Taxonomic and Phylogenetic Studies of Two Brackish Pleuronema Species (Protista, Ciliophora, Scuticociliatia) from Subtropical Coastal Waters of China, with Report of a New Species

The genus Pleuronema Dujardin, 1841, with nearly 40 morphospecies, is one of the largest genera in the well-known subclass Scuticociliatia. In the present study, two Pleuronema species were collected from subtropical coastal waters of the East China Sea. The morphology and molecular phylogeny were investigated using modern standard methods. Pleuronema ningboensis n. sp. is mainly characterized by an elliptical body in outline with the right ventrolateral side straight, 16–22 somatic kineties, 3–5 preoral kineties, and the posterior end of the membranelle 2a hook-like. An improved diagnosis of Pleuronema orientale Pan et al., 2015 was provided: body size in vivo usually 90–135 × 45–85 μm, right ventrolateral side convex, 36–51 somatic kineties, 1–5 preoral kineties, one to three spherical macronuclei, membranelle 2a arranged in a zig-zag pattern in middle portion, posterior region hook-like, both membranelle 1 and membranelle 3 composed of three rows of basal bodies. The small subunit ribosomal DNA (SSU rDNA) of two species is sequenced, and their molecular phylogeny is analyzed. The new species Pleuronema ningboensis n. sp. clusters with P. grolierei KF840519, P. setigerum JX310015, P. paucisaetosum KF206430, and P. cf. setigerum KF848875, basically in accord with the morphological characteristics.


Introduction
Ciliated protozoa (ciliates) are an extremely diverse and ubiquitous group with more than 10,000 nominal species from a wide range of habitats [1]. They are considered a major link in microbial food webs and play an important role in energy flow and material circulation in aquatic environments [2][3][4][5]. The subclass Scuticociliatia is a speciose assemblage of ciliates that are found in various aquatic and terrestrial habitats [6,7]. Some scuticociliates are well known for their facultative parasitism and cause serious diseases of aquatic organisms [8][9][10][11]. Actually, most ciliates assigned to this subclass are free-living with significant ecological functions regarding their nutrition style, behavior, and many other biological characteristics [12][13][14][15][16].
Pleuronematida is one of four orders within the subclass Scuticociliatia and most Pleuronematida species are free-living [1]. They are mainly characterized by an expansive oral region with a prominent, curtain-like paroral membrane [17]. As the representative genus of this order, Pleuronema is a speciose and cosmopolitan genus comprising nearly 40 known morphospecies which can be found in various aquatic environments and mainly distributed in the intertidal zone [18][19][20][21][22][23]. In the past decade, about 10 new species have been discovered, indicating a high species diversity of this genus, and a further study on the biodiversity of this genus remains to be explored [23][24][25][26].
In the present work, two Pleuronema species, Pleuronema ningboensis n. sp. and P. orientale  were collected from the subtropical coastal wetlands in China. They were investigated both in vivo and by using silver staining methods. In addition, phylogenetic analysis was performed based on the SSU rDNA sequences to reveal the evolutionary relationships of Pleuronematidae.

Sample Collection, Observation, and Identification
Samples of subtropical brackish waters were collected from coastal wetlands in Ningbo, China ( Figure 1). Pleuronema ningboensis n. sp. was collected on 13 November 2019 from an intertidal sand (29 • 46 33 N, 121 • 57 47 E). The water temperature was 19 • C, and the salinity was 19‰. Pleuronema orientale was collected on 7 May 2020 from a ditch on Meishan Island (29 • 77 63 N, 121 • 95 20 E). The water temperature was 24 • C, and the salinity was 4.5‰. For Pleuronema ningboensis n. sp., several holes (diameter and depth were about 10 cm) were excavated on the beach when at low tide. After water gradually seeped into the holes, water and sand at the bottom of the holes were mixed and collected. An approximately 400 mL water sample was placed into a 500 mL bottle. For P. orientale, approximately 400 mL of well-stirred brackish water sample with humus was placed into a 500 mL bottle. Samples were then maintained in the laboratory for about one week as raw cultures in Petri dishes, and individuals were directly isolated using micropipettes for live observation and protargol staining.
Living cells were observed under a bright field and differential interference contrast microscopy (Leica DM2500) at 100-1000× magnification. The ciliature and nuclear apparatus were revealed with the protargol staining method [27]. Counts, measurements, and drawings of stained specimens were performed at 1000× magnification. Terminology and systematics follow Lynn [1] and Gao et al. [28], respectively.

