Redescription of Parasonderia Vestita (Kahl, 1928) Comb. Nov. (Ciliophora

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European Journal of Protistology 49 (2013) 106–113

Redescription of Parasonderia vestita (Kahl, 1928) comb. nov. (Ciliophora, Plagiopylida), with notes on its phylogeny based on SSU rRNA gene Yuan Xua, Chen Shaob, Miao Miaoc, Weibo Songa,∗ aLaboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China bThe Key Laboratory of Biomedical Information Engineering, Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China cCollege of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Received 15 November 2011; received in revised form 9 March 2012; accepted 9 March 2012 Available online 6 July 2012

Abstract

During faunistic studies on psammophilic ciliates along the coast of Qingdao, China, a population of the poorly known species Parasonderia vestita (Kahl, 1928) comb. nov. (basionym: Plagiopyla vestita Kahl, 1928) was found and investigated using silver staining methods. These revealed that its oral ciliature is rather unique and composed of polykinetids comprising four parts: prebuccal polykineties, postbuccal polykineties, parabuccal polykineties, and intrabuccal polykineties. Both the pre- and the postbuccal polykineties are, as extensions of the somatic kineties, spiraling and extending from cell surface into buccal cavity. The somatic kineties are composed mainly of dikinetids with some monokinetids and trikinetids inserted. They are separated into two types: four circle kineties and 24–28 bipolar kineties. The circle kineties start from the side of oral region and connect with the opposite section at the end of the body. Phylogenetic analysis based on SSU rRNA gene sequence data indicates that P. vestita falls into the core part of the order Plagiopylida and groups with an environmental sequence with high support and forms a sister clade with Lechriopyla mystax, Plagiopyla frontata and P. nasuta. © 2012 Elsevier GmbH. All rights reserved.

Keywords: Marine ciliate; Parasonderia; Plagiopylida; SSU rRNA gene; Taxonomy

Introduction When Parasonderia Fauré-Fremiet, 1973 was erected it was invalid due to the failure to ﬁx a type species (Aescht Members of the ciliate order Plagiopylida can be isolated 2001; ICZN 1999, Article 13.3). It is characterized by “oral from both marine and freshwater habitats and are especially kineties, as extensions of body kineties, entering round oral common in anaerobic salt-marsh and interstitial biotopes opening, spiraling or twisting to right before ending near (Corliss 1979; Kahl 1931b). According to Lynn (2008), this cytostome” (Lynn and Small 2002). According to Fauré- assemblage includes three families: Trimyemidae, Plagiopy- Fremiet (1973), the species assigned to Parasonderia have lidae, and Sonderiidae. The genus Parasonderia, together a “primitive” type of oral area, i.e. it is elliptical to circu- with Sonderia, Sonderiella, and Oncosonderia, belongs to lar shape, and does not extend as far across the cell as that the family Sonderiidae. of other plagiopylids. Jankowski (2007) re-established it as a new genus and designated P. kahli (Fauré-Fremiet, 1973) Jankowski, 2007 as the type species. Although Fauré-Fremiet ∗Corresponding author. (1973) described the somatic infraciliature of P. kahli,hedid E-mail address: [email protected] (W. Song). not give any detailed information about the oral structure.

