New Contributions to the Diversity of Hypotrichous Ciliates: Description of a New Genus and Two New Species (Protozoa, Ciliophora)

Ciliated protists (ciliates) are extremely diverse and play important ecological roles in almost all kinds of habitats. In this study, two new hypotrichs, Wilbertophrya sinica n. g. and n. sp. and Bakuella xianensis n. sp., from China are investigated. Wilbertophrya n. g. can be separated from related genera mainly by the combination of lacking a buccal cirrus, pretransverse cirri, and caudal cirri, while possessing frontoterminal cirri. Analyses based on morphological and molecular data confirm the validity of the species, W. sinica n. sp., which is characterized as follows: body 50–115 μm × 15–35 μm in vivo; midventral complex comprises four or five cirral pairs only and terminates above mid-body; three frontal, two frontoterminal cirri, and two to four transverse cirri; about 15 macronuclear nodules; colorless cortical granules sparsely distributed. Another new species, B. xianensis n. sp., was isolated from a freshwater wetland and is defined as follows: body 115–150 μm × 40–65 μm in vivo; about 70 macronuclear nodules; dark-brownish cortical granules in groups; midventral complex comprises 8–12 cirral pairs forming a row that terminates posteriorly in mid-body region and two or three short midventral rows that are continuous with the row of midventral pairs; three frontal, four to six frontoterminal, and three to five fine transverse cirri; three bipolar dorsal kineties. Phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequence data suggest that the new genus Wilbertophrya n. g. belongs to an isolated clade, which might represent an undescribed taxon at the family level, whereas B. xianensis n. sp. groups with several congeners and members of other related genera are within the core urostylids.

Here, we document two hypotrichs from inland China: a novel form that represents a new genus and a new species (Wilbertophrya sinica n. g., n. sp.) found in a puddle in a forest in Tibet and a freshwater species, Bakuella xianensis n. sp., isolated from a wetland in the Qinling Mountains area near Xi'an. Both species were characterized based on morphological observations of specimens in vivo and following silver staining. In addition, their molecular phylogeny was analyzed based on small subunit ribosomal DNA (SSU rDNA) sequence data.

Sampling, Cultivation, and Morphological Studies
Wilbertophrya sinica n. g., n. sp. was collected on April 22, 2017, from a small puddle in a forest in Bomi County, Linzhi (29 • 39 N; 94 • 21 E), Tibet, where the altitude is about 3,000 m above sea level (Figures 1A,C).
Bakuella xianensis n. sp. was collected on May 25, 2019, from a well-vegetated freshwater pond located in Chanba National Wetland Park,Xi'an (34 • 31 N;109 • 01 E), near the junction of the River Ba and River Wei (Figures 1A,B).
Uni-protistan cultures were established in Petri dishes containing rice grains to support the growth of bacteria, which served as a food source for the ciliates. The species was accurately identified based on its morphology in vivo. Moreover, no other urostylids morphotypes were present in the protargol preparation. We assure the accuracy of that identification for molecular studies, even though we were unable to establish clonal cultures.
Ciliate cells were observed in vivo using bright field and differential interference contrast microscopy. The protargol silver staining method according to Wilbert (1975) was used to reveal the infraciliature. Counts and measurements of stained specimens were performed at a magnification of ×1,000. Drawings of stained specimens were performed at ×1,250 magnification with the aid of a camera lucida. Terminology is mainly according to Berger (2006), and the systematic classification follows Lynn (2008).

DNA Extraction, PCR Amplification, and Sequencing
Single cells of each species 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. Genomic DNA extraction was performed with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the modified manufacturer's protocol (1/4 of suggested volume for each solution) (Chen et al., 2019;Lu et al., 2020). PCR amplification of the SSU rDNA was performed using Q5 Hot Start high fidelity DNA polymerase (NEB, Ipswich, MA, United States) to minimize the possibility of amplification errors. One cycle of initial denaturation at 98 • C for 30 s, followed by 18 cycles of amplification (98 • C, 10 s; 69-51 • C touchdown, 30 s; 72 • C, 1 min), and another 18 cycles (98 • C, 10 s; 51 • C, 30 s; 72 • C, 1 min), with a final extension of 72 • C for 5 min. Sequencing of PCR products was performed bidirectionally on an ABI 3700 sequencer using two PCR primers 18S-F (5 -AAC CTG GTT GAT CCT GCC AGT-3 ) and 18S-R (5 -TGA TCC TTC TGC AGG TTC ACC TAC-3 ) (Medlin et al., 1988).

