Cell-division pattern and phylogenetic analyses of a new ciliate genus Parasincirra n. g. (Protista, Ciliophora, Hypotrichia), with a report of a new soil species, P. sinica n. sp. from northwest China

Ciliated protists, a huge assemblage of unicellular eukaryotes, are extremely diverse and play important ecological roles in most habitats where there is sufficient moisture for their survivals. Even though there is a growing recognition that these organisms are associated with many ecological or environmental processes, their biodiversity is poorly understood and many biotopes (e.g. soils in desert areas of Asia) remain largely unknown. Here we document an undescribed form found in sludge soil in a halt-desert inland of China. Investigations of its morphology, morphogenesis and molecular phylogeny indicate that it represents a new genus and new species, Parasincirra sinica n. g., n. sp. The new, monotypic genus Parasincirra n. g. is defined by having three frontal cirri, an amphisiellid median cirral row about the same length as the adoral zone, one short frontoventral cirral row, cirrus III/2 and transverse cirri present, buccal and caudal cirri absent, one right and one left marginal row and three dorsal kineties. The main morphogenetic features of the new taxon are: (1) frontoventral-transverse cirral anlagen II to VI are formed in a primary mode; (2) the amphisiellid median cirral row is formed by anlagen V and VI, while the frontoventral row is generated from anlage IV; (3) cirral streaks IV to VI generate one transverse cirrus each; (4) frontoventral-transverse cirral anlage II generates one or two cirri, although the posterior one (when formed) will be absorbed in late stages, that is, no buccal cirrus is formed; (5) the posterior part of the parental adoral zone of membranelles is renewed; (6) dorsal morphogenesis follows a typical Gonostomum-pattern; and (7) the macronuclear nodules fuse to form a single mass. The investigation of its molecular phylogeny inferred from Bayesian inference and Maximum likelihood analyses based on small subunit ribosomal DNA (SSU rDNA) sequence data, failed to reveal its exact systematic position, although species of related genera are generally assigned to the family Amphisiellidae Jankowski, 1979. Morphological and morphogenetic differences between the new taxon and Uroleptoides Wenzel, 1953, Parabistichella Jiang et al., 2013, and other amphisiellids clearly support the validity of Parasincirra as a new genus. The monophyly of the family Amphisiellidae is rejected by the AU test in this study. The critical character of the family Amphisiellidae, i.e., the amphisiellid median cirral row, might result from convergent evolution in different taxa. Amphisiellidae are not monophyletic.

