Parasitology International 59 (2010) 549–555

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Parasitology International

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Phylogenetic consideration of two genera, and Boveria (Protozoa, Ciliophora) based on 18 S rRNA gene sequences

Feng Gao a, Xinpeng Fan a, Zhenzhen Yi a,b, Michaela Strüder-Kypke c, Weibo Song a,⁎ a Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, University of China, Qingdao 266003, China b Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China c Department of Integrative Biology, University of Guelph, Guelph, ON, Canada NIG 2W1 article info abstract

Article history: Many are facultative parasites of aquatic organisms and are among the most problematic Received 22 January 2010 taxa regarding their systematic relationships. The main reason is that most , especially taxa in Received in revised form 14 May 2010 the order Thigmotrichida have similar morphology and have not been studied yet using molecular methods. Accepted 9 July 2010 In the present work, two scuticociliate genera, represented by two rare parasitic species, Philasterides Available online 15 July 2010 armatalis (order ) and Boveria subcylindrica (order Thigmotrichida), were studied, and phylogenetic trees concerning these two genera were constructed based on their 18 S rRNA gene sequences. Keywords: fi Ciliophora The results indicate that: 1) Philasterides forms a sister group with Philaster, supporting the classi cation that Phylogeny these two genera belong to the family Philasteridae; 2) it is confirmed that the nominal species, Philasterides Morphology dicentrarchi Dragesco et al., 1995 should be a junior synonym of as revealed by both Philasterida previous investigations and the data revealed in the present work; and 3) the poorly known form B. Thigmotrichida subcylindrica, the only member in the order Thigmotrichida, of which molecular data are available so far, 18 S rRNA gene always clusters with Cyclidium glaucoma, a highly specialized scuticociliate, indicating a sister relationship between the orders Thigmotrichida and . © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Chatton and Lwoff [21,22], Fenchel [23], and Raabe [24–28], and no molecular information is available for any species of this order. Scuticociliates are found in a variety of habitats with great Phylogenetic analyses based on molecular data (e.g., 18 S rRNA and biodiversity regarding their life styles, structure, behaviour and RFLP) have been successfully used to identify and separate scutico- many other biological characters [1–11]. Although scuticociliates ciliate species, however, only few representative pleuronematids, and were separated into different groups by different taxonomists [12– no thigmotrichid species have been examined by molecular techni- 15], three scuticociliate orders, namely Philasterida, Pleuronematida ques so far [29–35]. Thus, there are still large gaps in our knowledge and Thigmotrichida, are generally well recognized [14,16]. about these taxa. The Philasterides is characterized by a Tetrahymena-like As a new contribution, we isolated and sequenced 18 S rRNA gene silverline system, the bipartite paroral membrane, and three well- of Philasterides armatalis, a philasterid species, and of Boveria defined oral membranelles, which are arranged in a Paranophrys-like subcylindrica, a thigmotrichid species. The main goal is to provide pattern [17,18]. This genus was originally assigned to the family molecular evidence for the phylogenetic assignments of the genus Philasteridae based on its morphological and morphogenetic features Philasterides and the order Thigmotrichida. The present study also [17–19], but recently transferred to the family provides photomicrographs and a brief redescription of the morphol- according to its stomatogenesis pattern [20]. However, the phyloge- ogy of both species. netic placement of Philasterides, especially its relationships to other morphologically similar genera, such as Philaster, has never been 2. Materials and methods assessed using molecular data. The order Thigmotrichida, characterized by a highly differentiated 2.1. Ciliate collection and identification thigmotactic somatic ciliature and a subequatorial oral region [16], has received relatively little attention since the monographic works of B. subcylindrica was isolated from the mantle cavity of the marine mollusk, Pinna pectinata on March 3 rd, 2009 from Qingdao (36° 04′ N; 120° 23′ E), China, by flushing the gills of hosts with seawater. P. ⁎ Corresponding author. Tel./fax: +86 532 8203 2283. armatalis was collected on June 26th, 2008 from the coastal area, the E-mail address: [email protected] (W. Song). same place as above. Microscopical observations and silver

1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.07.002 550 F. Gao et al. / Parasitology International 59 (2010) 549–555 impregnations are according to Wang et al. [9] and Wilbert [36]. replicates was constructed using PAUP* v. 4.0b10 [46] by means of Terminology and systematics are mainly following Lynn [16]. a heuristic search with all characters coded as unordered.

