Integrative Studies on a New Ciliate Campanella sinica n. sp. (Protista, Ciliophora, Peritrichia) Based on the Morphological and Molecular Data, With Notes on the Phylogeny and Systematics of the Family Epistylididae

During an investigation on freshwater peritrichs, a new colonial sessilid ciliate, Campanella sinica n. sp., was isolated from aquatic plants in an artificial freshwater pond in Qingdao, China. Specimen observations of this species were performed both in vivo and using silver staining. C. sinica n. sp. is characterized by the appearance of the mature colony, which is up to 2 cm high and contains more than 1,000 zooids, the asymmetric horn-shaped zooids, strongly everted and multi-layered peristomial lip, the slightly convex peristomial disc, and the well-developed haplokinety and polykinety, which make more than four circuits of the peristome before descending into the infundibulum. The small subunit ribosomal DNA (SSU rDNA), 5.8s rDNA and its flank internal transcribed spacers (ITS1-5.8s rDNA-ITS2), and large subunit ribosomal DNA (LSU rDNA) are sequenced and used for phylogenetic analyses which reveal that the family Epistylididae Kahl, 1933 is non-monophyletic whereas the genus Campanella is monophyletic and nests within the basal clade of the sessilids. The integrative results support the assertion that the genus Campanella represents a separate lineage from other epistylidids, suggesting a further revision of the family Epistylididae is needed. We revise Campanella including the transfer into this genus of a taxon formerly assigned to Epistylis, which we raise to species rank, i.e., Campanella ovata (Nenninger, 1948) n. grad. & n. comb. (original combination Epistylis purneri f. ovata Nenninger, 1948). In addition, we provide a key to the identification of the species of Campanella.

Recent molecular phylogenetic analyses have cast doubts on the validity, monophyly, and/or membership for several peritrich genera and families, for example, the family Epistylididae Kahl, 1933, species of which are distributed among several separate clades in gene trees (Miao et al., 2001(Miao et al., , 2004Sun et al., 2016;Zhuang et al., 2018). According to Lynn (2008), the family Epistylididae comprises 11 genera and is mainly characterized by the sessile trophont that: (1) is attached to substrate via either a non-contractile stalk or the scopula; (2) has an everted peristomial lip and a slightly projecting peristomial disc; and (3) has an oral ciliature that makes more than one turn around the peristome before entering the infundibulum (Lynn, 2008;Irwin and Lynn, 2015;Lu et al., 2020). One of the most distinctive genera of epistylidids is Campanella Goldfuss, 1820. Campanella is colonial with a non-contractile stalk and large zooids that have a multilayered peristomial lip, an oral ciliature that makes several circuits around the peristome before descending into the infundibulum and a reticulate silverline system (Nenninger, 1948;Yu et al., 1995;Lynn, 2008). Three valid species of Campanella have been described, namely Campanella umbellaria (Linnaeus, 1758) Goldfuss, 1820 (the type species), C. hanchuansensis Yu et al., 1995 and C. purneri (Nenninger, 1948) Yu et al., 1995, but only C. umbellaria has been studied using modern methods (Miao et al., 2004;Shi et al., 2004;Wang et al., 2011). Nevertheless, phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequences have challenged the traditional classification of Campanella suggesting that this genus may not belong to the family Epistylididae (Miao et al., 2004;Utz et al., 2010;Sun et al., 2016).
In the present study, we describe a new species, C. sinica n. sp., isolated from a freshwater pond in Qingdao, China, based on observations of specimens both in vivo and following silver staining. The phylogeny of this species based on SSU rDNA, ITS1-5.8S rDNA-ITS2, and large subunit ribosomal DNA (LSU rDNA) sequences is also analyzed, offering new insights into the evolutionary relationships of sessilids. Moreover, we reevaluate the classification of epistylidid-like species based on the morphological and phylogenetic results. In addition, the taxonomy and nomenclature of the species of Campanella are reviewed and a key to their identification is supplied.