DNA Extraction, PCR Amplification, and Gene Sequencing
For each species, five to ten cells were selected from pure cultures and washed five times with filtered in situ water (0.22 µm, Millex-GP filter unit) to exclude contamination. Then cells were placed into three Eppendorf tubes with one and multiple individuals. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the optimized manufacturer's protocol. The SSU rDNA was amplified using the 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

Phylogenetic Analyses
Two new SSU rDNA sequences from the present work and 33 reference sequences obtained from the National Center for Biotechnology Information (NCBI) GenBank database were selected for the phylogenetic analyses. The accession number of each sequence was provided to the right of species names in the phylogenetic tree (also listed in Table  S1). Four Histiobalantiidae, namely Falcicyclidium plouneouri (FJ868181), Falcicyclidium fangi (FJ868185), Acucyclidium atractodes (FJ868182), Hippocomos salinus (JX310012), and one Eurystomatellidae, Wilbertia typica (FJ490551) were selected as out-group species (Table  S1). A total of 35 sequences were aligned using MUSCLE implemented in GUIDANCE with default parameters [30]. The ends of the resulting alignments were trimmed manually using the program BioEdit v7.0.5.3 [31]. Maximum likelihood (ML) analysis was conducted on the CIPRES Science Gateway server, using the RAxML-HPC2 located on XSEDE v8.2.9 [32], with the GTR + I + G model. Support for the best ML tree was calculated from 1000 bootstrap replicates. Bayesian inference (BI) analysis was performed on CIPRES Science Gateway with MrBayes on XSEDE v3.2.7a [33] using the GTR + I + G model selected by MrModeltest v2.2 [34]. The BI analysis was run for 10 6 generations, with trees sampled

Phylogenetic Analyses
Two new SSU rDNA sequences from the present work and 33 reference sequences obtained from the National Center for Biotechnology Information (NCBI) GenBank database were selected for the phylogenetic analyses. The accession number of each sequence was provided to the right of species names in the phylogenetic tree (also listed in Table S1). Four Histiobalantiidae, namely Falcicyclidium plouneouri (FJ868181), Falcicyclidium fangi (FJ868185), Acucyclidium atractodes (FJ868182), Hippocomos salinus (JX310012), and one Eurystomatellidae, Wilbertia typica (FJ490551) were selected as out-group species (Table S1). A total of 35 sequences were aligned using MUSCLE implemented in GUIDANCE with default parameters [30]. The ends of the resulting alignments were trimmed manually using the program BioEdit v7.0.5.3 [31]. Maximum likelihood (ML) analysis was conducted on the CIPRES Science Gateway server, using the RAxML-HPC2 located on XSEDE v8.2.9 [32], with the GTR + I + G model. Support for the best ML tree was calculated from 1000 bootstrap replicates. Bayesian inference (BI) analysis was performed on CIPRES Science Gateway with MrBayes on XSEDE v3.2.7a [33] using the GTR + I + G model selected by MrModeltest v2.2 [34]. The BI analysis was run for 10 6 generations, with trees sampled every 100th generation, with the first 2500 trees discarded as burn-in. The tree topologies were visualized via MEGA v7.0 [35] and TreeView v.1.6.6 [36].

Etymology
This species-group name, ningboensis, refers to the area (Ningbo, China) where the sample was collected.