Among these, the order Stichotrichida Fauré-Fremiet, 1961 is one of the most confused and diverse ciliate groups in terms of both its taxonomy and phylogeny [18]. One of its largest families, Amphisiellidae Jankowski, 1979, is characterised by the possession of an amphisiellid median cirral row derived from two or three, rather than one, frontoventral-transverse cirral anlagen. Most amphisiellids occur in terrestrial habitats, although some are marine [15,19,20]. In the present study, we present a new amphisiellid collected from sludge soil in a flood drain in Lanzhou, China (Fig. 1). Observations of its morphology and morphogenesis, both in vivo and after protargol staining, demonstrate that it represents a novel genus, Parasincirra n. g., of the family Amphisiellidae. The SSU rDNA of the new isolate was sequenced and its molecular phylogeny was analyzed.
Diagnosis Amphisiellidae with elongate body. Three frontal cirri. Amphisiellid median cirral row about same length as adoral zone. One short frontoventral cirral row. Cirrus III/2 and transverse cirri present. One right and one left marginal row. Three dorsal kineties. Caudal cirri and buccal cirrus lacking.
Type species Parasincirra sinica n. sp. Etymology Composite of the Greek prefix para-(close to; related; deviating) and suffix (-sincirra) of the genus name Hemisincirra Hemberger, 1985. This indicates that Parasincirra has a cirral pattern similar to that of Hemisincirra. Feminine gender.
Remarks We do not assign Hemisincirra interrupta (Foissner, 1982) Foissner in Berger, 2001 andH. vermicularis Hemberger, 1985 to our new genus Parasincirra although both species also lack a buccal cirrus. The main reasons are that either the ontogenetic or the molecular information are unknown for these two species.
Type material One protargol-stained slide (no. MJY2017043001B) with the holotype specimen and two paratype slides (no. MJY2017043001A, C) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.
Etymology The species-group name sinica means the species was first discovered in China.
Morphological description Body 90-160 μm × 20-40 μm in vivo (n = 6) with a ratio of length to width of about 3.5:1-7.5:1; protargol-stained cells 120 μm × 30 μm on average with a ratio of length to width of about 4:1. Generally slender, almost fusiform to vermiform, non-contractile but highly flexible, and thus cell outline variable, i.e., sigmoidal or curved (Fig. 2d). Anterior end narrowly rounded and posterior end more or less tapered to form a pointed tail that is more flexible and contractile than the rest of the cell (Fig. 2a, d, g, h); tail unrecognisable in protargol preparations (Fig. 2e, f, i). Dorsoventrally flattened up to 2:1. Usually four (2-6) macronuclear nodules arranged along or slightly left of mid-line, behind buccal vertex; one to three, on average two, micronuclei attached, or near to, macronuclear nodules. Macronuclear nodules ellipsoidal, about 9-19 μm × 4-10 μm after protargol staining (Fig. 2j). Micronuclei about 2.9 μm × 2.4 μm after protargol staining. One contractile vacuole measuring about 13 μm in diameter in diastole, positioned near left margin, contracting at intervals of 10 s (Fig. 2g, h). Cortical granules colourless, globular, about 0.5 μm in diameter, distributed around dorsal ciliary organelles, also visible in protargol preparations (Fig. 2c, j, m). Cytoplasm colourless to greyish, often packed with numerous small lipid droplets. Locomotion mainly by slowly crawling on substrate and debris, sometimes jerking back and forth. When suspended, cells often swim continuously in circles.
Infraciliature as shown in Fig. 2b, e, f, i-l. Most somatic cirri relatively fine with cilia about 12-16 μm long. Constantly three relatively stout frontal cirri in an almost transverse pseudo-row immediately behind several distal adoral membranelles, cilia about 15 μm long. Amphisiellid median cirral row (ACR) short and consisting of four cirri; commences at about the level of the rightmost frontal cirrus (about 6% down length of body), or slightly lower, terminates at about level of buccal vertex (about 21% down length of body). Parabuccal cirrus (cirrus III/2) located at the level of the middle region of the paroral and endoral. Frontoventral row lies between the parabuccal cirrus and the ACR, invariably composed of two cirri; commences at about the level of the second cirrus in the ACR (about 8% down length of body) and terminates ahead of the third cirrus in the ACR (about 10% down length of body). Three, rarely two or four, slightly subterminal transverse cirri, cilia of which are about 16 μm long. Constantly one left and one right marginal row with 34-52 and 34-53 cirri, respectively (Table 1). Right marginal cirral row begins dorsolaterally at anterior end of cell Three dorsal kineties arranged in Gonostomum-pattern, with cilia about 3 μm in length, composed of about 13, 15 and 15 dikinetids, respectively, and arranged in a gradient; that is, kinety 3 commences apically, kinety 2 starts slightly behind kinety 3, while kinety 1 starts slightly behind kinety 2. Each terminates at the posterior end of the body (Fig. 2f, j). Adoral zone of membranelles (AZM) shaped as in other amphisiellid species, terminates 11-20% (average about 16%) down length of body, comprising 14-19 membranelles. Cilia of distal membranelles about 13 μm long. Buccal cavity small, endoral and paroral bending strongly and optically intersecting with each other at their lower or middle regions (Fig. 2b, e, i, k).

Morphogenesis during binary fission Stomatogenesis
Cortical morphogenesis in Parasincirra sinica n. sp. mainly occurs in two zones: an anterior field for the proter and a posterior field for the opisthe. In the opisthe, the first evidence of stomatogenesis during cell division is the appearance of groups of basal bodies on the cell surface, i.e., the opisthe's oral primordium, which is located in the end of the ACR, indicating that parental basal bodies are incorporated in the primordium (Fig. 3a). These groups subsequently merge by further proliferation of basal bodies forming a single anarchic field. Subsequently the new adoral membranelles organise posteriad (Figs. 3c, 5e). The anlage for the undulating membranes (anlage I) is formed to the right of the oral primordium (Figs. 3c, 5e). Later, the left frontal cirrus develops from the anterior end of the UM-anlage (Figs. 4g, 5j). During the later stages, the differentiation of membranelles is completed forming the new oral structure for the opisthe. Subsequently, the UM-anlage gives rise to the leftmost frontal cirrus and the new endoral and paroral (Figs. 4a, b, 5n).
In the proter, several of the proximal membranelles dedifferentiate into sparsely distributed basal bodies which then differentiate into membranelles ( Fig. 3c-e). The parental undulating membranes dedifferentiate into UM-anlage. The basic development of the UM-anlage then follows a similar pattern to that in the opisthe (Figs. 3b-e, g, 4a, 5d, e, m).

