One Freshwater Species of the Genus Cyclidium, Cyclidium Sinicum Spec. Nov. (Protozoa; Ciliophora), with an Improved Diagnosis of the Genus Cyclidium

NOTE Pan et al., Int J Syst Evol Microbiol 2017;67:557–564 DOI 10.1099/ijsem.0.001642

One freshwater species of the genus Cyclidium, Cyclidium sinicum spec. nov. (Protozoa; Ciliophora), with an improved diagnosis of the genus Cyclidium

Xuming Pan,1 Chengdong Liang,1 Chundi Wang,2 Alan Warren,3 Weijie Mu,1 Hui Chen,1 Lijie Yu1 and Ying Chen1,*

Abstract The morphology and infraciliature of one freshwater ciliate, Cyclidium sinicum spec. nov., isolated from a farmland pond in Harbin, northeastern China, was investigated using living observation and silver staining methods. Cyclidium sinicum spec. nov. could be distinguished by the following features: body approximately 20–25Â10–15 µmin vivo; buccal field about 45– 50 % of body length; 11 somatic kineties; somatic kinety n terminating sub-caudally; two macronuclei and one micronucleus; M1 almost as long as M2; M2 triangle-shaped. The genus Cyclidium is re-defined as follows: body outline usually oval or elliptical, ventral side concave, dorsal side convex; single caudal cilium; contractile vacuole posterior terminal; adoral membranelles usually not separated; paroral membrane ‘L’-shaped, with anterior end terminating at the level of anterior end of M1; somatic kineties longitudinally arranged and continuous. Phylogenetic trees based on the SSU rDNA sequences showed that C. sinicum spec. nov. clusters with the type species, Cyclidium glaucoma, with full support. Cyclidium is not monophyletic with members of the clade of Cyclidium+Protocyclidium+Ancistrum+Boveria.

INTRODUCTION During a survey of the freshwater ciliate fauna in northeastern China, one scuticociliates was isolated and observed in Scuticociliates are common inhabitants of freshwater, brack- vivo and after silver staining. In addition, the molecular phy- ish and marine habitats [1–18]. Investigations over the past logeny of Cyclidium sinicum spec. nov., was investigated on c. 20 years have demonstrated that the subclass Scuticocilia- the basis of SSU rDNA sequence data. The diagnosis of the tia Small, 1967, is much more diverse than was previously genus Cyclidium is emended based on current observations. assumed [19–24]. Recent investigations in China have shown a high diversity of marine scuticociliates, but few reports about freshwater species [8, 11, 13, 25–33]. METHODS C. sinicum spec. nov. was collected on 26 Oct 2015 from a The well-known scuticociliate genus Cyclidium comprises more than 40 species isolated from terrestrial, marine and farmland pond (44 87¢ 14.7† N 127 09¢ 12.0† E) in Harbin, – Heilongjiang province, northeastern China (water tempera- freshwater habitats [34 39]. Three species have been recorded from China, all from the northern China seas, ture 14 C, pH 7.3; Fig. 1). About 0.4 l water was collected – namely Cyclidium citrullus Cohn, 1865, Cyclidium glaucoma from 0.1 0.5 m below the surface using a sampling bottle. Ciliates were maintained in habitat water in Petri dishes as [40] and Cyclidium varibonneti Song, 2000 [16, 22, 24, 39]. Cyclidium and Cylidium-like species are generally recog- raw cultures at room temperature (approx. 25 C) with rice nized by having a conspicuously truncated apical end, non- grains added as food to enrich the growth of bacteria. separated membranelles and a prominent, wing-like paroral Isolated cells were observed and photographed in vivo membrane [17, 36]. All members of the genus Cyclidium are using differential interference contrast microscopy. The small and have high degree of morphological similarity in protargol method used to reveal the infraciliature follow- vivo; therefore, their infraciliature as revealed by silver staining the protocol of Wilbert [42]. The protargol was ing is of great importance for species identification [22, 24, made according to the method of Pan et al. [9]. Silver 41]. Many nominal species, however, are insufficiently carbonate [43] and Chatton-Lwoff silver nitrate described and/or lack gene sequence data [24, 41]. [44] stains were also were used to reveal the

Author affiliations: 1College of Life Science and Technology, Harbin Normal University, Harbin 150025, PR China; 2Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, PR China; 3Department of Life Sciences, Natural History Museum, London SW7 5BD, UK. *Correspondence: Ying Chen, [email protected] Keywords: Cyclidium; Cyclidium sinicum spec. nov.; SSU rDNA sequence; new species. Abbreviations: BI, Bayesian inference; ML, maximum-likelihood. The GenBank/EMBL/DDBJ accession number for the SSU rDNA sequence of Cyclidium sinicum is KX853100.

