Morphology, Morphogenesis, and Molecular Phylogeny of a New Freshwater Ciliate, Quadristicha subtropica n. sp. (Ciliophora, Hypotrichia)

The morphology and the regulation of cortical pattern associated with the cell size, division, and phylogenetic position of a new hypotrichous ciliate, Quadristicha subtropica n. sp. collected from a freshwater pond in southern China, were investigated. Quadristicha subtropica n. sp. is characterized as follows: size in vivo 60–115 μm × 25–45 μm; 19–21 adoral membranelles; buccal cirrus near anterior end of endoral and paroral; cirrus IV/3 at about level of buccal vertex; right marginal row begins ahead of buccal vertex; 11–16 right and 12–19 left marginal cirri; and dorsal cilia about 5 μm long. The basic morphogenetic process in Q. subtropica n. sp. is consistent with that of the type species, Quadristicha setigera. Phylogenetic analyses based on small subunit ribosomal DNA sequence data reveal that the systematic position of Q. subtropica n. sp. is rather unstable with low support values across the tree and the genus Quadristicha is not monophyletic.


MATERIALS AND METHODS
Quadristicha subtropica n. sp. was isolated from a freshwater pond in Peninsula Lake Park in Wanning, China (18°41′03″N; 110°24′02″E), on September 12, 2018. Some bark and rotten leaves were taken together with water from the sampling site. Cells were cultured at the room temperature in the laboratory with mineral water (Nongfu Spring), enriched with rice. Although we failed to establish a clonal culture, no other oxytrichid morphotypes were present in the protargol preparations. Therefore, we are confident that the morphological, morphogenetic, and molecular studies reported here deal solely with the same species.
Cells were studied in vivo using a high-power oil immersion objective and differential interference contrast. Protargol (Wilbert, 1975) was used to reveal the infraciliature. Measurements of silvered specimens were performed with the imaging software Frontiers in Microbiology | www.frontiersin.org cellSens Entry (Olympus). Drawings of live specimens are based on photographic records, and those of impregnated cells were made with a camera lucida. For clarity, parental cirri are shown only by outline, whereas new ones are shaded. Terminology follows Berger (1999) and Foissner (2016).

DNA Extraction, PCR Amplification, and Sequencing
The genomic DNA extraction, PCR amplification, and gene sequencing were carried out according to Wang et al. (2021b).

Phylogenetic Analyses
The SSU rDNA sequence of Quadristicha subtropica n. sp. was aligned with sequences of 70 other hypotrich species downloaded from GenBank database for phylogenetic analyses. Euplotid species were used as the outgroup taxa. Phylogenetic analyses were carried out according to Wang et al. (2021b).

Type Material
The protargol slide (registry no. GQ2018091201A) with the holotype specimen ( Figures 1E,F, 2F) and four paratype slides (registry no. GQ2018091201B-E) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China. A paratype slide (registry no. GQ2018091201F) with protargol-stained specimens is deposited in the Laboratory of Protozoology, Ocean University of China.

Etymology
The Latin adjective subtropicus, −a, −um (masc., fem., neut.) recalls the fact that the type material was found in a subtropical area of China.
Eighteen frontal-ventral-transverse cirri: three slightly enlarged frontal cirri (FC) near distal portion of adoral zone of membranelles (AZM), cilia about 19 μm long in vivo; buccal cirrus (BC) near anterior end of paroral; four frontoventral cirri, cirrus III/2 slightly ahead of level of cirrus VI/3, cirrus IV/3 at about level of buccal vertex; three postoral ventral cirri located behind buccal vertex, with cirrus IV/2 arranged anterior to level of cirrus V/4, distance between cirri V/3 and V/4 slightly shorter than that between cirri V/3 and V/2; two pretransverse ventral cirri, cirrus VI/2 located between the levels of cirri II/1 and III/1, distance between cirri V/2 and VI/2 slightly shorter than that between cirri V/2 and V/3; five transverse cirri (TC) located about three-quarters down length of body, bases distinctly enlarged, cilia about 23 μm long in vivo and slightly protruding beyond posterior cell margin (Figures 1D,E, 2F,H,I). Marginal cirri are disposed in two rows, on the right and left of the cell, respectively, composed of 11-16 and 12-19 cirri, respectively, in life about 16 μm long; left marginal row commences at level of buccal vertex and terminates subcaudally, while right marginal row (RMR) commences slightly below level of buccal vertex (Figures 1E, 2F).