Development of the frontoventral-transverse cirri
The development of the somatic ciliature begins with the formation of the frontoventral-transverse cirral anlagen (FVT-anlagen). Initially, the FVT-anlagen appear as a small group of basal bodies (Fig. 3a). The Table 1 Morphometric characteristics of Parasincirra sinica n. sp ACR amphisiellid median cirral row, AZM adoral zone of membranelles, CV coefficient of variation in %, DK dorsal kineties, FVR frontoventral cirral row, HT holotype specimen, M median, Max maximum, Mean arithmetic mean, Min minimum, n sample size, PBC parabuccal cirri, SD standard deviation a All data are based on protargol-stained specimens. Measurements in µm parental frontoventral cirri disaggregate and appear to in the formation of the FVT-anlagen. Later, five FVTanlagen are formed to the right of the UM-anlage in the proter as primary primordia (Figs. 3b, 5d). Then, the FVT-anlagen fragment in the middle to form two sets of anlagen, one set for the proter and the other for the opisthe (Figs. 3d, e, 5h, i). Subsequently, cirri segregate from anterior to posterior in the following manner: anlage I develops the frontal cirrus I/1 (leftmost frontal cirrus); anlage II produces the middle frontal cirrus; anlage III generates a parabuccal cirrus and the rightmost frontal cirrus; anlage IV contributes two cirri forming the short frontoventral cirral row; anlage V produces the posterior two cirri in the ACR; anlage VI forms the anterior two cirri in the ACR; and anlagen IV-VI produce one transverse cirrus each (Figs. 4a, b, 5m, p). Finally, the new cirri move to their final positions.

Development of marginal rows and dorsal kineties
Within every parental marginal row a few cirri near the anterior end, and a few others below the mid-body, differentiate to form two separate anlagen. The dorsal kineties develop by intrakinetal basal body proliferation, i.e. two anlagen develop in each parental row. Subsequently, the new marginal cirral rows and dorsal kineties develop and replace the old ones (Figs. 3d-h, 4a-c, 5g, h, j, m).

Division of nuclear apparatus
The nuclear apparatus divides in the usual way for hypotrichs hence no need to describe this process in detail (Figs. 3f, h, 4c, 5k).

SSU rDNA gene sequence and phylogenetic analyses
The 18S rDNA gene sequence of Parasincirra sinica n. sp. (GenBank accession number: MN472864) is 1731 bp long and has a G + C content of 45.70%. Phylogenetic trees inferred from the SSU rDNA sequences using two different methods (ML and BI) show similar topologies. Therefore, only the topology of the ML tree is presented with nodal support from both methods (Fig. 7). Molecular phylogenetic analyses result in a clade containing four polytomies represented by Parasincirra sinica n. sp., two Uroleptoides species and Parabistichella variabilis Jiang et al., 2013 with high support (83% ML, 1.00 BI, Fig. 7). They also confirm the polyphyly of other amphisiellids including species belonging to the type genus Amphisiella Gourret and Roeser, 1888. The monophyly of the family Amphisiellidae is rejected by the AU test (p < 0.05) based on SSU-rDNA dataset ( Table 2).