001642 ã 2017 IUMS

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CHINA

(b)

Harbin b c d

China

Seoul

South Korea Yellow Sea (a) (d) 200 km

Fig. 1. Map and sampling site. (a) Map showing collecting sites. (b–d) Three photographs showing the same farmland pond in Harbin, Heilongjiang province, northeastern China (44 87¢ 14.7† N 127 09¢ 12.0† E). infraciliature and argyrome, respectively. Counts and by Modeltest v.3.4 [50]. The reliability of internal branches measurements of stained specimens were performed at was estimated by bootstrapping with 1000 replicates. Bayes- magnifications of Â100–1250. Drawings were made with ian inference (BI) analysis was performed with MrBayes the help of a drawing device. Systematics and terminol- v3.2.3 [51] via the CIPRES Science Gateway using the GTR ogy are mainly according to those of Lynn [45]. +I+G model selected by MrModeltest v.2.0 [52]. The chain length of Markov chain Monte Carlo simulations was Genomic DNA of C. sinicum spec. nov. was extracted, using 1 000 000 generations with a sampling frequency of 100 gen- the DNeasy Tissue kit (Qiagen), from about five to seven erations. The first 25 % of sampled trees was discarded as cells that had been starved overnight. The amplification of burn-in. Phylogenetic trees were visualized with TreeView SSU rDNA was performed with universal primers, Euk A v.1.6.6 [53] and MEGA v.4 [54]. and Euk B [46]. Purified PCR products of the appropriate size were inserted into the pMD18 T vector (Takara Biotech- RESULTS AND DISCUSSION nology) and transformed into Escherichia coli competent cells, and products from transformed clones were sequenced Subclass Scuticociliatia Small, 1967. on an ABI-PRISM 3730 automatic sequencer (Applied Bio- Family Cyclidiidae Ehrenberg, 1838. systems) using M13 forward and reverse primers. Other sequences used in the study were obtained from the Gen- Genus Cyclidium O. F. Müller, 1773. Bank database (accession numbers as shown in Fig. 4). Cyclidium sinicum spec. nov. (Fig. 2; Table 1). Sequences were aligned using Clustal W implemented in BioEdit 7.0 [47] enabling pairwise analysis. Uronema mari- Diagnosis num and Paranophrys magna were used as the outgroup Body about 20–25Â10–15 µm in vivo; buccal field about taxa. Maximum likelihood (ML) analysis was conducted 45–50 % of body length; 11 somatic kineties; somatic kinety using RAxML-HPC2 on XSEDE (8.1.11) [48, 49] via the n (SKn) extending to posterior sub-terminally; two macro- CIPRES Science Gateway website (http://www.phylo.org/ nuclei and one micronucleus; M1 almost as long as M2; M2 sub_sections/portal), using the GTR+I+G model as selected triangle-shaped; paroral membrane consisting of rows of

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Ma SK1

(b) (c)

CV (d) (e) (f)

(a)

(i) (j) (k)

(n) M2 (h)

Ma M3 (l) (m) M1 M2 Ma PM M1 Mi M3 M2 Ma Ma Ma Sc M3 PM (g) (o) (p) (q) (r) Sc

Fig. 2. Morphology and infraciliature of Cylidium sinicum spec. nov. from life (a, d, h–o) and after silver nitrate- (f) and protargol- staining (b, c, e, g, p–r). (a, h) Ventral views of a representative individual; arrowhead in (a) marks caudal cilium and in (h) depicts paroral membrane, arrow in (a) marks paroral membrane and in (h) shows caudal cilium. (b, c) Infraciliature in ventral (b) and dorsal view (c) of the holotype specimen. (d) Lateral view, to show contractile vacuole. (e) Different shapes of macronuclei, also showing nucleoli. (f) Detail of argyrome. (g, r) Oral ciliature of the holotype specimen. (i–o) Different individuals; arrowheads in (l, n) mark the apical plate, in (m) shows caudal cilium and in (o) exhibit somatic cilia, arrow in (n) marks contractile vacuole and in (m) shows paroral membrane. (p, q) Anterior parts, to show macronuclei (arrowheads in p) and micronucleus. CV, contractile vacuole; M1–3, membranelles 1, 2 and 3; Ma, macronucleus; Mi, micronucleus; PM, paroral membrane; Sc, scutica; SK1, somatic kinety 1. Bars, 8 µm (g, p, q), 10 µm (a–d, h–o).

basal bodies forming a zig-zag pattern; scutica composed of Type locality two pairs of kinetosomes positioned posterior of cytostome; A farmland pond near Harbin (44 87¢ 14.7† N 127 09¢ freshwater habitat. 12.0† E), northeastern China.