Phylogenetic Analyses and Sequence Comparison With Related Species
In order to perform phylogenetic analyses, the SSU rDNA sequences of W. sinica n. g., n. sp. and B. xianensis n. sp. were aligned with other related sequences downloaded from the National Center for Biotechnology Information (NCBI) Database (accession numbers shown in Figure 2) on the GUIDANCE2 server 1 with default parameters (Sela et al., 2015). Representative species of the subclass Euplotia Jankowski, 1979, were selected as the outgroup. The aligned sequences were manually edited using the program BioEdit 7.2.5 (Hall, 1999), resulting in a final alignment with 1,772 sites. Both maximum likelihood (ML) and Bayesian inference (BI) analyses were performed on the final alignment. The ML analysis was performed with 1,000 rapid bootstrap replicates using RAxML-HPC2 on XSEDE v8.2.12 (Stamatakis, 2014) on CIPRES Science Gateway with the GTRGAMMA model (Miller et al., 2010). The BI analysis was computed by MrBayes on XSEDE 3.2.6 (Ronquist et al., 2012) on CIPRES Science Gateway with the GTR + I + G model selected by MrModeltest 2.2 (Nylander, 2004). Markov chain Monte Carlo (MCMC) simulations were run for 10,000,000 generations with sampling every 100 generations. The first 10,000 trees were discarded as burn-in. Seaview v.4.3.3 (Gouy et al., 2010) and MEGA v.6 (Tamura et al., 2011) were used to visualize the tree topologies.

ZooBank registration.
Wilbertophrya n. g.: urn:lsid:zoobank.org:act:61BC94E 2-17BA-48D8-9219-921267C0919B Diagnosis: Urostylid with a continuous adoral zone; three clearly differentiated frontal cirri; midventral complex composed only of cirral pairs arranged in a zigzag pattern; frontoterminal and transverse cirri present; buccal, pretransverse, and caudal cirri absent; one marginal row on each side, the anterior end of the left marginal row not curved rightward; three dorsal kineties. four or five midventral pairs in anterior half of cell; three frontal, two frontoterminal, and two to four transverse cirri; the left marginal row is composed of 13-21 cirri, and right marginal row is composed of 14-25 cirri; three bipolar dorsal kineties; freshwater habitat.
Type material: One protargol-stained slide (No. MJY2017042201-3) with the holotype specimen ( Figure 3I) circled in ink and two paratype slides (No. MJY2017042201-1, 2) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China.
Etymology: The species-group name sinica recalls that this species was first discovered in China.
Description (Figures 3A-N and Table 1). Body size 50-115 µm × 15-35 µm in vivo. Cell outline variable, generally elliptical to elongate-elliptical, right cell margin slightly concave, left margin slightly convex; usually widest in front of mid-body; dorsoventrally flattened; slightly flexible but non-contractile (Figures 3A, B, G). Nuclear apparatus composed of about 15 ellipsoidal nodules and one to four, on average two, micronuclei up to 2 µm in diameter (Figures 3F, L). Contractile vacuole about 7 µm in diameter, located slightly ahead of mid-body near left body margin,  Table 1). In living cells, cilia of distal membranelles about 12 µm in length, and those of proximal membranelles are about 6 µm long. The distal end of the adoral zone extends only slightly onto right body margin forming a question mark shape as in other urostylids. Paroral longer than endoral, both generally straight and optically intersect at their upper region (Figures 3D, E). Three slightly enlarged frontal cirri about 8 µm in length, rightmost one located behind the distal end of the adoral zone ( Figure 3D, pink area, Figure 3E, double arrowheads). Two frontoterminal cirri behind the distal end of the adoral zone ( Figure 3E, arrowheads); buccal cirrus lacking (Figures 3D, E); two to four slightly enlarged transverse cirri, up to 12 µm long in vivo. Pretransverse cirri absent. Midventral complex composed of four or five pairs of cirri, terminating ahead of mid-body ( Figure 3E, arrows). Left and right marginal rows composed of 13-21 and 14-25 cirri, respectively, with the anterior end of the left marginal rows not curved rightward ( Figure 3E).
Description of Bakuella xianensis n. sp.  located ahead of mid-body; dark-brownish cortical granules arranged in groups, about 0.7 µm across; about 23-33 adoral membranelles; midventral complex composed of 8-12 cirral pairs, terminating at about mid-body; two or three short ventral rows continuous with midventral pairs; three frontal, four to six frontoterminal, and three to five fine transverse cirri; one right and one left marginal rows with 24-38 and 22-33 cirri, respectively; three bipolar dorsal kineties; freshwater habitat.
Type material: One protargol-stained slide (No. MJY2019052501-1) with the holotype specimen ( Figure 4B) circled in ink and two paratype slides (No. MJY2019052501-2, 3, 4) were deposited in the Laboratory of Protozoological   Figures 4B-D,K-M. The adoral zone is about 40 µm long, occupying about 1/3 of body length, and composed of about 28 membranelles on average ( Table 1). Endoral and paroral prominent in vivo, that is, long and curved, optically intersecting at the paroral's mid-region. Most somatic cirri are relatively fine with cilia about 10-15 µm long. Constantly three relatively stout frontal cirri (FC, Figure 4D), apically positioned. Invariably one buccal cirrus, about level of endoral's mid-region (Figure 4B, arrow). Four to six fine frontoterminal cirri (Figure 4B, arrowheads). Three to five transverse cirri form roughly V-shaped (Figures 4B,L), protruding beyond rear end of body. Cilia of which is 13-15 µm long. Of the 18 examined specimens, 12 had one pretransverse cirrus, while the remainder lacked the cirrus. Midventral complex terminates at about posterior 1/4 of body length, composed of about 8-12 cirral pairs forming a zigzag row that extends to about mid-region of the cell, and two, sometimes three, short ventral rows that are continuous with a row of midventral pairs (Figures 4B, D,  VR and arrows, inset). One right and one left marginal rows composed of 24-38 and 22-33 cirri, respectively (Table 1); the right marginal row commences slightly ahead of level of buccal cirrus, always with two pairs of basal bodies ahead of its anterior end (Figures 4C,M, arrows). Three bipolar dorsal kineties arranged in typical Gonostomum pattern (Figures 4C,M).

