plesiomorphies of this family/group and added in the diagnosis of the family.

DATA AVAILABILITY STATEMENT
The data presented in the study are deposited in the GenBank database, accession number OK173050.

AUTHOR CONTRIBUTIONS
QG and QT collected the samples and carried out almost all of the experiments (preparations, illustrations, micrographs, etc.). All authors did the identification of the species and wrote the manuscript. All authors contributed to the article and approved the submitted version.