Taxonomy and Molecular Phylogeny of Two New Urostylid Ciliates (Protozoa: Ciliophora) From Chinese Wetlands and Establishment of a New Genus

Hypotrich ciliates with evolutionary novelties are continually being discovered, challenging the current taxonomic system and attracting increased attention. In the present work, two new urostylid ciliates, Heterobakuella bergeri gen. nov., sp. nov. and Anteholosticha perezuzae sp. nov., isolated from Chinese wetland samples, were identified based on morphology and 18S rRNA gene sequences. Heterobakuella gen. nov. is defined by three frontal cirri, single buccal cirrus, one parabuccal cirrus, midventral complex composed of cirral pairs and one cirral row, one left and two right marginal cirral rows, transverse and pretransverse cirri present, caudal and frontoterminal cirri absent. Heterobakuella can be easily distinguished from the morphologically most similar genus, Apobakuella, mainly by the single buccal cirrus (vs. one buccal cirral row) and one parabuccal cirrus (vs. several parabuccal cirral rows originated from different anlagen). Phylogenetic analyses show that H. bergeri branches within the clade formed by Bergeriella ovata, Monocoronella carnea, Anteholosticha gracilis, and Neourostylopsis spp., rather than the clade represented by Apobakuella. The other species, A. perezuzae, is mainly characterized by a distinctly slender body shape with an average length:width ratio about 7, distinctively shaped biconcave and greenish cortical granules, as well as one or two pretransverse cirri. Phylogenetic analyses indicate the genus Anteholosticha is non-monophyletic.

More studies, based on morphologic, molecular, and ecologic data, are thus urgently needed to tackle these problems. In this work, two novel wetland urostylid ciliates, Heterobakuella bergeri gen. nov., sp. nov. and Anteholosticha perezuzae sp. nov., are reported and their phylogenetic positions are discussed.

Sample Collection
Heterobakuella bergeri gen. nov., sp. nov. was collected on November 12, 2019 from a small brook that flows into the Weishan Lake Wetland, administrated by the city of Jining, Shandong Province, China (34 • 46 14 N, 117 • 12 56 E) ( Figure 1A), when the water temperature was 15 • C. Anteholosticha perezuzae sp. nov. was isolated from the sample collected from a brackish water stream that originates from a small lake wetland near Tangdao Bay, Qingdao, China (35 • 56 18 N, 120 • 12 44 E) ( Figure 1B) on March 27, 2017, where the water temperature was 15 • C and salinity was 8 . In each case, sampling water (up to 20 cm deep) was first stirred, then about 300 ml of water including sediments, mud, and rotten plants was collected and transferred to our laboratory. Next, the sample was divided into several aliquots that were used to establish raw cultures in Petri dishes (diameter = 9 cm). All raw-culture samples were maintained at room temperature (about 23 • C). Because the two species we studied could be easily distinguished from other species present in the same Petri dish by body shape and color, the confusion with other species was avoided.

Morphological Observation
Live specimens were observed under the microscope (Olympus BX53; Olympus Corporation, Tokyo, Japan) with magnifications ranging from 100× to 1000×. Protargol impregnation was performed to reveal ciliary patterns and nuclear apparatus (Wilbert, 1975). Drawings were based on free-hand sketches with the help of drawing devices. Classification and terminology mainly follow Berger (2006).