Phylogeny Based on Small Subunit Ribosomal DNA Sequence Data
The SSU rDNA sequences of Wilbertophrya sinica n. sp. and Bakuella xianensis n. sp. were deposited to GenBank with accession numbers, length, and guanine-cytosine (GC) content as follows: MT809485, 1,762 bp, 45.40% and MT819382, 1,770 bp, 44.18%, respectively. W. sinica and nine morphologically related isolates generated a nucleotide matrix with 204 unmatched sites (Figure 5A), while B. xianensis and five related taxa formed the table with 57 unmatched sites ( Figure 5B).
Phylogenetic trees inferred from the SSU rDNA sequences using ML and BI had similar topologies; therefore, only the ML tree is shown with nodal support from both methods (Figure 2). W. sinica n. sp. clustered with Anteholosticha monilata (KJ958488) with high support (87% ML, 1.00 BI), which together grouped with four sequences of Extraholosticha sylvatica (MG603613, MG603614, KJ958490, and MN160327) with full support (Figure 2). This group was sister to the clade composed of Eschaneustyla lugeri (KY874005) and Holostichides heterotypicus (KY231187). Consequently, a comparison was made among these seven and two other morphologically similar species of W. sinica n. sp., namely, Adumbratosticha tetracirrata (KJ958491) and Caudikeronopsis marina (KR612270) (Figure 5A). The sequence differences between W. sinica n. sp. and these nine "related" taxa are as follows: 15 bp for A. monilata, 16-33 bp for the four sequences of E. sylvatica, 63 bp for E. lugeri, 67 bp for H. heterotypicus, 84 bp for A. tetracirrata, and 126 bp for C. marina.