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Table 1. Morphometric data based on silver staining specimens of Cyclidium sinicum spec. nov All measurements in µm. CV, coefficient of variation (%); M, Median; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of specimens; SD, standard deviation.

Character Min Max Mean M SD CV n

Body, length 21 32 28.3 27 5.7 21.4 15 Body, width 12 16 13.2 13 1.2 9.1 15 Buccal field, length 10 15 12.2 12 1.1 9.1 13 Buccal field, width 3 5 3.6 4 0.3 7.5 13 Somatic kineties, number 11 11 11 11 0 0 13 Macronucleus, number 2 2 2 2 0 0 13 Basal bodies in somatic kinety 1, number 11 12 11.4 11 0.4 3.8 14 Basal bodies in somatic kinety n, number 12 13 12.6 13 0.5 4.6 14

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

(k) (l) (m) (n)

Fig. 3. Various species of the genus Cyclidium in vivo (h, k, n) and after silver nitrate (a, b, e, g) and protargol staining (c, d, f, i, j, l, m). (a–d) C. glaucoma Müller [40] (a, b from Berger and Thompson [55]; c, d from Song and Wilbert [24]). (e) Cyclidium plouneouri Dragesco, 1963 (from Dragesco [56]). (f) C. plouneouri sensu Wilbert, 1986 (from Wilbert [57]). (g) Cyclidium borrori [58] (from Small and Lynn [58]). (h–j) C. sinicum (present work). (k, l) C. varibonneti Song, 2000 (from Song [22]). (m, n) C. citrullus Cohn, 1865 (from Song and Wil- bert [24]). Bars, 5 µm (d), 10 µm (a–c, e, h–n), 20 µm (f, g).

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Type slides h). Buccal field about 45–50 % of body length, with promi- – The slide with the protargol-stained holotype specimen is nent paroral membrane on ventral side (Fig. 2i o). Somatic deposited in the Natural History Museum, London, UK, cilia and cilia of paroral membrane about 7 µm long, caudal – with registration NHMUK.2016.10.15.1. A paratype speci- cilium about 12 µm long (Fig. 2i o). Cytoplasm colourless, men in a slide is deposited in the Laboratory of Protozool- containing several to many large (approximately 3 µm in ogy, Ocean University of China, with registration number diameter) bacteria-filled food vacuoles and variable-sized PXM-2015102602. (0.5–1 µm) refringent granules (Fig. 2a, h, o). Two globular macronuclei approximately 5 µm in diameter; one micronu- Etymology cleus approximately 2 µm across (Figs 2c, e, p–r). Contrac- The name ‘sinicum’ recalls the fact that this species was first tile vacuole located near posterior end of cell, approximately found in China: si¢ni.cum. N.L. neut. adj. sinicum pertaining 4 µm in diameter (Fig. 2n). Movement moderately fast, to China. rotating clockwise about main body axis, sometimes Description motionless for short periods. Body size 20–25Â10–15 µm in vivo, ellipsoidal with an api- Invariably 11 bipolar somatic kineties extending from near cal plate about 1/4 body width (Fig. 2a, h, n, l). Laterally flat- anterior apical plate to posterior contractile vacuole. All tened about 3 : 1 with right side concave and left side convex somatic kineties (SK1Àn) about equal length, comprising (Fig. 2d). Pellicle smooth, extrusomes not observed (Fig. 2a, loosely spaced monokinetids; somatic kinety 1 (SK1)

Fig. 4. Phylogenetic tree inferred from SSU rDNA sequences, showing the position of Cyclidium sinicum (in bold type). Numbers at nodes represent the bootstrap values of ML out of 1000 replicates and the posterior probability of BI. Fully supported (100 %/1.00) branches are marked with filled circles. Bar, five substitutions per 100 nucleotide positions.