Type Materials
Two slides with protargol-stained specimens have been deposited in the Laboratory of Protozoology, Ningbo University, including one slide (registration number: YTT-20191113-2-1) with the holotype specimen circled in black ink and one slide (registration number: YTT-20191113-2-2) with paratype specimens. Another two slides with protargol-stained paratype specimens (registration number: YTT-20191113-2-3, 2-4) have been deposited to the Laboratory of Protozoology, Ocean University of China.  Figure 3D). Cytoplasm transparent to greyish, containing several food vacuoles, refractile globules, and crystals usually concentrated in posterior of cell ( Figure 3E). Single contractile vacuole dorsally located about 80% down length of cell, approximately 12-15 µm in diameter when fully expanded, pulsating at interval of about 35-40 s ( Figure 3C). One spherical to ellipsoidal macronucleus positioned in body center slightly anteriorly ( Figure 3B), diameter of about 24 µm after protargol staining ( Figure 3J, Table 1). Somatic cilia densely packed, about 9 µm long. Ten to seventeen prolonged caudal cilia, each about 20 µm in length, projecting radially from posterior end of cell (Figures 2A and 3E). Movement moderately fast, rotating around main body axis and occasionally resting on substrate for some time.    Sixteen to twenty-two somatic kineties (SK) extending entire length of cell to form small, bald apical plate at anterior end of cell ( Figure 2D, Table 1). Each with densely spaced dikinetids in anterior 60% and monokinetids in posterior 40% (Figures 2C,D, and 3J). Three to five preoral kineties (PK) located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 60-70% down length of cell ( Figures 2C and 3F, Table 1).

SSU rDNA Sequence
The sequence of Pleuronema ningboensis n. sp. was deposited in GenBank with accession number OQ591738. The length and G + C content of the sequence are 1658 bp and 43.00%.

Pleuronema orientale Pan et al., 2015
This species was originally collected from Hangzhou Bay, China (30 • 35 33.8 N, 122 • 04 59.1 E) and detailed described by Pan et al. [24] based on living cells and protargol staining specimens. As a new contribution, here we describe a subtropical population and provide some new information.

Morphological Description of Ningbo Population
Living cell about 90-105 × 45-60 µm, elliptical or oval in outline. Anterior end slightly narrowed in some individuals, posterior end rounded, right ventrolateral and dorsal sides convex ( Figures 4A and 5A-C). Buccal field occupies 75% of body length, PM prominent ( Figures 4A and 5A,D). Pellicle rigid and slightly notched, beneath which extrusomes closely arranged, about 6 µm in length ( Figure 4A). Cytoplasm usually colorless and transparent, containing numerous food vacuoles, refractile globules, and crystals that are usually ventrally distributed ( Figures 4A and 5A,B). Single contractile vacuole dorsally located approximately 80% down length of body, about 10 µm across when fully expanded, pulsating at interval of about 20-40 s ( Figure 5B,C). One to three macronuclei (usually one spherical macronucleus, in 23 out of 25 cells examined), diameter approximately 32 µm after protargol staining (Figures 4C-E,G and 5F,K, Table 1). Micronucleus not detected. Somatic cilia densely packed, about 10 µm in length; 10-13 prolonged caudal cilia, each 20-25 µm in length ( Figures 4A and 5A). Locomotion moderately fast while rotating about main body axis, sometimes drifting or lying motionless on debris for short periods.
Thirty-six to fifty-one somatic kineties extending over entire length of cell to form small, bald apical plate at anterior end of body ( Figure 4G, Table 1). Kineties consist of dikinetids in anterior 75% of cell and monokinetids in posterior 25% ( Figures 4F,G and 5K). One to five preoral kineties located to left of buccal field, commencing near anterior end of cell and terminating posteriorly 75-80% down length of cell ( Figures 4F and 5E,H, Table 1).
Anterior 20% of M1 three-rowed while rest two-rowed ( Figures 4B and 5G). Anterior 20% and posterior 35% of M2a two-rowed, basal bodies in mid-portion arranged in zigzag pattern; posterior portion of M2a hook-like ( Figures 4B and 5J); M2b V-shaped, basal bodies arranged in several single-rowed groups, each group composed of about six basal bodies ( Figures 4B and 5J). M3 three-rowed and closely packed, posterior end of rightmost row slightly separated from other rows by diverging rightwards (Figures 4B and 5I). PM prominent, with basal bodies arranged in zig-zag pattern, occupying 75% of body length ( Figures 4A,B,F, and 5J).