Stomatogenesis
Opisthe: The earliest cortical morphogenetic event is the apokinetal appearance of a small patch of basal bodies (kinetosomes) in irregular arrangement, the oral primordium (OP; Figures 3A-I, 4A-D, 5A-M). Subsequently, a long and narrow oral primordium is formed (Figures 3B,C, 5B,C). The membranelles of the opisthe's adoral zone organize in a posterior direction. Simultaneously, the anlage for the undulating membranes (UM-anlage) is formed to the right of the oral primordium as a streak of basal bodies (Figures 3D,E, 5D,E). Later, the membranelles of the opisthe's adoral zone are organized completed and the anterior end of the newly built adoral zone bends to the right, forming the new oral structure. It is suggested that the leftmost frontal cirrus is generated from the anterior end of the undulating membrane-anlage (= anlage I). Subsequently, the undulating membrane-anlage of both the proter and the opisthe is separated from which the endoral and paroral are formed (Figures 3H, 4A,C, 5H).
Proter: The parental AZM is retained by the proter, so changes to the oral structure are confined to the paroral membranes and endoral membranes. The UM-anlage is formed Frontiers in Microbiology | www.frontiersin.org by the dedifferentiation of the parental undulating membranes.
In subsequent stages, the basic development of the UM-anlage follows a similar pattern to that in the opisthe (Figures 3F,H, 4A,C, 5F,H,J,M).

Development of Cortical Ciliature
Along with the organization of the membranelles of the opisthe's adoral zone, division continues with the formation of the development of the frontoventral-transverse cirral anlagen (FVT-anlagen). We failed to obtain specimens in the stage between those as shown in Figures 3E,F and hence were unable to determine the origin of anlagen II to VI. We speculate that FVT-anlagen I and II in the opisthe develop de novo, and cirri IV/3, IV/2, and V/4 contribute the formation of the FVT-anlagen. Five thread-like anlagen are formed in both proter and opisthe (Figures 3F, 5F). Subsequently, cortical morphogenesis proceeds with the cirral segregation from these streaks. After migration and differentiation, 17 cirri will be formed from each group, whereas the remaining one (the leftmost frontal cirrus) may derive from the undulating membraneanlage (Figures 3H, 4A,C, 5H,J,M). Finally, the constant 18 cirri are formed within the anlagen I-VI as follows: 1, 3, 3, 3, 4, and 4 cirri. The anteriormost marginal cirri and some cirri near the prospective division furrow of the marginal rows disaggregate to form the marginal anlagen [left marginal anlagen (LMA) and right marginal anlagen (RMA); Figures 3F, 5F]. The new marginal cirri then develop and replace the old ones (Figures 3H,  4A,C, 5H,J,M).
New dorsal kineties are formed in a typical Urosomoida pattern. Firstly, within dorsal kineties 1, 2, and 3, basal bodies are proliferated to form dorsal kineties anlagen (DKA) at two sites above and below the prospective division furrow (Figures 3G,I, 4B,D, 5G). In the later stage, a gap is always present in posterior portion of kinety 1 (Figure 4D). Subsequently, above the anteriormost portion of the proter's and opisthe's Frontiers in Microbiology | www.frontiersin.org right marginal primordia a short streak of paired basal bodies develops, viz. the anlage for the shortened dorsal kinety 4 (Figures 3H, 5H). The posterior ends of the new dorsal kineties 1, 2, and 3 commence with the differentiation of caudal cirri (Figures 3I, 4B,D, 5L).

Division of Nuclear Apparatus
The nuclear apparatus divides in the usual way and hence requires no further comment (Figures 3G,I, 4B,D, 5I,K).