Thus the oral structure, which is one of the most important 5-AACCTGGTTGATCCTGCCAGT-3 and Euk B, 5- diagnostic characters of genera within the family Sonderi- TGATCCTTCTGCAGGTTCACCTAC-3. idae, was hitherto unknown. The final alignment of 1434 characters and 76 taxa, Furthermore, molecular data for the class Plagiopylea, to including 35 environmental sequences (Alexander et al. which the genus Parasonderia is assigned, are limited to only 2009; Behnke et al. 2010; Stoeck et al. 2007; Takishita 31 small subunit (SSU) rRNA gene sequences, 22 of which et al. 2007, 2010; Zuendorf et al. 2006), was used to con- belong to unidentified environmental isolates (Behnke et al. struct phylogenetic trees according to the methods reported 2010; Dunthorn et al. 2008; Embley et al. 1995; Lefèvre et al. by Foissner and Stoeck (2011) and Zhang et al. (2011). 2008; Lefranc et al. 2005; Luo et al. 2011; Lynn and Strüder- Briefly, the Bayesian (BI) analysis was performed with Kypke 2002; Shinzato et al. 2007; Stoeck et al. 2007; Zhang MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using et al. 2010). The molecular phylogeny of Parasonderia has GTR + I (= 0.2556) + G (= 0.5089) as the best model selected never been investigated. by the program MrModeltest v.2.0 (Nylander 2004). The During a survey of the ciliate fauna of the Yellow Sea chain length for our analysis was 1,000,000 generation with coastal waters of northern China, a population of the poorly trees sampled every 100 generations; the first 25% were known Parasonderia vestita (Kahl, 1928) comb. nov. was discarded as burn-in. Maximum-likelihood bootstrapping isolated giving the opportunity to investigate its morphol- analyses were carried out with 100 replicates using RAxML ogy in detail. The SSU rRNA gene was also sequenced and with the setting as described in Stamatakis et al. (2008).ML phylogenetic analyses were performed in order to assess its analyses were conducted online on the CIPRES Protal V 2.0 systematic position within the Plagiopylea. (http://www.phylo.org). Symbiodinium pilosum and Proro- centrum micans were selected as outgroup in the analyses.

Material and Methods Results Ciliates were isolated on 5 November 2010 from the intertidal zone near a sewage outfall at Qingdao (36◦03N, Parasonderia vestita (Kahl, 1928) comb. nov. 120◦19E), China. The water temperature was 16 ◦C and the salinity 20‰. Samples were taken from an anaerobic sapro- Remarks: This organism was first reported by Kahl (1928) pel of a 20 cm-deep hole, where the sand was black in color as Plagiopyla vestita, but provided only a schematic figure and with strong smell of hydrogen sulfide. Living cells were and a brief description on its living morphology. Later, he studied in the laboratory using bright field and differential transferred it to Sonderia (Kahl 1931b). Based on the cur- interference contrast microscopy (100× to 1000× magnifica- rent studies, an improved diagnosis and a redescription are tions). The infraciliature was revealed with both the protargol supplied here. and the Chatton–Lwoff methods (Song and Wilbert 1995; Improved diagnosis (based on the description in Kahl Wilbert 1975). Counts and measurements of stained speci- 1928 and data in the present paper): Marine Parasonderia mens were performed at a magnification of 1000×. Drawings about 35–75 ␮m long, 35–45 ␮m wide in vivo with bacte- were made with the help of a camera lucida. Terminology and ria on cell surface. Four circle kineties on cell margin; 13 systematics are according to Corliss (1979) and Lynn (2008). or 14 somatic kineties on ventral side and 11–14 on dorsal Since some structures found in this organism are revealed side; 14–17 prebuccal, six parabuccal, 12 or 13 postbuccal for the first time, the following terms are introduced here polykineties. Single ellipsoidal macronucleus. Single con- (Figs 10–12): Circle kineties (CK). Kineties starting from tractile vacuole near posterior end of cell. right or left side of oral region and connecting at poste- Deposition of voucher slide: A voucher slide with rior end of body. Prebuccal polykineties (PrBP). Parallel protargol-impregnated specimens is deposited in the Labora- kineties or kinety fragments located on upper side of buccal tory of Protozoology, Ocean University of China, Qingdao, field, extending into buccal cavity and ending near cytophar- China with registration number XY10110501. ynx. Postbuccal polykineties (PoBP). Opposite to PrBP Description of the Qingdao population (Figs 1–34 and (see above), fragments of kineties, also arranged in parallel, Table 1): Cell size in vivo mostly about 65 ␮m × 35 ␮m. located on lower side of buccal field, terminating in buc- Body elliptical with length:width ratio about 2:1; dorsoven- cal cavity. Parabuccal polykineties (PaBP). Several short trally flattened about 2:1 but dorsal surface clearly convex polykineties located on right side of buccal cavity. Intrabuc- (Figs 1–3, 13–15). Oral cavity with an oval opening, dom- cal polykineties (IBP). Kineties located deep within buccal inant and positioned sub-apically (Figs 1, 2, 13, 18, 23). cavity (likely close to cytopharynx). Extrusomes curved bar-shaped, about 10–12 ␮m long, scat- For molecular analyses, DNA extraction, PCR ampli- tered within cytoplasm (Figs 1, 2, 4, 21, 24), readily extruded fication, SSU rRNA gene cloning and sequencing were when suitably stimulated (Figs 9, 22). Cytoplasm transpar- performed according to Zhang et al. (2010). Primers ent and colorless, often containing several food vacuoles used for SSU rRNA gene amplification were: Euk A, with bacteria inside (Figs 1, 6, 19). Several refracting 108 Y. Xu et al. / European Journal of Protistology 49 (2013) 106–113