SSU rDNA Sequence
The SSU rDNA sequence of the Ningbo population of Pleuronema orientale has been deposited in the GenBank database with the accession number, length, and G + C content as follows: OQ591739, 1606 bp, 43.71%.

SSU rDNA Sequences and Phylogenetic Analyses
Phylogenetic trees inferred from SSU rDNA sequences using BI and ML analyses generated similar topologies. Therefore, only the ML tree is presented here, with supporting values from both algorithms ( Figure 6).

SSU rDNA Sequence
The SSU rDNA sequence of the Ningbo population of Pleuronema orientale has been deposited in the GenBank database with the accession number, length, and G + C content as follows: OQ591739, 1606 bp, 43.71%.

SSU rDNA Sequences and Phylogenetic Analyses
Phylogenetic trees inferred from SSU rDNA sequences using BI and ML analyses generated similar topologies. Therefore, only the ML tree is presented here, with supporting values from both algorithms ( Figure 6). The scale bar corresponds to five substitutions per 100 nucleotide positions. All branches are drawn to scale. The systematic classification mainly follows Gao et al. [28]. Pleuronema coronatum (JX310014) (marked with red triangle) deviates from the other three P. coronatum sequences; unfortunately, available information is not sufficient to determine its identity. Pleuronema clades of 'coronatum-type' and 'marinum-type' groups are marked with yellow and blue background colors, respectively.  [28]. Pleuronema coronatum (JX310014) (marked with red triangle) deviates from the other three P. coronatum sequences; unfortunately, available information is not sufficient to determine its identity. Pleuronema clades of 'coronatum-type' and 'marinum-type' groups are marked with yellow and blue background colors, respectively. Two newly sequenced species fall in the genus Pleuronema. The Ningbo population of Pleuronema orientale (OQ591739) clusters with P. puytoraci (KF840520) and the Hangzhou Bay population of P. orientale (KF206429) with full support (ML/BI, 100/1.00). Pleuronema ningboensis n. sp. (OQ591738) groups with the clade of P. grolierei (KF840519), P. setigerum (JX310015), P. paucisaetosum (KF206430) and P. cf. setigerum (FJ848875) with high support (ML/BI, 89/1.00), then the clade of the five species above clusters with the P. coronatum clade, but the support value is quite low (ML/BI, 22/0.73).

Discussion
The genus Pleuronema was established by Dujardin in 1841. It is a speciose and cosmopolitan genus comprising nearly 40 nominal species found in various aquatic environments [23][24][25][26]37,38]. The living cell size, ciliature, and especially the structure of the oral apparatus are the most important criteria for species separation within the genus [4,20]. Based on the structure of M2a, Wang et al. suggested that members of Pleuronema could be divided into two groups: 'coronatum-type' with the posterior end of M2a hook-shaped, 'marinum-type' characterized by the straight posterior end of M2a [39]. This view was widely accepted by later researchers and largely supported by the phylogenetic tree (Figure 6) [26]. In the present study, in terms of the structure of oral apparatus, both species belong to the 'coronatum-type' group.

Comparison of Pleuronema ningboensis n. sp. with Related Congeners
In the previous studies, more than twenty species have hook-shaped M2a and belong to the group of 'coronatum-type'. Some of them can be clearly distinguished from Pleuronema ningboensis n. sp. according to the significant morphological differences, for exam-