DNA Extraction, PCR, and Sequencing
Five cells of each species were isolated from Petri dishes and washed several times using filtered and sterilized site water. Using micropipettes, cells were transferred into three 1.5-ml eppendorf tubes, two tubes with one cell each and the third with three cells. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, United States) according to the manufacturer's protocol with 25% of the suggested reagent volumes as described by Lu et al. (2020). The 18S rRNA genes of Heterobakuella bergeri gen. nov., sp. nov. and Anteholosticha perezuzae sp. nov. were amplified with the same forward primer 82F (5 -GAAACTGCGAATGGCTC-3 ) (Jerome et al., 1996) but different reverse primers, 5.8SR (5 -TACTGATATGCTTAAGTTCAGCGG-3 ) (Wang J. et al., 2017) for H. bergeri gen. nov., sp. nov. and 18SR (5 -TGATCCTTCTGCAGGTTCACCTAC-3 ) (Medlin et al., 1988) for A. perezuzae sp. nov. To minimize amplification errors during PCR, we used Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs, United States) as recommended by . The thermocycler program was set according to Bai et al. (2020). The PCR products were sequenced bidirectionally in the Tsingke Biological Technology Company (Qingdao, China) using the PCR primers and three internal primers, i.e., Pro + B (5 -GGTTAAAAAGCTCGTAGT-3 ), 900F (5 -CGATCAGATACCGTCCTAGT-3 ), and 900R (5 -ACTAGGACGGTATCTGATCG-3 ) for both species (Wang J. et al., 2017).

Phylogenetic Analyses
To infer the phylogenetic position of the two newly obtained species, we downloaded 18S rRNA gene sequences of 77 other ciliates from the National Center for Biotechnology Information (NCBI) database 1 , including 62 "core urostylids, " seven oxytrichids, three Arcuseries species, as well as the outgroup taxa composed of two Uncinata and three Holosticha species. GenBank accession numbers are shown in Figure 6. After combining all of the sequences, the alignment was carried out with the MUSCLE algorithm on the webserver GUIDANCE 2 2 with default parameters (Sela et al., 2015). The primer sequences were manually trimmed using BioEdit v.7.0 (Hall, 1999). The maximum likelihood (ML) analysis was done using RAxML-HPC2 (XSEDE v.8.2.12) on the CIPRES Science Gateway server 3 with 1,000 bootstrap replicates and the GTRGAMMA model of nucleotide substitution (Stamatakis, 2014). Bayesian inference (BI) analysis was carried out using MrBayes (Ronquist et al., 2012) on CIPRES Science Gateway (XSEDE v.3.2.6), with the GTR + I + G nucleotide substitution model selected under Akaike Information Criterion (AIC) by jModelTest 2 (Darriba et al., 2012). Four Markov chain Monte Carlo (MCMC) simulations were run for 1,000,000 generations with the first 2,500 sampled trees discarded as burn-in. Convergence was assessed using RWTY . SeaView v.4.6.1 (Gouy et al., 2010) and MEGA 6.0 (Tamura et al., 2013) were used to visualize the tree topologies.
For further comparison of the 18S rRNA gene sequences among Heterobakuella bergeri gen. nov., sp. nov. and other seven phylogenetically related species, the program BioEdit v.7.0 (Hall, 1999) was used to calculate the number of unmatched nucleotides and the pairwise identities, using "sequence difference count matrix" and "sequence identity matrix" options, respectively. After manually removing identical nucleotides, a nucleotide matrix with 84 unmatched sites is shown in Figure 7.

Subclass Hypotrichia Stein, 1859
Order Urostylida Jankowski, 1979 Genus Heterobakuella gen. nov. Diagnosis. Urostylids with three clearly differentiated frontal cirri, one buccal and one parabuccal cirrus. Midventral complex composed of cirral pairs and one cirral row. One left and more than one right marginal cirral rows. Transverse and pretransverse cirri present. Caudal and frontoterminal cirri lacking.
Etymology. Composite of the Greek prefix hetero + (different) and the generic name Bakuella. This alludes to the fact that the new genus is different from the "core bakuellid genus" Bakuella.
Type species. Heterobakuella bergeri sp. nov. Species assignable. The type species only. Heterobakuella bergeri gen. nov., sp. nov. Diagnosis. Size usually 90-130 × 30-45 µm in vivo. Body outline long-ellipsoidal and usually with the anterior part wider than the posterior one. Cortical granules yellow-brown, arranged along cirral rows and dorsal kineties. Adoral zone composed of about 25 membranelles. Seven to eleven transverse cirri, two pretransverse cirri, seven to twelve midventral cirral pairs, and a single cirral row composed of four to eleven cirri. One left and two right marginal cirral rows. Three complete dorsal kineties. About 29 macronuclear nodules. Freshwater habitat.
Dedication. We dedicate this new species to our eminent colleague, Prof. Helmut Berger (Consulting Engineering Office for Ecology, Salzburg, Austria) in recognition of his contributions to ciliatology.