Systematic Position of Wilbertophrya n. g. and Comparison With Related Taxa
The possession of three clearly differentiated frontal cirri and a midventral complex composed of midventral pairs only places Wilbertophrya n. g. unequivocally in the family Holostichidae (sensu Berger, 2006). Based on the following combination of features, that is, single left and right marginal rows, a continuous adoral zone, presence of frontoterminal cirri, clearly differentiated frontal cirri, and midventral cirral pairs arranged in a zigzag pattern, five genera in the family Holostichidae should be compared with Wilbertophrya n. g., namely, Anteholosticha Berger, 2003;Arcuseries Huang et al., 2014;Afrothrix Foissner, 1999;Acuholosticha Li et al., 2017;andHolosticha Wrzesniowski, 1877 (Berger, 2003;Huang et al., 2014;Li et al., 2017). All five of these genera differ from Wilbertophrya n. g. in having a buccal cirrus and pretransverse cirri, whereas both these structures are lacking in the new genus (Berger, 2006;Huang et al., 2014;Li et al., 2017). Furthermore, Acuholosticha possesses caudal cirri, whereas these are absent in Wilbertophrya n. g. (Berger, 2006; Figure 6; Table 2). The validity of the new genus is also supported by the molecular data ( Figure 5A).
The systematic position of Wilbertophrya n. g. remains unclear considering that the family assignment though morphological information supports its possible assignment to the family Holostichidae. The SSU rDNA tree reveals that Wilbertophrya groups with seven representatives of four genera: Anteholosticha, Extraholosticha, Eschaneustyla, and Holostichides (Figure 2). It is most closely related to A. monilata (KJ958488), the identification of which needs to be verified since there is another sequence with the same name but which occupies a different position in the tree (Figure 2, double arrowheads). Previous molecular phylogenetic analyses suggest that the genus Anteholosticha is not monophyletic and that most of its nominal species belong to the Urostylidae + Pseudokeronopsidae + Pseudourostylidae complex, which is the sister group of the clade that includes Wilbertophrya (Figure 2; Shao et al., 2011;Li et al., 2017;Luo et al., 2018;Xu et al., 2020).
In the SSU rDNA tree (Figure 2), the new genus clusters with several representatives of the genus Extraholosticha followed by E. lugeri + H. heterotypicus. This large clade is separated from other sequences at a deep level, suggesting that it might represent an undefined group at about family level. In terms of its morphology, however, the genus Wilbertophrya should be assigned to the family Holostichidae (see above). But as revealed in previous molecular studies, the Holostichidae complex (e.g., Holostichidae + Urostylidae + Pseudokeronopsidae) is a so-called melting pot of taxa, the evolutionary relationships of which cannot be resolved using phylogenetic analyses of single-gene sequence data (Bernhard et al., 2001;Yi and Song, 2011;Zhao et al., 2015;Luo et al., 2018;Jung and Berger, 2019;Paiva, 2020;Wang et al., 2021b;   Xu et al., 2020). Similarly, it is difficult to place Wilbertophrya n. g. into any known family based on SSU rDNA sequence data. Since ontogenetic information is unavailable for the new genus, we suggest that Wilbertophrya n. g. should be regarded as incertae sedis within the order Urostylida pending the availability of further data. Wilbertophrya n. g. is a monotypic genus, and thus, the type species, W. sinica n. sp., can be separated from its most "similar" morphospecies, that is, members of the genera Adumbratosticha, Periholosticha, Afrothrix, and Acuholosticha (see Figure 6) by the same combination of features that define the genus, i.e., the absence of the buccal cirrus and pretransverse cirri, and the presence of sparsely distributed cortical granules and the conspicuously short midventral rows (Berger, 2003;Li et al., 2017;Shao et al., 2020). The validity of W. sinica n. sp. as a distinct species is also firmly supported by the molecular data, which demonstrates its considerable difference from other taxa ( Figure 5A).
Including B. xianensis n. sp., SSU rDNA sequence data are available for only six species of Bakuella. These are grouped into three neighboring clades in the SSU rDNA tree (Figure 2). B. xianensis n. sp. is placed within the core clade of the genus. It is noteworthy, however, that some Bakuella sequences group with non-bakuellid genera, e.g., nominal isolates of genera such as Urostyla, Diaxonella, and Anteholosticha, with high support. This is consistent with previous reports (Chen et al., 2010(Chen et al., , 2013 and indicates that none of the families Bakuellidae, Urostylidae, and Holostichidae is monophyletic; and the systematics of each requires further investigation.

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 in the article/ supplementary material.

AUTHOR CONTRIBUTIONS
JM carried out the live observation and protargol impregnation. TZ was responsible for DNA amplification and sequencing, and the molecular phylogenetic analyses. JM, TZ, and CS performed the manuscript draft. CS and WS performed the manuscript review and editing. All authors helped to revise the manuscript, and read and approved the final manuscript.