Sampling and Observation
Campanella sinica n. sp. was collected between 27 March and 27 July 2020 from aquatic plants growing in an artificial freshwater pond (N36°03ꞌ45ꞌꞌ; E120°20ꞌ10ꞌꞌ) in Ocean University of China, Qingdao, China (Figure 1, inset). The water and sediment in the pond came from the Lake Weishan wetland (N34°43ꞌ59ꞌꞌ; E117°9ꞌ22ꞌꞌ). Live cells were observed and measured under a Zeiss AXIO Imager D2 microscope (Zeiss, Germany). The ciliature and silverline system were revealed by the protargol staining method and dry silver nitrate staining method, respectively (Klein, 1958;Foissner, 2014). The protargol powder was synthesized following the method of Pan et al. (2013). All measurements were performed at 400-1,000× magnifications. Drawings of live cells, ciliature, and silverline system were based on direct observations and photomicrographs. Classification and terminology are mainly according to Warren (1986) and Lynn (2008), respectively.

Phylogenetic Analyses
Phylogenetic analyses were performed both with single-gene datasets of SSU rDNA, ITS1-5.8S rDNA-ITS2, and LSU rDNA separately, and with a concatenated dataset of all three genes. In addition to the newly obtained sequences, other sequences downloaded from the GenBank database (for accession numbers, see Figures 1, 2), selected based on earlier studies Zhuang et al., 2018), were used in the phylogenetic analyses. The boundary of the ITS1-5.8S rDNA-ITS2 was identified according to the methods described by Sun et al. (2010Sun et al. ( , 2013. A total of 53 SSU rDNA sequences including Campanella sinica n. sp., 48 peritrichs and four hymenostomatids as outgroup taxa were used to construct the SSU rDNA trees, while the analyses of ITS1-5.8S rDNA-ITS2, LSU rDNA, and concatenated data contained 28, 26, and 19 taxa, respectively (Figures 1, 2). Before constructing the trees, the selected sequences were aligned by MAFFT and further refined by Guidance 2 (https://guidance.tau.ac.il/ver2/; Katoh and Standley, 2013;Sela et al., 2015). The final alignments used for subsequent phylogenetic analyses comprised 1,605 sites for the SSU rDNA, 521 sites for the ITS1-5.8S rDNA-ITS2, 1,749 sites for the LSU rDNA, and 3,702 sites for the concatenated (SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA) dataset.
The best-fit models for maximum likelihood (ML) and Bayesian inference (BI) analyses were calculated by ModelFinder under Bayesian Information Criterion (Table 1; Kalyaanamoorthy et al., 2017). The concatenated sequences were treated with partitioned analyses to give the best-fit models for different regions.
Maximum likelihood analysis was carried out by IQ-TREE v2.0 with 10000 Ultrafast bootstrap replicates (Minh et al., 2020). BI analysis was carried out by MrBayes v3.2.7 (Ronquist et al., 2012). Markov chain Monte Carlo simulations were run for 1,000,000 generations with sampling every 100 generations. The first 25% of trees were discarded as burn-in. The run continued until the SD of split frequencies was below 0.01, and the effective sample size was > 200 (Rambaut et al., 2014). Phylogenetic trees were viewed by MEGA v.7 (Kumar et al., 2016)  The SSU rDNA and ITS1-5.8S rDNA-ITS2 sequences of C. sinica n. sp. were separately aligned with closely related taxa by MAFFT v.7 with L-INS-I strategy (Katoh and Standley, 2013  The ends of alignments were refined by eye. The final comparisons were edited and viewed by Bioedit v.7 (Hall, 1999) and TBtools (Chen et al., 2020a). The nucleotide differences between each of the new sequences and those species with high sequence similarities are shown in Figure 3.