Taxonomy and Morphological
Description of Anteholosticha perezuzae sp. nov.

Phylogenetic Analyses Based on 18S rRNA Gene Sequences
The GenBank accession number, length, and G + C content of the 18S rRNA gene sequences of the two species are as follows: Heterobakuella bergeri gen. nov., sp. nov., MW692986, 1,595 bp, 45.39%; Anteholosticha perezuzae sp. nov., MW692987, 1,618 bp, 48.75%. Topologies of the maximum likelihood (ML) and Bayesian inference (BI) trees are nearly congruent; thus, only the ML tree is shown with support values from both algorithms (Figure 6). In Figure 6, Heterobakuella bergeri gen. nov., sp. nov. and Anteholosticha perezuzae sp. nov. are included in the "core urostylids" with strong statistical support.
In the 18S rRNA gene tree, Heterobakuella bergeri gen. nov., sp. nov. falls within a group including Bergeriella ovata, Monocoronella carnea, Anteholosticha gracilis, and Neourostylopsis spp. with full support. However, the position of H. bergeri gen. nov., sp. nov. within this well-supported clade is not robust, as without support in the ML tree (44%) and having an incongruent topology in the BI tree. In the ML tree, H. bergeri gen. nov., sp. nov. is sister to a cluster comprising B. ovata, M. carnea, and A. gracilis without support (44%). This poorly resolved cluster is then sister to the strongly supported clade (97%) including Neourostylopsis species with full support. While in the BI analysis, H. bergeri gen. nov., sp. nov. is placed as a polytomy with B. ovata, M. carnea, and a clade comprising A. gracilis, Neourostylopsis spp., respectively (insert in Figure 6).
Anteholosticha perezuzae sp. nov. does not cluster with any congeners in ML and BI analysis, and clusters with Apourostylopsis sinica (Shao et al., 2008) Chen et al., 2013 (EU220227) without support in ML analysis (46%) and with strong support in BI analysis (0.98). This sister cluster groups with a clade comprising two Metaurostylopsis species (M. struederkypkeae and M. antarctica) with weak support in ML analysis (55%) and high support in BI analysis (0.98). Then, the forming sister group among them clusters together with another clade comprising Thigmokeronopsis spp. and two populations of Apokeronopsis ovalis. Other Anteholosticha species are scattered throughout the tree as shown in Figure 6, again indicating the non-monophyly of Anteholosticha.