Comparison with similar genera
Amphisiellidae were divided into three groups by Berger (2008) [19]. Group I comprises the marine taxa  [19]. With reference to the general infraciliature, Hemisincirra resembles Parasincirra n. g., however, the type . a-d Ventral views of early dividers, to indicate the oral primordium (arrows in a-c), the formation of frontoventral-transverse cirral anlagen and undulating membranes starting to dedifferentiate (arrowhead). Note the old frontal cirri remain intact (arrows in d). e, f Ventral views of later dividers, to show the oral primordia starting to differentiate into membranelles (arrow), formation of undulating membranes anlagen in the proter (arrowhead), and frontoventral-transverse cirral anlagen starting to separate (f ). g-i Ventral views of later dividers, to show the dedifferentiation of membranelles at the proximal end of the old adoral zone of membranelles (arrow), the intrakinetally formed anlagen for the marginal rows (arrowhead), and stretched marginal anlagen and frontoventral-transverse cirral anlagen (h, i). j-l Ventral (j) and dorsal (k, l) views of a middle divider, to show frontoventral-transverse cirral anlagen differentiating into cirri (j), dorsal kineties anlagen (arrowheads), the old dorsal dikinetids (arrows) and the macronuclear nodules fusing into a single mass (k). m, n Ventral views of a late divider, arrows show transverse cirri migrating into their final positions in the opisthe (m) and proter (n). Note the undulating membranes anlagen longitudinally splitting into parorals and endorals. o Dorsal view, to show the newly formed dorsal kineties. p Ventral view, to demonstrate transverse cirri (arrow) migrating into their final positions. Scale bars: 15 μm  [19]. Afroamphisiella can be distinguished from Parasincirra n. g. by the presence (vs. absence) of a buccal cirrus and the absence (vs. presence) of transverse cirri [19]. Cossothigma can be separated from the new genus by its trachelostylid (vs. elliptical to elongate-elliptical) body shape and trachelostylid oral apparatus (vs. in Oxytricha-pattern), and the probable presence (vs absence) of caudal cirri [19]. Mucotrichidium differs from the new genus in possessing a buccal cirrus, postperistomial cirrus and caudal cirri, all of which are absent in Parasincirra n. g. [19]. Terricirra can be separated from Parasincirra n. g. by the presence (vs. absence) of a buccal cirrus, while Tetrastyla can be separated from Parasincirra n. g. by the absence (vs. presence) of parabuccal cirri [19].
Parasincirra sinica n. sp. can be separated from Uroleptoides longiseries by its lack of a buccal cirrus (vs. present in the latter) and having fewer cirri in the ACR (4 vs. 24-54 in the latter) [19].

Phylogenetic analyses
Molecular phylogenetic analyses did not resolve the relationship of the four polytomies represented by Parasincirra sinica n. sp., Uroleptoides magnigranulosa, U. longiseries and Parabistichella variabilis (Figs. 6, 7). Taxonomically, P. sinica n. sp. has the critical character of the family Amphisiellidae, i.e., the ACR that originates from two separate anlagen, and apparently it should be assigned in this family (exactly, group II in Amphisiellidae) [18,19]. However, the long ventral row is formed by just a single anlage in Uroleptoides longiseries and Parabistichella variabilis, hence they should not be assigned to Amphisiellidae. Whether Uroleptoides magnigranulosa is correctly assigned to the family Amphisiellidae needs further clarification [19,24,25]. The close relationship between these four species is supported by each having three enlarged frontal cirri, one marginal cirral row on each side and cortical granules present. Nevertheless, their close relationship not represented in the SSU rDNA tree might be due to poor taxon sampling. The phylogenetic relationship between Parasincirra sinica n. sp. and its most morphologically similar genera, Lamtostyla and Hemisinicirra, also needs further investigation due to the remote position of P. sinica n. sp. in the SSU rDNA tree and the lack of information of the latter, e.g., the ontogenetic process has not been characterised for the type species of either.

Conclusions
It is noteworthy that the positions of other amphisiellid species, even members of the type genus Amphisiella, are not well resolved well in the SSU rDNA tree (Fig. 7), which is consistent with previous studies [22,26,27]. Members of the Amphisiellidae are placed at two different parts of the phylogenetic tree even though they all possess the critical character of the family, i.e., the development of the ACR from two separate anlagen. Furthermore, the monophyly of the family Amphisiellidae is rejected by the AU test (p < 0.05) based on SSU-rDNA datasets. A composite row, resembling ACR, is formed in Kahliella matisi Vďačný et al., 2010, which belongs to the oxytrichine hypotrichs, and in Hemiholosticha pantanalensis Vďačný and Foissner, 2019, which belongs to the psilotrichid hypotrichs [28,29]. These observations indicate that the ACR might be homoplastic. Further studies are needed to clarify the systematic position and evolution of species within the family Amphisiellidae.