Diagnosis
Mature colony up to 2 cm high with up to 1,000 zooids and dichotomously branched stalk. Zooids asymmetric horn-shaped, A C B FIGURE 2 | The ML trees inferred by ITS1-5.8S rDNA-ITS2 region (A), LSU rDNA sequence (B), and the concatenated data of SSU rDNA, ITS1-5.8S rDNA-ITS2, and LSU rDNA sequences (C). In each tree, Campanella sinica n. sp. is in red and indicated by an arrow. Numbers given at nodes of branches are the UF values for ML analysis and PP values for BI analysis. The scale bar corresponds to ten, two, and five substitutions per 100 nucleotide positions in (A-C), respectively.

Deposition of Slides
One protargol slide with the holotype specimen circled in ink (registration number: WT2020072201-01; Figure 4N), and two "dry" silver nitrate slides with paratype specimens (registration numbers: WZ2020052701-01, 02) were deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, China.

Etymology
The species-group name "sinica" refers to the country where the sample was collected.

Phylogenetic Analyses
The GenBank accsssion numbers, lengths, and GC contents of the newly obtained rDNA sequences (SSU rDNA, ITS1-5.8S rDNA-ITS2, and LSU) are shown in Table 3.
In all the cases, the ML and BI trees have similar topologies, therefore, only the ML tree is presented for each gene or region. In the SSU rDNA tree, the order Sessilida is monophyletic, whereas the families Epistylididae, Opisthonectidae Foissner, 1976, Vorticellidae Ehrenberg, 1838, and Zoothamniidae Sommer, 1951 were non-monophyletic (Figure 1) a fully supported clade (ML/BI, 100/1.00) that was sister to a clade comprising two epistylidids (Epistylis galea Ehrenberg, 1831 and Pseudepistylis songi Peng et al., 2007), three opercularids (Opercularia microdiscum Fauré-Fremiet, 1904, O. allensi Stokes, 1887, andPropyxidium sp.), and the opisthonectid Telotrochidium cylindricum (Figure 1). The above-mentioned species formed the basal clade within the Sessilida with maximal support (ML/ BI, 100/1.00). The remaining sessilids formed a clade with maximal support (ML/BI, 100/1.00). Within this clade, loricate species of the family Vaginicolidae formed the basal branch, albeit with low support (ML/BI, 66/0.68). Species of the family Zoothamniidae were divided into separate clades. The majority of epistylidids (core Epistylis species) nested within Zoothamniidae (Figure 1), while E. anastatica (Linnaeus, 1767) Ehrenberg, 1830 and another epistylidid (Rhabostyla commensalis Möbius, 1888) nested within the Vorticellidae, and E. galea Ehrenberg, 1831 grouped with the operculariids, although the identity of this latter sequence awaits verification (Figure 1).    The topology of the ITS1-5.8S rDNA-ITS2 tree was broadly similar to that of the SSU rDNA tree except the position of family Vaginicolidae, which clustered with the Vorticellidae to form the crown group of sessilids rather than branching basally. Three colonial species, Campanella, C. sinica n. sp., and Propyxidium taradigradum Van der Land, 1964, formed the basal clade of the Sessilida. The remaining sessilids clustered into similar groups as those in the SSU rDNA tree. Each of the families Zoothamniidae, Epistylididae, Operculariidae, and Vorticellidae was non-monophyletic (Figure 2A).
In the LSU rDNA tree, Campanella sinica n. sp. and P. songi formed a strongly supported clade (ML/BI, 98/1.00) which was basal within the Sessilida (Figure 2B). The topology of the concatenated sequence tree was similar to that of the SSU rDNA tree, supporting the assertion that Campanella and Epistylis are distantly related. Epistylis chrysemydis Bishop and Jahn, 1941 nested within the Zoothamnidae (Figure 2C).
We also compared nucleotide differences of SSU rDNA and ITS1-5.8S rDNA-ITS2 sequences between C. sinica n. sp. and other closely related taxa (Figure 3). The SSU rDNA sequence most similar to C. sinica n. sp. was Campanella sp. (KU363248) with one variable site, followed by C. umbellaria (AF01524) with 13 variable sites. The GC contents of the SSU rDNA sequences of Campanella species, Epistylis gelea, and P. songi ranged from 41 to 43% (Figure 3A).