DISCUSSION
Comparison of Heterobakuella bergeri gen. nov., sp. nov. With Similar Taxa and Basis for the Erection of the Genus Seven urostylid genera with a continuous adoral zone, three frontal cirri, and midventral complex composed of cirral pairs and row(s) should be compared with Heterobakuella gen. nov., namely, Apobakuella, Bakuella, Holostichides, Metaurostylopsis, Monourostylopsis Song et al., 2020, Neobakuella, andParagastrostyla (Berger, 2006;Li et al., 2011;Jiang et al., 2013;Song et al., 2020;Figure 8). Of these, Apobakuella is most similar to Heterobakuella gen. nov. in terms of the cirral pattern. Hitherto, Apobakuella, with the type species A. fusca, is monotypic (Jiang et al., 2013). Morphologically, Heterobakuella bergeri gen. nov., sp. nov. can be distinguished from A. fusca by the smaller body size (90-130 × 35-45 µm vs. 150-210 × 50-60 µm), cortical granules (one type vs. two types), buccal cirri (one vs. three to nine), parabuccal cirri (one vs. five to nine arranged in two or three rows), fewer midventral cirral rows with unpaired cirri in posterior portion(s) (one vs. four to nine), and habitat (freshwater vs. brackish). In addition, Heterobakuella gen. nov. can be easily separated from Holostichides and Paragastrostyla by transverse cirri (present vs. absent in Holostichides and Paragastrostyla) (Berger, 2006;Zhu et al., 2019), and from the other four genera by marginal cirral rows (a single left and two right rows vs. a single marginal cirral row on each side in Bakuella; two or more marginal cirral rows on each side in Metaurostylopsis; more than one left and single right marginal cirral rows in Monourostylopsis and Neobakuella), frontoterminal cirri (absent vs. present in Bakuella, Metaurostylopsis, Monourostylopsis, and Neobakuella) (Li et al., 2011;Jo et al., 2015;Lu et al., 2016;Moon et al., 2020;Song et al., 2020;Zhang et al., 2020b). Generic classification of hypotrichs is traditionally based on characteristics of the ciliature and nuclear apparatus in morphostatic and dividing cells (for reviews, see Berger, 1999Berger, , 2006Berger, , 2008Berger, , 2011. Our new species distinctly differs from the morphologically most similar genus, Apobakuella, as mentioned previously. In addition, the phylogenetic trees based on 18S rRNA gene sequences show our new species is distant from A. fusca, the sequences of these two species differing in 68 nucleotide positions. Thus, because our new species cannot be assigned to any existing genus, we erect the new genus, Heterobakuella.
Familial Classification of Heterobakuella gen. nov.
Although Lynn (2008) merged all genera within Bakuellidae and most genera of two other families (e.g., Holostichidae and Urostylidae) from Berger's (2006) classification into a single large family, Urostylidae, we prefer Berger's (2006) classification since all genera assigned to Urostylidae sensu Lynn (2008) are paraphyletic and require further study and possibly further subdivision. The monophyly of Bakuellidae has recently been challenged in a number of reports (Lynn, 2008;Yi et al., 2008a,b;Chen et al., 2011;Dai and Xu, 2011;Foissner, 2016;Lyu et al., 2018), and additional data are needed to better define the family. Heterobakuella bergeri gen. nov., sp. nov. has morphological characteristics typical for Bakuellidae, including three frontal cirri and a midventral complex composed of pairs and row(s) of unpaired cirri (Berger, 2006). However, H. bergeri gen. nov., sp. nov. is distant from the well-known bakuellids including Apobakuella, Bakuella, Diaxonella, and Neobakuella in the 18S rRNA gene tree. Morphogenetic features of H. bergeri gen. nov., sp. nov. are, as yet, unknown. For these reasons, a confident familial assignment of Heterobakuella cannot be made at the current state of knowledge and we consider the genus as incertae sedis in Urostyloidea.
Anteholosticha perezuzae sp. nov. can be distinguished from A. grisea (Kahl, 1932) Berger, 2003, because the new species possesses conspicuous greenish cortical granules (about 1.0-1.5 µm in diameter) along the marginal cirral rows, while cortical granules were not mentioned in any population of A. grisea (Berger, 2006, page 332). As noted by Berger (2006), Kahl was a very good observer, thus we prefer to accept cortical granules are lacking. Anteholosticha grisea invariably displays a blackish body color due to food vacuoles containing ingested rhodobacteria (Berger, 2006, page 332), while A. perezuzae sp. nov. shows a brownish body at low magnifications. In addition, A. grisea is more likely confined to the freshwater sapropel, while A. perezuzae sp. nov. is from a brackish stream.
According to Berger (2006), the taxonomy of Anteholosticha violacea is rather complicated. At least six populations were reported and the possibility that they belong to different species could not be excluded, thus Berger simply accepted Kahl's data (Kahl, 1928(Kahl, , 1932Berger, 2006). Anteholosticha perezuzae sp. nov. differs distinctly from Kahl's (1928) population by frontal cirri (three enlarged frontal cirri vs. six to seven) and from Kahl's (1932) second population by dorsal bristles (3-4 µm long vs. at least 8 µm long).