Sample collection, isolation, and culturing
Sludge soil samples were collected from the upper 10 cm layer within a flood drain in Lanzhou (36º 3′ N; 103º 49′ E), China on 30 April 2017 (Fig. 1). Samples were dried at room temperature (about 24 °C) immediately after collection in order to preserve them. Several months later, ciliates were induced to excyst from the soil samples by employing the non-flooded Petri dish method [30]. Ciliate cells were then isolated using micropipettes and nonclonal cultures were established at room temperature in Petri dishes containing mineral water (Nongfu Spring) with rice grains added in order to stimulate the growth of bacteria as food source for the ciliates. We identified only one species, and relied on in vivo morphological characteristics to assure the accuracy of that identification for all downstream analyses, even though we were unable to establish clonal cultures. No other stichotrichid morphotypes were present in the protargol preparations.

Morphology and morphogenetic studies
Live observations were carried out using bright field and differential interference contrast microscopy (Olympus BX53), photographed using a digital camera and figures were made by Photoshop. Protargol staining was used to reveal the ciliature and the nuclear apparatus [31]. The protargol reagent was synthesized following the protocol of Pan et al. (2013) [32]. Counts and measurements of stained specimens were performed at a magnification of 1000×. Drawings of protargol-stained cells were made with the assistance of a drawing device (camera lucida). To illustrate the changes that occurred during morphogenesis, parental structures are depicted by contour whereas new structures are shaded black [33,34]. Terminology is according to Berger (2008) [19] and the systematic classification follows Lynn (2008) [18].

DNA extraction, PCR amplification, and gene sequencing
Single cells of Parasincirra sinica n. sp. were isolated from cultures, washed several times with distilled water using a micropipette in order to remove potential contamination, and then transferred to 1.5 mL microfuge tubes with a minimum volume of water. DNA extraction was performed with the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions with minor modifications [35,36]. PCR amplification and sequencing of the SSU rDNA were performed according to Sheng et al. (2018) [37] using high fidelity Takara Ex Taq DNA polymerase (Takara Ex Taq; Takara Biomedicals) to minimise the possibility of amplification errors. The PCR products were purified using Geneclean (BIO 101 Inc., La Jolla, CA, USA) and sequenced bidirectionally on the ABI 3700 sequencer (GENEWIZ Biotechnology Co., Ltd., Beijing, China).

Phylogenetic analyses and topology testing
The SSU rDNA sequence of the new species, together with 54 representative taxa downloaded from the Gen-Bank database, were used in the phylogenetic analyses.  Wilbert et al., 2005) were selected as putative outgroups. All sequences were aligned using the GUIDANCE web server (http://guida nce.tau.ac.il/) [38]. The resulting alignment was manually edited using the program BioEdit 7.0 [39]. Both Maximum likelihood (ML) and Bayesian inference (BI) analyses were performed on the final alignment under the best-fit nucleotide substitution model of GTR + Γ that was selected by jModelTest ver. 2.1.7 [40]. The ML analysis was performed using RAxML-HPC2 on XSEDE v8.2.12 on the online server CIPRES Science Gateway [41], with 1000 rapid bootstrap replicates and a subsequent thorough ML search. Bayesian inference was computed with MrBayes on XSEDE 3.2.6 [42], Fig. 6 a-j Diagram of the infraciliature, and formation patterns of ventral cirri (dotted lines connecting cirri that develop from the same cirral streaks, arrows mark the buccal cirri) (a, c, e-j) and dorsal ciliature (b, d).  [19, 22, 24-26, 47, 48] running four Markov chains sampling every 100 generations for a million generations and discarding the first 25% of trees as burn-in. The majority rule consensus tree was produced from the remaining samples with each node labelled with its posterior probability. SeaView v.4 [43] and MEGA v5 [44] were used to visualise the tree topologies.
The approximately unbiased (AU) test [45] was performed to assess the monophyly of species of the family Amphisiellidae that possess an amphisiellid median cirral row. The constrained ML tree was generated based on SSU rDNA sequences. The site likelihoods for the resulting constrained topology and then on-constrained ML topology were calculated using PAUP and then analyzed in CONSEL [46].