Morphology and Phylogeny of Four New Vorticella Species (Ciliophora: Peritrichia) from Coastal Waters of Southern China

Four new species of Vorticella, V. parachiangi sp. n., V. scapiformis sp. n., V. sphaeroidalis sp. n., and V. paralima sp. n., were isolated from coastal brackish waters of southern China. Their morphology, infraciliature, and silverline system were investigated based on observations of specimens both in vivo and following silver staining. Vorticella parachiangi sp. n. is distinguished by: a J‐shaped macronucleus; a single dorsally located contractile vacuole; a two‐rowed infundibular polykinetid 3, in which row 1 is shorter than row 2; 21–31 silverlines between peristome and aboral trochal band, 6–11 between aboral trochal band and scopula. Vorticella scapiformis sp. n. is characterized by its conspicuously thin and irregularly edged peristomial lip; a J‐shaped macronucleus; a single, ventrally located contractile vacuole; row 1 of the infundibular polykinetid 3 proximally shortened; 18–25 silverlines between peristome and aboral trochal band, 8–12 between aboral trochal band and scopula. Vorticella sphaeroidalis sp. n. can be identified by its small, sub‐spherical zooid; a C‐shaped macronucleus; a ventrally located contractile vacuole; an aboral trochal band adjacent to the scopula; 16–18 silverlines between persitome and aboral trochal band, two between aboral trochal band and scopula. Vorticella paralima sp. n. can be identified by its ovoidal zooid; a J‐shaped macronucleus; a dorsally positioned contractile vacuole; rows 1 and 2 of the infundibular polykinetid 3 proximally shortened; 26–35 silverlines from peristome to aboral trochal band, and 7–13 from aboral trochal band to scopula. The SSU rDNA genes of these four species were sequenced and their phylogeny was analyzed.

described in the 250 yr since it was created. Noland and Finley (1931) published the first comprehensive revision of the genus, listing 220 species or named varieties that had been assigned to the genus by 1930, and reduced its membership to 95 species following the elimination of many that obviously belonged to other peritrich genera, to other ciliate taxa, or to animal phyla (e.g. rotifers, cnidarians, crinoids). An additional 113 species, varieties, or forms of Vorticella were described in the period between Noland and Finley's paper and publication of the next major revision of the genus by Warren (1986) who excluded over 100 dubious species, reducing the total membership of the genus to 86 species. Many new species of Vorticella have since been described, raising the total to more than 100 again (e.g. Song 1997;Sun et al. 2006Sun et al. , 2009Sun et al. , 2017. The specialized morphology of Vorticella reflects its sessile, suspension-feeding lifestyle. The cell body, or zooid, is usually shaped like an inverted bell but may be cylindroid, conical or sub-spherical. The exposed part of the oral region is the peristome, a generally flattened surface with a thickened border or lip and a raised epistomial disc in the center. Two bands of cilia, the haplokinety and polykinety, run counterclockwise around the peristome and beat to create a vortex that carries suspended particles into the oral opening or infundibulum. The shape and size of the zooid, width of the peristome, height and contour of the epistomial disc and thickness of the peristomial lip have been used as taxonomic characters. Species identification of Vorticella is, however, frequently very difficult because of its variable zooid shape and size and its highly contractile nature resulting in the creation of numerous species and varieties of doubtful taxonomic validity (Itabashi et al. 2002;Noland and Finley 1931;Warren 1986).
In the last 50 yr, silver staining methods have been used to reveal additional characters for species circumscription and identification. These include the transverse pellicular ridges that encircle the zooid and can be visualized either as silverlines by staining with silver nitrate or directly by scanning electron microscopy, and the structure of the infundibular polykineties within the infundibulum, which can be revealed by staining with protargol. These characters are now routinely used in the determination of species (Sun et al. 2006(Sun et al. , 2017. More recently, gene sequence information has also been included in species descriptions and redescriptions (Ji et al. 2015;Sun et al. 2012). Phylogenetic studies based on gene sequence data derived from carefully identified species have shown that Vorticella is a paraphyletic group (Gao et al. 2016;Sun et al. 2012Sun et al. , 2013. In the present study, four new solitary vorticellids were discovered during a survey of ciliate diversity in coastal waters of southern China. Detailed investigations of the specimens, both in vivo and following silver staining (protargol and silver nitrate), and analysis of their small subunit (SSU) rDNA gene sequences revealed them to be new members of the genus Vorticella.

Sample collection
Vorticella parachiangi sp. n. was collected on 14th March 2010 from a mangrove wetland on Techeng Island (21°16 0 N; 110°44 0 E) in Zhanjiang, Guangdong Province, China, at a water temperature of 25°C, a salinity of 25&, and a pH of about 7.
Vorticella scapiformis sp. n. was collected on 21st March 2010 from a sewage discharge outlet on Donghai Island (21°02 0 N; 110°34 0 E) in Zhanjiang, Guangdong Province, China, at a water temperature of 25°C, a salinity of 12&, and a pH of about 9.
Vorticella sphaeroidalis sp. n. was collected on 5th May 2011from a small ditch near the coast (23°38 0 N; 116°68 0 E) of Shantou, Guangdong Province, China, at a water temperature of 23°C, a salinity of 5&, and a pH of about 7.