Figs. 1–12. Morphology of Parasonderia vestita comb. nov. from life (1–4, 6, 7, 9) and after protargol impregnation (5, 8, 10–12). 1. Ventral view of a typical individual. 2. A cell with different body shapes. 3. Left lateral view, to show dorsoventrally ﬂattened body. 4.Viewof deep level within anterior part, showing distribution of extrusomes (arrowheads) and narrowed buccal cavity. 5. View from inside to ventral surface, to show oral ciliature, namely, prebuccal, parabuccal, postbuccal, and intrabuccal polykineties. 6. Food vacuoles with bacteria inside. 7. Bacteria on cell surface. 8. Part of prebuccal polykineties, marking their curving from cell surface to inside of buccal cavity. 9. Posterior part, marking extruded extrusomes and bacteria on cell surface. 10. Ventral side, to indicate oral ciliature (arrowhead marks anterior part of ﬁrst circle kinety on right side composed of monokinetids). 11, 12. Infraciliature of ventral and dorsal sides of same specimen. B, bacteria; CK, circle kineties; CV,contractile vacuole; Ex, extrusomes; FV,food vacuole; IBP,intrabuccal polykineties; Ma, macronucleus; Mi, micronucleus; PaBP, parabuccal polykineties; PoBP, postbuccal polykineties; PrBP, prebuccal polykineties. Scale bars = 40 ␮m (1–3); 15 ␮m (10); 30 ␮m (11, 12). granules (about 2–5 ␮m in diameter), mostly packed in pos- vacuole ca. 8 ␮m in diameter, located near posterior end terior part of body (Figs 1, 2, 14, 15, 19). Cell surface of cell (Figs 1, 2, 14, 15). Somatic cilia about 10 ␮m long. always entirely covered by dense lawn of bacteria, curved, Locomotion by rotating slowly around main body axis. ca. 2–3 ␮m long (Figs 4, 7, 9, 16, 17, 21). Single ellipsoidal Oral infraciliature composed of four types of macronucleus, about 15–18 ␮m long, located below oral area polykineties: 14–17 prebuccal, six parabuccal, 12 or (Figs 1, 2, 12, 20, 26–28). One micronucleus accompanied 13 postbuccal, and several intrabuccal polykineties anteriorly with macronucleus (Fig. 12). Single contractile (Figs 5, 8, 10, 26, 28, 29, 32, 34). Somatic infraciliature Y. Xu et al. / European Journal of Protistology 49 (2013) 106–113 109

Figs. 13–25. Photomicrographs of Parasonderia vestita comb. nov. from life (13–24) and after silver nitrate impregnation (25). 13–15. Ventral views of different individuals, showing buccal field (arrowhead in 13) and contractile vacuole (arrow in 14 and 15). 16, 17. Cell surface covered by dense lawn of curved bacteria. 18. Buccal field deep within cell. 19. Food vacuole with bacteria inside (arrow) and spherical fat globules (arrowheads). 20. Macronucleus. 21. Anterior end, showing extrusomes (arrowheads) and layer of bacteria. 22. Extruded extrusomes (arrowheads). 23. Buccal field. 24. Mid-region of cell, showing extrusomes (arrowheads). 25. Ciliature of ventral side. B, bacteria; BF, buccal field; Ma, macronucleus. Scale bars = 40 ␮m (13–15); 25 ␮m (25).