561 Pan et al., Int J Syst Evol Microbiol 2017;67:557–564 comprises 11 or 12 monokinetids (Figs 2b, c, p–r). Membra- Phylogenetic analyses nelle 1 composed of two longitudinal rows of monokinetids; The topologies of the SSU rDNA trees reconstructed using M2 triangle-shaped, almost as long as M1 and composed of BI and ML analyses were almost identical; therefore, only about six horizontally oriented rows; membranelle 3 small, the ML tree is presented here with support values from both ‘ ’ two-rowed (Fig. 2g, r). Paroral membrane L -shaped, algorithms (Fig. 4). The phylogenetic relationships of pleu- extending to about 45 % of body length (Fig. 2g, r). Scutica ronematids have previously been examined using SSU comprises four kinetosomes arranged in two groups, located rDNA, ITS1-5.8S-ITS2 and LSU rDNA gene sequences [8, near posterior end of paroral membrane (Fig. 2g, r). No sil- 31]. The present study revealed similar relationships among ver nitrate preparations of sufficient quality were obtained families within the order Pleuronematida: i.e. Pleuronemati- to allow observation of the entire argyrome or contractile dae, Histiobalantiidae, Eurystomateliidae and Ctedoctemati- vacuolar pore (Fig. 2f). dae are all monophyletic, whereas Cyclidiidae is non- SSU rDNA sequence data monophyletic because several of its members group with thigmotrichids (represented by species of the genus Ancis- The SSU rDNA sequence of Cyclidium sinicum spec. nov. trum and Boveria subcylindrica). These include Cyclidium has been deposited in the GenBank database with the acces- marinum and C. varibonneti, which cluster with full support sion number, length and DNA G+C content as follows: with the clade comprising Protocyclidium citrullus and the KX853100, 1679 bp (not including Euk A and Euk B primer thigmotrichids Ancistrum sp., Ancistrum crassum and B. sites), 45.44 %. subcylindrica. With the addition of one new sequence, Remarks and comparison the genus Cyclidium is still non-monophyletic with other pleuronematids and thigmotrichids grouped within it. C. It is widely accepted that the most important criteria for spe- sinicum and C. glaucoma form a fully supported clade that cies identification and separation in the genus Cyclidium are is sister to the clade comprising C. marinum and C. varibon- the structure of membranelles 1–3, body size and shape, the neti with full support. This finding supports the placement length of the buccal field relative to the body length, the of the novel species in the genus Cyclidium. presence of extrusomes, the number of somatic kineties, the number of macronuclei, the termination position of somatic The genus Cyclidium is a well-known genus to which many – kineties in posterior end of somatic kineties SKn and SKnÀ1, forms have been assigned [16, 24, 34 39]. Morphological and the habitat [34–38, 41]. data based on observations of specimens both in vivo and after silver staining are, however, available for relatively few With respect to its body shape and size, prominent paroral species so many might have been misidentified. Moreover, membrane and freshwater habitat, C. sinicum most closely with the application of molecular techniques in taxonomy, resembles C. glaucoma [40], C. varibonneti Song, 2000, and further descriptions and comparisons based on combina- Cyclidium bonneti [38]. It can be easily separated from other tions of morphological and molecular data of species of the congeners (Fig. 3). genus Cyclidium are needed. Hence, an improved diagnosis Although C. sinicum and C. glaucoma share a number of of the genus Cyclidium is supplied here, based both on pre- features such as small body size, ellipsoidal body shape and vious studies and the present study. two pairs of kinetosomes in the scutica, the former can be Improved diagnosis of genus Cyclidium separated from the latter by the following combination of characters: the number of macronuclei (two vs one in C. Body outline usually oval or elliptical, truncated to form an glaucoma); the number of somatic kineties (invariably 11 vs apical plate, ventral side concave, dorsal side convex; single 8–10 in C. glaucoma); the length of the buccal field relative caudal cilium; contractile vacuole posterior terminal or sub- to the body length (45–50 % vs 55–65 % in C. glaucoma); terminal; membranelles usually non-separated; paroral ‘ ’ and the relative lengths of membranelles 1 and 2 (M2 and membrane L -shaped, with the anterior end terminating at M1 about equal length vs M2 longer than M1 in C. glau- or before anterior end of M1; somatic kineties longitudinally coma) ([24, 39]; Figs 3a–d, h–i). arranged and continuous. C. sinicum can be clearly distinguished from C. varibonneti Song, 2000, by the length of the buccal field relative to the Funding information body length (45–50 % vs 75 % in C. varibonneti), the This work was supported by the Natural Science Foundation of China (project number: 31501844). arrangement of kinetids in the somatic kineties (each kinety composed of monokinetids only vs each kinety composed of Acknowledgement dikinetids in anterior half of body and monokinetids in pos- We thank Dr Xinglong Cai, a PhD student of the Harbin Normal Univer- terior half in C. varibonneti) and habitat (freshwater vs sity, for sample collection. marine in C. varibonneti) ([22]; Fig. 3h–i, k, l). Conflicts of interest C. sinicum can be distinguished from C. bonneti [38] by The authors declare that there are no conflicts of interest. the number of somatic kineties (invariably 11 vs 14–16 in References C. bonneti) and the length of the buccal field relative to 1. 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