Morphological studies
Ciliates were observed in vivo using bright field and differential interference contrast microscopy (Nikon 80i, Japan). The infraciliature and silverline system were revealed by the protargol method following Wilbert (1975) and the Chatton-Lwoff silver nitrate-stain method following Song and Wilbert (1995), respectively. Counts and measurements of stained specimens were performed at 1,000X magnification. Drawings of stained specimens were conducted by hand with the help of a camera lucida at 1,000X magnification. Classification and terminology are according to Lynn (2008).
Phylogenetic trees were constructed by two methods: Bayesian inference (BI) and maximum likelihood (ML). Fifty-six taxa were used for phylogenetic analyses, including the four that were newly sequenced and 52 previously sequenced obtained from the NCBI GenBank database. The SSU rDNA of three Vorticella species sequenced by Sun et al. (2013), namely, V. chlorostigma, V. striata and V. natans, were removed from the alignment after preliminary analysis because of their short lengths. The hymenostomes Tetrahymena bergeri and T. corlissi were selected as outgroup taxa. Sequences were aligned with the ClustalW Multiple alignment in BioEdit 7.2.5 (Hall 1999) and then refined by removing ambiguous gaps at both termini of the alignment and in the highly variable V4 and V9 regions. The length of the final alignment was 1,616 bp.
The tree topologies were inferred, using the best-fit model GTR+I+G selected by AIC criterion in MrModeltest 2.2 (Nylander 2004). A 50% majority-rule Bayesian inference tree was constructed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The chain length for the analysis was 1,000,000 generations with trees sampled every 100 generations; the first 25% of trees were discarded as burn-in . The Maximum likelihood trees inferred from the sequences were constructed with the PhyML 3.0 program (Guindon et al. 2005) with a parametric bootstrap base frequency of 1,000.
Stalk up to 350 lm long and 4 lm in diameter, surface smooth without attached bacteria. Stalk spasmoneme about 2 lm in diameter, with a string of light gray thecoplasmic granules (ca. 0.5 lm in diameter) sparsely distributed along its length (Fig. 2F).
Haplokinety and polykinety make approximately 1¼ turns around peristome and extend into infundibulum where they separate and make a further turn. Polykinety transforms into three infundibular polykineties (P1-3) at lower half of infundibulum. P1 and P2 are composed of three distinct rows of kinetostomes. Arrangement of P3 unclear due to limited number of specimens and improper specimen orientation for observation. P3 very likely tworowed (Fig. 5C, D). Aboral trochal band dikinetidal, encircles zooid at 88% distance from peristome to scopula (Fig. 5D, 6G).
Stalk approximately 80-120 lm long and 4 lm in diameter, surface smooth with a few attached bacteria and algae. Spasmoneme about 2 lm in diameter; thecoplasmic granules not observed (Fig. 7B).
Haplokinety and polykinety make approximately 1½ turns around peristome and extend into infundibulum where they separate and make another turn. Polykinety transforms into three infundibular polykineties (P1-3) at lower half of infundibulum. P1-3 three-rowed. P1 and P3 converge at curvature of the latter. Row 3 of P3 extends to the end of infundibulum where it terminates near the end of P1. Rows 1 and 2 of P3 are half the length of row 3, terminating together with P2 (Fig. 7D, 8G). Germinal kinety lies in distal portion of infundibulum parallel to haplokinety. Epistomial membrane near entrance of infundibulum (Fig. 7E). Aboral trochal band dikinetid, encircles zooid at 75% distance from peristome to scopula (Fig. 7D, 8F).
Phylogenetic trees constructed with ML and BI analyses had almost identical topologies, therefore only the ML tree is shown here (Fig. 9). In all phylogenetic analyses, the three new species clustered with species of Vorticella and Pseudovorticella within the family Vorticellidae. Within the Vorticella-Pseudovorticella subclade, V. parachiangi sp. n. is sister group to V. scapiformis sp. n. with maximal support (100% ML, 1.00 BI), V. sphaeroidalis sp. n. and Vorticellides astyliformis cluster together with high support (95% ML, 1.00 BI) and are sister to the Vorticellides aquadulcis-complex with differential support (99% ML, 0.53 BI). Vorticella paralima sp. n. and Pseudovorticella species form a clade that is sister to the grouping of V. parachiangi sp. n. and V. scapiformis sp. n.