generally composed of dikinetids, with some monokinetids similarity with an environmental sequence RM1-SGM04 and trikinetids inserted (Figs 11, 12, 27, 30). Four closed (accession no. AB505461). Phylogenetic trees inferred from circle kineties separating somatic kineties into 13 or 14 SSU rRNA gene sequence data using two methods (BI and ventral and 11–14 dorsal ones (Figs 11, 12, 26, 27, 31). ML) generate similar topologies; hence topology of BI tree Anterior part of the ﬁrst circle kineties on right side and is presented here (Fig. 35). Both BI and ML analyses recover always composed of monokinetids (Figs 10, 33, 34, arrows). the class Plagiopylea as a fully supported monophyletic group. In both trees, Parasonderia vestita and the environmental sequence RM1-SGM04 form a small, well-supported Phylogenetic position of Parasonderia vestita clade (1.00 BI, 95% ML) that is basal to the clade contain- comb. nov. (Fig. 35) ing species in the family Plagiopylidae. The environmental sequences from the Norwegian and Japanese studies form The SSU rRNA gene sequence of Parasonderia vestita a large, maximally supported clade (1.00 BI, 100% ML) comb. nov. (accession no. JN857941) is 1725 nucleotides in together with the Parasonderia clade and the Plagiopylidae length and has a GC content of 42.78%. It shares about 93% clade. 110 Y. Xu et al. / European Journal of Protistology 49 (2013) 106–113

Figs. 26–34. Photomicrographs of Parasonderia vestita comb. nov. after protargol impregnation. 26, 27. Ventral and dorsal view of same specimen as shown in Figs 11, 12, showing prebuccal polykineties, macronucleus and mono/trikinetids (arrowheads in 27) within somatic kineties. 28, 29, 32. Detailed views of oral ciliature, to show prebuccal (28, 29), postbuccal (28) and intrabuccal polykineties (32). 30. Detail of somatic kineties, indicating the mono/trikinetids (arrowheads). 31. View of posterior end of cell to show the circle kinety (arrow). 33, 34. View of anterior body portion, showing the monokinetids in anterior part of ﬁrst circle kinety (arrow), parabuccal, prebuccal, and postbuccal polykineties. IBP, intrabuccal polykineties; Ma, macronucleus; PaBP, parabuccal polykineties; PoBP, postbuccal polykineties; PrBP, prebuccal polykineties. Scale bars = 30 ␮m. Y. Xu et al. / European Journal of Protistology 49 (2013) 106–113 111

Table 1. Morphometric data of the Chinese population of Parasonderia vestita comb. nov.

Charactera Min Max Mean SD CV n

Body, length (␮m) 52 79 63.4 6.9 11.0 20 Body, width (␮m) 24 39 32.7 4.0 12.3 20 Anterior body end to prebuccal polykineties (␮m) 2 6 3.9 1.0 26.3 15 Oral apparatus, length (␮m) 9 12 10.3 1.1 10.8 15 Anterior body end to macronucleus, distance (␮m) 13 25 19.5 2.9 14.7 15 Somatic kineties on ventral side, number 13 14 13.7 0.5 3.6 20 Somatic kineties on dorsal side, number 11 14 12.2 1.0 7.8 20 Circle kineties, number 4 4 4.0 0 0 20 Prebuccal polykineties, number 14 17 15.2 1.0 6.3 20 Postbuccal polykineties, number 12 13 12.7 0.5 3.9 20 Parabuccal polykineties, number 6 6 6.0 0 0 20 Macronucleus, width (␮m) 15 17 17.7 1.7 9.4 15 Micronucleus, diameter (␮m) 3 4 3.4 0.5 14.9 15

a All data based on protargol-impregnated specimens. Abbreviations: CV, coefﬁcient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of specimens investigated; SD, standard deviation of the mean.