Review of Campanella Species
Campanella umbellaria (Linnaeus, 1758) Goldfuss, 1820 is commonly found in eutrophic freshwaters (Linnaeus, 1758;Foissner et al., 1992). It is characterized by its symmetrical inverted bell-shaped zooid and horseshoe-shaped macronucleus (Foissner et al., 1992 ; Figures 6A,B). According to Sládeček and Sládečková (1974) and Foissner et al. (1992), the synonyms of this species are as follows: Hydra umbellaria Linnaeus, 1758, Epistylis flavicans Ehrenberg, 1838, Epistylis grandis Ehrenberg, 1838, Epistylis tincta Stokes, 1887, andEpistylis liebmanni Nenninger, 1948. It is noteworthy that no illustration of E. tincta was provided in the original description (Stokes, 1887). Kahl (1935) considered that E. tincta should belong to the genus Campanella and mentioned the difficulty in separating E. tincta from C. umbellaria. Yu et al. (1995) also accepted that E. tincta and E. liebmanni should belong to the genus Campanella but either overlooked or rejected the assertion that they are junior synonyms of C. umbellaria, referring to them as C. tincta and C. liebermanni (which is presumably a misspelling of "liebmanni"), respectively. Based on the descriptions provided by Stokes (1887) and Nenninger (1948), we agree with Sládeček and Sládečková (1974) and Foissner et al. (1992), who suggested that E. tincta and E. liebmanni (and as a consequence C. tincta and C. liebermanni) are junior synonyms of C. umbellaria. Nevertheless, variations are recognized in different populations of C. umbellaria, so it is possible that cryptic species might exist among these populations (Sládeček and Sládečková, 1974;Foissner et al., 1992). Further investigations including both morphological and molecular methods therefore need to be carried out to accurately characterize such populations.
Besides E. liebmanni, Nenninger (1948) described other two taxa that she assigned to the genus Epistylis, i.e., E. purneri and E. purneri f. ovata, both of which should belong to the genus Campanella due to the presence in each of a multilayered peristomial lip and an oral ciliature that makes more than four circuits around peristome before descending into the infundibulum (Figures 6B-D). Epistylis purneri and E. purneri f. ovata are similar but they differ from each other as follows: (1) the zooid shape of E. purneri is cylindrical, whereas that of E. purneri f. ovata is pyriform and (2) the stalk of E. purneri has several conspicuous transverse annular bulges, whereas that of E. purneri f. ovata has smooth surface (Figures 6C,D). Consequently, E. purneri f. ovata should be elevated to species rank. Yu et al. (1995) established the name C. purneri for E. purneri although without providing any justification. Here, we establish Campanella ovata (Nenninger, 1948) n. grad. & n. comb for E. purneri f. ovata (original combination Epistylis purneri f. ovata Nenninger, 1948). Nenninger (1948) also reported a new sessilid species, Opisthostyla tritora, which resembles colony-founding individuals of C. umbellaria (Figure 6E). Opisthostyla Stokes, 1886 is characterized by its solitary lifestyle and long, non-contractile stalk that is curved near the point of attachment to its substrate and acts in a spring-like manner throwing the organism backward when the zooid contracts (Stokes, 1886;Shen and Gu, 2016). However, the stalk of O. tritora has neither the structure nor the behavior that characterizes Opisthostyla. Therefore, O. tritora was probably misidentified by Nenninger (1948) and should belong to the genus Campanella. Horst (2018) listed O. tritora in the synonyms of C. umbellaria but did not provide any comments on this misidentification. Due to the lack of important morphological data for O. tritora, further studies are needed to determine the correct taxonomy of this species. Yu et al. (1995) reported a new Campanella species, C. hanchuanensis, from a freshwater pond in Hubei Province, China ( Figure 6F). In the original description, evidence for the presence of a reticulate (vs. transverse) silverline system is ambiguous (Yu et al., 1995). However, in a subsequent report, Shen and Gu (2016) confirmed that the silverline system of C. hanchuanensis is reticulate. Although C. hanchuanensis closely resembles C. umbellaria, they can be separated as follows: (1) the stalk of C. hanchuanensis is curved, whereas that of C. umbellaria is straight and (2) the trochal band of C. hanchuanensis consists of six rows of kinetosomes with four compact middle rows and two separated peripheral rows, whereas that of C. umbellaria comprises two compact rows only (Stiller, 1941;Yu et al., 1995;Shen and Gu, 2016).