Phylogenetic Analyses of the Genus Heterobakuella and Anteholosticha
Considering the position and the specialized pattern of ventral cirri, viz., three frontal cirri and a midventral complex composed of cirral pairs and row(s), Heterobakuella might be closely related to the bakuellid taxa, especially to the morphologically similar genus Apobakuella. However, a close phylogenetic relationship between Heterobakuella and bakuellid taxa is not detected in the very conservative 18S rRNA gene tree, as these taxa do not form a monophyletic assemblage, and Heterobakuella branches off earlier than bakuellid taxa. Thus, their similar ventral cirral pattern likely represents a convergently evolved character, viz., the ventral ciliary pattern is analogous and not homologous. On the other hand, Heterobakuella groups together with Bergeriella, Monocoronella, Anteholosticha gracilis, and Neourostylopsis with the full support in 18S rRNA gene tree (Figure 6). Within this clade, Bergeriella, Heterobakuella, and Monocoronella are three monotypic genera with evolutionary novelties, and type species of which are B. ovata, H. bergeri, and M. carnea, respectively. Morphologically, H. bergeri can be easily distinguished from B. ovata by frontal cirri (three vs. 6-13) and postoral ventral rows (absent vs. present) (Liu et al., 2010), from M. carnea by frontal cirri arranged in monocorona (absent vs. present) (Chen et al., 2011), and from A. gracilis and Neourostylopsis spp. by midventral complex composed anteriorly of pairs and posteriorly of unpaired cirri (present vs. absent) (Berger, 2006;Chen et al., 2013). Thus, to understand their evolutionary relationship, more discoveries of further congeners and more genes must be awaited.
Because the genus Anteholosticha was established based only on a combination of plesiomorphies in the absence of obvious synapomorphies, Berger (2003Berger ( , 2006 had hypothesized the nonmonophyly of Anteholosticha. Subsequently, the non-monophyly of Anteholosticha has been reported in many previous studies (Park et al., 2013;Zhao et al., 2015;Fan et al., 2016;Chen et al., 2018Chen et al., , 2020Paiva, 2020;Jung et al., 2021). Our analyses also confirm that Anteholosticha species are scattered among different branches inside the core urostylid assemblage. Morphological and molecular data for the newly discovered species A. perezuzae sp. nov. is highly incongruent. Anteholosticha perezuzae clusters with Apourostylopsis sinica (EU220227) without support in ML analysis and with strong support in BI analysis (0.98). However, morphologically, A. perezuzae sp. nov. can be easily separated from A. sinica by body size (140-260 × 25-40 µm vs. 100-120 × 30 um in vivo), one type of cortical granules (vs. both two), number of midventral cirral pairs (17-31 vs. 11-17), number of left marginal cirral rows (one vs. three), and number of left marginal cirral rows (one vs. two) (Shao et al., 2008;Chen et al., 2013). Likewise, if the "good" apomorphies for inferring their evolutionary relationship cannot be found, this polyphyly might be better resolved with future analysis of additional taxa. Regarding this problem of taxonomic and phylogenetic resolution, a new classification system proposed by Paiva (2020) might give us some inspirations. According to Paiva (2020), the newly established taxon Kentrurostylida corresponds to the "core urostylids". Within the taxon, two secondary taxa Simplicitergida and Hispidotergida were established, based on three dorsal bristle rows and numerous dorsal bristles, respectively. Accordingly, A. antecirrata, A. gracilis, A. manca, A. multicirriata, and A. paramanca were assigned to Simplicitergida, while the other species, A. cf. azerbaijanica, A. foissneri, A. marimonilata, A. monilata, A. pseudomonilata, A. pulchra, A. songi, A. randani, and A. rectangula were placed in Hispidotergida. Under Paiva's (2020) classification, Anteholosticha perezuzae would be assigned to Simplicitergida. From the current work, it is obvious that the taxonomy and phylogeny of both bakuellid-like taxa and those species currently included in Anteholosticha remains in a state of flux.

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
YW designed and supervised the research study. WS, TZ, and JD drafted the manuscript. XL and WB revised and improved the manuscript. WS and TZ collected samples and performed staining. All authors contributed to the manuscript and approved the submitted version.