Discussion the type species of the genus, resembles P. vestita comb. nov. in body shape, the number of somatic kineties and the cau- Identification of the Qingdao population of dally positioned contractile vacuole. However, the former can Parasonderia vestita (Kahl, 1928) comb. nov. be separated from the latter by the shape of macronucleus (incomplete curved triangular blade vs. ellipsoidal) and the Kahl (1928) assigned this species to the genus Plagiopyla. striated band on the right side extending from the buccal area Later, he transferred it to Sonderia (Kahl 1931b). Nei- to the posterior end of the body (vs. not observed in P. vestita ther Fauré-Fremiet (1973) nor Jankowski (2007) formally comb. nov.) (Fig. 25; Fauré-Fremiet 1973). transferred Sonderia vestita to Parasonderia. Based on its Parasonderia cyclostoma (Kahl, 1931a) Jankowski, 2007 “primitive” type of oral area, which is oval instead of extend- resembles P. vestita in having a similar body size and shape, ing as far across the cell as in other plagiopylids, it should and a single ellipsoidal macronucleus. Nevertheless the two be transferred to the genus Parasonderia Jankowski, 2007: can be separated by the shape of the oral opening (circular Parasonderia vestita (Kahl, 1928) comb. nov. in P. cyclostoma vs. oval in P. vestita) and the presence of The Qingdao population matches the original description distinct caudal cilia in P. cyclostoma (vs. absent in P. vestita) in most characters: body shape; oval oral opening; having (Kahl 1931b). extrusomes; single macronucleus ellipsoidal and centrally located and single contractile vacuole near posterior end of Phylogenetic position of Parasonderia cell. Kahl (1928) described the cell as being enveloped in a ␮ 3 m thick layer of gelatinous substance which might be the According to Lynn (2008), the order Plagiopylida includes bacteria on cell surface. Kahl (1931b) therefore transferred three families: Trimyemidae, Plagiopylidae, and Sonderi- this species to Sonderia which differs from Plagiopyla in idae. Both BI and ML analyses recover the class Plagiopylea having bacteria on the cell surface. The only significant dif- as a fully supported monophyletic group and taxa belong- ference between the Qingdao population and that described ing to the order Plagiopylida, albeit many un-identified, fall by Kahl (1928) is the body length, the former being somewhat into the core part of this class. Our phylogenetic analyses ␮ ␮ larger (65–75 m vs. 35–40 m). Kahl (1931b), however, revealed that Parasonderia vestita and related environmen- reported that the size is rather variable and mentioned a tal sequences group together with sequences from the family ␮ length range of 50–100 m. Thus we believe that these fea- Plagiopylidae in one large, maximally supported clade. This tures are population-dependent and that these populations are may indicate that separation of the Sonderiidae from the Pla- conspecific. giopylidae is unwarranted although the monophyly of the family Trimyemidae seems to be strongly supported. Mean- while, the closest sequence to P. vestita is one from deep-sea Comparison between P. vestita comb. nov. and its sediments (Takishita et al. 2010). Similarly, environmental congeners sequences that cluster as basal relatives of the Parasonderia clade are from studies of anoxic regions from Norway, Mex- Since the genus Parasonderia was established, only two ico, USA, Denmark, and Japan which are the types of habitat species have been reported, namely P.kahli and P.cyclostoma typically inhabited by Parasonderia (Alexander et al. 2009; (Fauré-Fremiet 1973; Jankowski 2007). Parasonderia kahli, Behnke et al. 2010; Stoeck et al. 2007; Takishita et al. 2007, 112 Y. Xu et al. / European Journal of Protistology 49 (2013) 106–113

Fig. 35. BI tree inferred from the small subunit rRNA gene sequences showing the position of Parasonderia vestita comb. nov. (arrow). Numbers near branches represent BI posterior probabilities and non-parametric bootstrap values from ML. Black circles indicate full support in all analyses. Clades with a different topology in the ML tree are shown by an asterisk. Symbiodinium pilosum and Prorocentrum micans were the outgroup taxa. All branches are drawn to scale. The scale bar corresponds to ﬁve substitutions per 100 nucleotide position.

2010; Zuendorf et al. 2006). Thus, it seems that P. vestita Museum, UK) for improving the English of this manuscript. and its molecular relatives are characteristic of anoxic habi- Thanks are also due to Ms. Jie Huang (Laboratory of tats (Alexander et al. 2009; Behnke et al. 2010; Stoeck et al. Protozoology, OUC) for her very helpful suggestions on 2007; Takishita et al. 2007, 2010; Zuendorf et al. 2006). The phylogenetic tree’s construction. The manuscript was sub- SSU rRNA gene tree seems to indicate that plagiopylids and stantially improved by the anonymous reviews and by the sonderiids may comprise one large, diverse assemblage of additional suggestions by the monitoring editor. free-living taxa that inhabit anoxic environments.

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