Discussion
The genus Pleuronema was established by Dujardin in 1841. It is a speciose and cosmopolitan genus comprising nearly 40 nominal species found in various aquatic environments [23][24][25][26]37,38]. The living cell size, ciliature, and especially the structure of the oral apparatus are the most important criteria for species separation within the genus [4,20]. Based on the structure of M2a, Wang et al. suggested that members of Pleuronema could be divided into two groups: 'coronatum-type' with the posterior end of M2a hook-shaped, 'marinum-type' characterized by the straight posterior end of M2a [39]. This view was widely accepted by later researchers and largely supported by the phylogenetic tree ( Figure 6) [26]. In the present study, in terms of the structure of oral apparatus, both species belong to the 'coronatum-type' group.
Pleuronema ningboensis n. sp. closely resembles P. paucisaetosum in their small living cell size, similar ciliature pattern, and habitat. The former, however, can be distinguished from the latter by having different patterns of M3 (leftmost row shorter than other rows vs. three-rowed basically equal in length in P. paucisaetosum) and fewer somatic kineties (16-22. vs. 21-23 in P. paucisaetosum) [24]. The SSU rDNA sequence of P. ningboensis n. sp. is different from P. paucisaetosum (KF206430) by 19 bp with a sequence identity of 98.7% (Figure 7).
Pleuronema smalli was originally described by Dragesco [46] with the cell size after silver staining and ciliature pattern, but lack of morphological data of living cells. In terms of its small body size after silver staining and the number of preoral kineties, Pleuronema ningboensis n. sp. closely resembles the species P. smalli. The former, nevertheless, is different from the latter mainly by having fewer somatic kineties (16-22 vs. 28-36 in P. smalli) [26,46].

Comparison of Two Populations of Pleuronema orientale Pan et al., 2015
Pleuronema orientale was originally collected from a brackish water ditch of a marine island of Hangzhou Bay, China, and detailly described with morphology and molecular information [24]. The Ningbo population is consistent with the original description in most of the morphological features, such as the structure of the oral apparatus and the size of the living cell. There are some minor differences between the two populations, that is, the number of somatic kineties (36-51 in the present study vs. 42-50 in the original population), the number of preoral kineties (1-5 in the present study vs. 2-3 in the original population), and the number of macronuclei (1-3 in present study vs. 1 or 4 in the original population). In addition, the SSU rDNA sequence of the Ningbo population differs from the original population by only five unmatched nucleotides (99.7% in sequence identity). Consequently, we consider these variations to be population-dependent, and these two forms are conspecific.
As described above, two SSU rDNA sequences of Pleuronema orientale (OQ591739, KF206429) are very similar to P. puytoraci (KF840520), with two and three unmatched nucleotides, respectively. However, the morphological characteristics of P. orientale are quite different from the latter; that is, the former has more somatic kineties (36-51 in P. orientale vs. 28 or 29 in P. puytoraci), clearly separating them as two valid species [45,52]. Pleuronema puytoraci was originally reported by Grolière and Detcheva [52] with morphological and morphogenesis information. Pan et al. [45] provided detailed living characteristics and ciliary patterns based on a Hong Kong population, but unfortunately, they did not supply corresponding molecular data. Several years later, they supplied the SSU rDNA sequence of this species from another population without morphological information [23]. Considering the lack of corresponding morphological and molecular data from the same population, we doubt whether the current molecular information of P. puytoraci (KF840520) is correct. The morphological and molecular information of this species should be supplied based on the same population in the future.

Phylogenetic Relationships between Pleuronema and Schizocalyptra
The study of polyphyly of genus Pleuronema caused by Schizocalyptra nest within it was first discovered by Yi et al. [53]. This view was widely confirmed in most phylogenetic research based on the SSU rDNA sequence [23][24][25][26][54][55][56][57][58]. However, the phylogenetic analyses based on nuclear or mitochondrial data showed that the genus Schizocalyptra falls outside of Pleuronema [59,60]. In fact, the cell shape, the well-developed oral region with long paroral membranes, and three distinct membranelles M1-3 of Schizocalyptra are quite similar to that of Pleuronema [43]. It indicates that species in the genera Schizocalyptra and Pleuronema most likely evolved from a common ancestor. The present phylogenetic tree shows that Schizocalyptra nest within Pleuronema, consistent with previous studies based on SSU rDNA sequence data ( Figure 6). However, the support valve is low, and the phylogenetic position of Schizocalyptra is unstable. Given the low and inconsistent support for the phylogenetic position of the genera Schizocalyptra and Pleuronema in previous and existing studies, more morphological and molecular information is needed to further clarify their positions.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11061422/s1, Table S1: GenBank accession numbers of all sequences included in phylogenetic analyses.

Data Availability Statement:
The data presented in the study are deposited in the NCBI database repository, accession numbers: OQ591738 and OQ591739.

Conflicts of Interest:
The authors declare that they have no conflict of interest.