DISCUSSION
Commonly accepted characters for species circumscription in Vorticella include zooid shape and size, number and location of contractile vacuoles, shape and position of the macronucleus, oral morphology (appearance of peristomial lip and epistomial disc), oral infraciliature (especially structure of P3), features of the silverline system, and habitat . * In C, the initial (i.e. proximal) position of P3 is shown, but the terminal (i.e. distal) position and the number of kinetosomal rows is indeterminate. (Sun et al. 2006(Sun et al. , 2017. The four new species could each be separated from their congeners and other similar taxa based on combinations of these characters.

Comparison of Vorticella sphaeroidalis sp. n. with congeners
Although the structure of P3 could not be clearly observed, V. sphaeroidalis sp. n. can be easily distinguished from its congeners by the combination of its small and globular zooid, and the location of the aboral trochal band close to the scopula. Three congeners, V. parapulchella Sun et al., 2006;V. pulchella Sommer, 1951;and V. sinica Sun et al., 2009; have to be compared with the new species. Vorticella sphaeroidalis sp. n. resembles V. parapulchella in zooid size and number of silverlines. Nevertheless, the former differs from the latter in respect to the conspicuously more posterior position of its aboral trochal band, the globular (vs. barrel-shaped) zooid, and its brackish water (vs. marine) habitat (Sun et al. 2006).
Like V. sphaeroidalis sp. n., V. pulchella has widely spaced pellicular striations. However, the new species can be separated by its much shorter zooid size (20-30 lm vs. 40-46 lm) and more posterior position of the aboral trochal band (Sommer 1951).
Vorticella sinica resembles V. sphaeroidalis sp. n. in terms of zooid size and shape and widely spaced pellicular silverlines. However, V. sphaeroidalis sp. n. can be separated from the former by its single-layered (vs. double-layered) peristomial lip, very likely two-rowed (vs. three-rowed) P3, and the conspicuously more posterior position of its aboral trochal band ).
Vorticella hamata Ehrenberg, 1831 also resembles V. paralima sp. n. in terms of its zooid shape and size, habitat and number of silverlines. Nevertheless, the two can be separated by the shape of the macronucleus (Cshaped vs. J-shaped) (Song 1991).
The present form is similar to V. striata Dujardin, 1841 in terms of body shape and general appearance in vivo. However, the latter species can be clearly separated from V. paralima sp. n by its shorter zooid in vivo (22-40 lm vs. 35-50 lm), C-shaped (vs. J-shaped) macronucleus, and the number of silverlines between the aboral trochal band and the scopula (5-7 vs. 7-13) (Song 1991).

Phylogenetic positions of four Vorticella species within the Vorticellidae
In the past decade, several studies have suggested that the family Vorticellidae is a nonmonophyletic assemblage of morphologically diverse groups (Miao et al. 2004;Sun et al. 2012Sun et al. , 2013. Among the genera of Vorticellidae, Vorticella Linnaeus, 1767, Pseudovorticella Foissner and Schiffmann, 1974, and Epicarchesium Jankowski, 1985 were reported to be nonmonophyletic .
In this study, Vorticella species are distributed among two clades: the majority cluster with Pseudovorticella species in the family Vorticellidae whereas the remainder, including V. microstoma, V. infusionum, V. mayeri, V. sphaeroidalis sp. n. and Vorticellides species, group with the Astylozoidae, the sister group of the Vorticellidae. The topology of our tree is consistent with previous studies (Ji et al. 2015;Li et al. 2015;Sun et al. 2012Sun et al. , 2013.
Among the new species, V. paralima sp. n. groups with Pseudovorticella punctata first with maximal support and both cluster with two Pseudovorticella species with a strongly support. This indicates a very close phylogenetic relationship of V. paralima sp. n. with these three Pseudovorticella species. However, they can be clearly differentiated by the zooid shape and silverline system (Ji et al. 2003(Ji et al. , 2004(Ji et al. , 2006. Vorticella parachiangi sp. n. and V. scapiformis sp. n. cluster together with maximal support to form a group that is sister to the subclade of Pseudovorticella species and V. paralima sp. n. Vorticella sphaeroidalis sp. n. clusters with Vorticellides species to form a strongly supported subclade that is sister to the Astylozoidae. Foissner et al. (2010) transferred a few Vorticella species to the newly established genus Vorticellides Foissner et al., 2010 based on the possession of two epistomial membranes, a small barrel-shaped zooid (usually < 60 lm long), and a minute two-rowed P3. Molecular data also supported a close phylogenetic relationship between small, barrel-shaped species, such as V. astyliformis and V. microstoma, and certain stalkless peritrichs such as Opisthonecta spp. and Astylozoon enriquesi (Foissner et al. 2010). Thus, it is not surprising that V. sphaeroidalis sp. n. clusters with members of Vorticellides and Astylozoidae based on its small zooid (20-30 lm 3 15-25 lm), globular shape, and the structure of P3.