Comparison of Campanella sinica n. sp. With Morphologically Similar Taxa
Campanella sinica n. sp. closely resembles C. umbellaria from which it can be separated as follows: (1) the zooid of C. sinica n. sp. is asymmetrical horn-shaped, while that of C. umbellaria is symmetrical and inverted bell-shaped; (2) the stalk of C. sinica n. sp. is smooth, whereas that of C. umbellaria has transverse annular bulges; and (3) the P3 of C. sinica n. sp. makes less than half a circuit within the infundibulum, whereas that of C. umbellaria makes 1-1.5 circuits. Campanella sinica n. sp. can be easily separated from C. purneri and C. ovata n. grad. & n. comb. by its asymmetrical horn-shaped (vs. elongated cylindrical) zooid, and convex, slightly elevated (vs. conspicuously elevated) peristomial disc. Compared with C. hanchuanensis, C. sinica n. sp. has a straight (vs. curved) stalk, asymmetrical horn-shaped zooids (vs. symmetrical inverted bell-shaped zooids), and the trochal band consists of several compact rows (vs. four compact middle rows and two separated peripheral rows). Based on these differences, C. sinica n. sp. can be easily distinguished from C. hanchuanensis.

Key to the Identification of Campanella Species
The genus Campanella contains five valid species, i.e., C. umbellaria, C. purneri, C. ovata n. grad. & n. comb., C. hanchuanensis, and C. sinica n. sp. We here supply a key to their identification.