Phylogenetic Analyses Based on SSU rDNA Gene Sequences
The SSU rDNA sequence of Quadristicha subtropica n. sp. was deposited in GenBank with the accession number MZ338339. The length and GC content of the new sequence are 1,627 bp and 45.09%, respectively.
Phylogenetic trees using two different methods (ML and BI) had almost identical topologies; therefore, only the ML tree is presented with support values from both algorithms at the nodes (Figure 6). Quadristicha subtropica n. sp. nests within a poorly supported clade (ML/BI, 15/0.50) that also contains Heterogastrostyla salina Lu et al., 2020, Heterourosomoida lanceolata (Berger, 1999 Singh and Kamra, 2014, Heterourosomoida sinica Wang et al., 2020, Kleinstyla dorsicirrata Singh and Kamra, 2014, and Oxytricha lithofera Foissner, 2016. Quadristicha setigera, the only congener of Q. subtropica n. sp., clusters with Monomicrocaryon euglenivorum euglenivorum ) Foissner, 2016 with low support (ML/BI, 32/0.71). The two clades containing species of Quadristicha are sister groups. Given the low support values across the tree, the present phylogeny is far from robust despite the fact that several preliminary phylogenetic analyses were performed using different taxon samples and outgroup species.
Quadristicha subtropica n. sp. can be distinguished from M. halophilum by the locations of the rightmost frontal cirrus slightly (vs. distinctly) ahead of the level of the buccal cirrus, transverse cirri (subcaudal vs. caudal), cirrus IV/2 (ahead of vs. below) cirrus V/4 and the anterior termination of the RMR (below the level of cirrus VI/3 vs. ahead of the level of cirrus VI/4), and also the number of dikinetids in dorsal kinety 1 (8-12 vs. 18 or 21 in population from , data from drawings), Frontiers in Microbiology | www.frontiersin.org cilia of caudal cirri protruding rightward (vs. straight) and the habitat (fresh water vs. saline water) (Berger, 1999;Foissner, 2016).
Quadristicha subtropica n. sp. can be separated from M. sphagni by the length of the dorsal cilia (5 μm vs. about 15 μm), the ratio of body length to width in vivo (about 3:1 vs. about 5:1, data from drawing), the location of the transverse cirri (subcaudal vs. caudal), and the location of cirrus IV/3 (at about the level of the buccal vertex vs. ahead of the level of the buccal vertex) (Berger, 1999;Foissner, 2016).

Morphogenetic Comparison
Until now, the cortical morphogenesis of Q. subtropica n. sp. is the only detailed study within the genus Quadristicha, which proceeds in a similar way to other members of the family Oxytrichidea (Berger, 1999). Berger (1999) documented some middle and late stages of morphogenesis in Q. setigera. Based on these and the present data, the mid-to-late stages of the two congeners are consistent (Berger, 1999). The early stages of morphogenesis in Quadristicha are revealed here for the first time.

Phylogenetic Analyses
The genus Quadristicha comprises two species, namely, Q. setigera (type species) and Q. subtropica n. sp. both of which were included in the present phylogenetic analyses. Quadristicha subtropica n. sp. groups with H. salina, H. lanceolata, H. sinica, and K. dorsicirrata. The close relationship among these species is supported by several morphological features including: a flexible pellicle, two macronuclear nodules, one marginal cirral row on each side, two pretransverse cirri and five transverse cirri, and four dorsal kineties (Berger, 1999;Singh and Kamra, 2014;Foissner, 2016;Lu et al., 2020;Wang et al., 2021b).

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at https://www.ncbi.nlm. nih.gov/genbank/, MZ338339.

AUTHOR CONTRIBUTIONS
CS and QG collected the samples and carried out almost all of the experiments (preparations, illustrations, micrographs, etc.). AW was responsible for the language correction. JW did the identification of the species and revised the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Natural Science Foundation of China (project number: 32070428) and the Natural Science Foundation of Shaanxi province (2021JQ-307).