Phylogenetic Analyses of the Genus Campanella and Other Sessilids
Campanella has been reported from freshwater habitats worldwide (Nenninger, 1948;Sládeček and Sládečková, 1974;Shen and Gu, 2016). Species of Campanella are characterized by their colonial lifestyle, dichotomously branched non-contractile stalk, everted multilayered peristomial lip, and well-developed haplokinety and polykiney that make more than four circuits around the peristome before descending into the infundibulum. Traditionally, this genus has been assigned to the family Epistylididae because of the non-contractile stalk and the well-defined, everted peristomial lip (Corliss, 1979;Lynn, 2008). However, phylogenetic analyses based on molecular data have challenged this classification. Miao et al. (2004), for example, suggested that the genus Campanella should be independent of the family Epistylididae and probably represents a separate lineage based on phylogenetic analyses of SSU rDNA sequences. Nonetheless, this study was solely based on single gene analyses and did not provide morphological data to support this conclusion.
In the present study, Campanella nests within the basal clade of the sessilids and is distinctly separated from other epistylidids in all phylogenetic trees, which is consistent with the findings of Miao et al. (2004). Based on the present phylogenetic analyses, Campanella species show a close relationship with species of the family Operculariidae (Figures 1,  2A). However, operculariids are characterized by the absence of a peristomial lip, separating them from Campanella which has a conspicuous peristomial lip. Thus, the genus Campanella should not be assigned to the family Operculariidae. In two of the present phylogenetic trees, i.e., LSU rDNA and concatenated data, the closest relative of C. sinica n. sp. is the epistylidid Pseudepistylis songi, while in the SSU rDNA tree, these two species, along with another epistylidid, namely Epistylis galea, nest within the basal clade of the sessilids. Nevertheless, Campanella can be clearly separated from both P. songi and E. galea by its multi-layered (vs. single-layered) peristomial lip and the haplokinety and ploykinety which make more than four turns around the peristome (vs. less than two turns) before descending into the infundibulum (Foissner et al., 1992;Peng et al., 2007). Compared with all families within the order Sessilida, Campanella is morphologically distinguishable by its multi-layered peristomial lip and the unique oral ciliature. Thus, a combination of the morphological and molecular phylogenetic data suggests that the genus Campanella should belong to a separate family within the order Sessilida as proposed by Miao et al. (2004). However, the present and previous studies show that most families in the order Sessilida are non-monophyletic (Miao et al., 2004;Sun et al., 2012Sun et al., , 2013Sun et al., , 2016Zhuang et al., 2018). Furthermore, Epistylis galea, Pseudepistylis songi, and Rhabdostyla commensalis are also independent of core epistylidids in the present phylogenetic trees. Therefore, the establishment of new family-level taxa for genera such as Campanella is premature and should await a re-evaluation of the family Epistylididae based on detailed morphological and accurate molecular data.
Although at least 800 nominal species of sessilids have been reported from various aquatic environments worldwide, the taxonomy and classification of this group are confusing (Foissner et al., 2010;Sun et al., 2016;Wang et al., 2017b;Zhang et al., 2019;Zhou et al., 2019a,b). The order Sessilida is subdivided into 14 families based on phenotypic characters, such as lifestyle modes (solitary or colonial), stalk structures (with or without spasmoneme), lorica (presence or absence), and living habits (sessile or free-swimming) (Lynn, 2008). However, the validity of several of these families has been challenged in recent years following the application of molecular phylogenetic analyses, mainly based on SSU rDNA sequence data (Miao et al., 2001(Miao et al., , 2004Utz and Eizirik, 2007;Utz et al., 2010;Zhan et al., 2013;Jiang et al., 2019). Miao et al. (2001) provided the first analyses of phylogenetic relationships within the subclass Peritrichia based on SSU rDNA sequences. Subsequent molecular phylogenetic studies have suggested that family or genus assignments of many taxa should be reevaluated (Clamp and Williams, 2006;Utz and Eizirik, 2007;Williams and Clamp, 2007). Members of the basal clade of the Sessilida assemblage (e.g., Campanella umbellaria, Opercularia microdiscum, and Propyxidium sp.) are thought likely to possess the plesiomorphic characters of sessilids . The results of the present study support the findings of previous studies that the family Epistylididae is polyphyletic and should be divided into several groups and/or various of its members should be re-assigned to other families (Sun et al., 2012(Sun et al., , 2013Zhou et al., 2019b;Lu et al., 2020). The clustering pattern in the phylogenetic trees suggests that the major epistylidid group (core Epistylis) evolved from species of the family Zoothamniidae, which indicates that these underwent loss of the stalk spasmoneme. It is noteworthy that in the tree based on the concatenated dataset, E. chrysemydis is nested within the Zoothamnidae thus supporting this hypothesis ( Figure 2C). Zhuang et al. (2018) reported a population of E. chrysemydis with a hollow stalk containing a central bundle of fibers that is similar to the spasmoneme of Zoothamnium. Species of Campanella have a hollow stalk suggesting that stalks with this structure may also represent an ancestral trait of the sessilids. Furthermore, based on their gene sequence similarities, Campanella spp. (represented by SSU rDNA sequence KU343248 and ITS1-5.8S rDNA-ITS2 sequence GU586188) and C. sinica n. sp. are probably conspecific, although it is not possible to verify this due to the lack of vouchered specimens or morphological data for the former taxa.

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/ (MW969624, MW969627, and MW969625).

AUTHOR CONTRIBUTIONS
ZW, LL, and CW conceived and designed the paper. ZW and TW carried out the live observations, protargol staining, and phylogenetic analyses. ZW, TW, BL, AW, YC, XZ, SA-F, LL, and CW wrote the paper. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We thank Mr. Changjun Mu (Weishan Special Aquaculture Base) and Mr. Ya Wang (Weishan Fishery Development Service Center) for the institutional support. We are also grateful to the editor and reviewers for their constructive suggestions.