2.2. DNA extraction, PCR amplification and sequencing 3. Results

Total genomic DNA was extracted from cells using REDExtract-N-Amp 3.1. Morphological redescription of P. armatalis Song, 2000 (Fig. 1) Tissue PCR Kit (Sigma, St. Louis, USA) as described by Yi et al. [37].ThePCR amplifications of 18 S rRNA genes were performed with primers Euk A Considering the morphology and infraciliature, the current popula- (5′-AAC CTG GTT GAT CCT GCC AGT-3′)andEukB(5′-TGA TCC TTC TGC tion corresponds well to the populations described by Song [47] AGG TTC ACC TAC-3′) [38]. PCR amplification, cloning and sequencing (Fig. 1 I–K) and Hu et al. [19] (Fig. 1L, M). Therefore, our description were done according to Yi et al. [31]. concentrates only on those features differing from the previous report. Cells approximately 60–70×20–25 μminvivo(Fig. 1A, B), generally thicker than the populations reported previously with 2.3. Sequence availability and phylogenetic analyses asymmetric body shape when viewed from ventral. Cytoplasm mostly full of large globular granules (about 3 μm in diameter), which Besides the newly sequenced 18 S rRNA genes, all other sequences indicates that the cells might be newly left from the host (Fig. 1A–G). used in the present analysis were obtained from the NCBI GenBank Always several to many sparsely distributed biconcave crystals database (Table 1). Three Colpodea species were selected as the detected (Fig. 1G). terminally located at posterior outgroup for all analyses. All available 18 S rRNA gene sequences of end of cell, formed slowly and then contracted rapidly. Locomotion the subclass Scuticociliatia were included in our phylogenetic generally hastily and swimming helically ahead or motionless for a analyses. However, Schizocaryum sp. EC13, Cyclidium citrullus and long while when feeding on surface of debris. Infraciliature and buccal Entorhipidium pilatum were excluded due to their limited sequence apparatus (Fig. 1H) as described by Song [47] and Hu et al. [19]. length (441 bp, 836 bp and 1376 bp, respectively). The secondary structure-based 18 S rRNA gene sequence alignment of downloaded from the European Ribosomal 3.2. Morphological redescription of B. subcylindrica Stevens, 1901 Database [39] wasusedasthe‘“seed” alignment to build a profile (Fig. 2) hidden Markov model (HMM) using Hmmer Package version 2.3.2 [40];theHMMprofile obtained was then used to create an Our isolation resembles the original redescription very well alignment of the 56 sequences we assembled using Hmmalign [24,48], thus only some features are emphasized/documented here. – – μ within the package. The masked regions that could not be aligned Body usually 40 60×20 25 m in vivo with both ends conspicu- unambiguously were removed from the initial alignment using ously truncated. Cytoplasm colourless and hyaline, often with many to – Bioedit 7.0.0 [41].Thefinal alignment of 1704 characters was used numerous food granules/vacuoles (Fig. 2A D). Somatic cilia about μ μ to construct phylogenetic trees according to Yi et al. [42].The 10 m in length, while cilia in buccal area about 30 minlength(Fig. 2A, program MrModeltest v.2 [43] selected the GTR+I (=0.3430)+G D, arrow). Contractile vacuole located near the posterior cell end, about μ (=0.4870) as the best model with AIC criterion, which was then 10 mindiameter(Fig. 2D, arrowhead). Locomotion by rotating around used for both Bayesian inference (BI) and maximum likelihood long axis of body. Isolated cells can be maintained in Petri dish for about (ML). The MrBayes 3.1.2 program [44] was used for Bayesian 1 h. Infraciliature (Fig. 2F, K, L) corresponds well with previous reports analyses, and four simultaneous chains were run for 1,500,000 [24,48]. Number of somatic kineties (SK) about 27. generations sampling every 100 generations. The first 4000 trees were discarded as burn-in. All trees remaining after the discarding 3.3. Phylogenetic analyses were used to calculate posterior probabilities (PP) using a majority rule consensus. A ML tree was constructed with the PhyML V2.4.4 The two new 18 S rRNA gene sequences have been deposited in the program [45]. The reliability of internal branches was assessed GenBank database with the length and the accession numbers as using a nonparametric bootstrap method with 1000 replicates. A follows: P. armatalis, 1758 bp, FJ848877; B. subcylindrica,1768bp, maximum parsimony (MP) tree incorporating 1000 bootstrap FJ848878.

Table 1 List of the 18 S rRNA nucleotide sequences used in the present work.

Species Accession number Species Accession number Species Accession number

Anophryoides haemophila U51554 Gymnodinioides pitelkae EU503534 Pleuronema coronatum AY103188 Anoplophrya marylandensis AY547546 Histiobalantium natans AB450957 Pleuronema sinica EF486864 Bresslaua vorax AF060453 Homalogastra setosa EF158848 Pseudocohnilembus hargisi AY212806 Bryometopus atypicus EU039886 Hyalophysa lwoffi EU503538 Pseudocohnilembus marinus Z22880 Cardiostomatella vermiformis AY881632 Lembadion bullinum AF255358 Pseudocohnilembus persalinus AY551906 Cohnilembus verminus Z22878 Mesanophrys carcini AY103189 Pseudovorticella sinensis DQ845295 Colpoda inflata M97908 Metanophrys similis AY314803 Schizocalyptra aeschtae DQ777744 Cyclidium glaucoma Z22879 Miamiensis avidus AY550080 Schizocalyptra similis EU744177 Cyclidium plouneouri U27816 Miamiensis avidusa AY642280 Schizocalyptra sinica FJ156106 Cyclidium porcatum Z29517 Ophryoglena catenula U17355 Schizocaryum dogieli AF527756 Dexiotrichides pangi AY212805 Paramecium tetraurelia AB252009 Tetrahymena bergeri AF364039 Entodiscus borealis AY541687 Paranophrys magna AY103191 Thyrophylax vorax AY541686 Entorhipidium triangularis AY541690 Paratetrahymena sp. EU744176 elegans AY103190 Entorhipidium tenue AY541688 Parauronema longum AY212807 Uronema marinum DQ867074 Epistylis chrysemydis AF335514 Parauronema virginianum AY392128 Uronemella filificum EF486866 Eurystomatella sinica FJ012143 Philaster apodigitiformis FJ648350 Vampyrophrya pelagica EU503539 Frontonia lynni DQ190463 Plagiopyliella pacifica AY541685 Zoothamnium alternans DQ662855 Glaucoma chattoni X56533 Pleuronema czapikae EF486863

a First reported at the name of Philasterides dicentrarchi in Kim et al. [53]. F. Gao et al. / Parasitology International 59 (2010) 549–555 551

Fig. 1. Photomicrographs of Philasterides armatalis from live cells (A–G) and after staining by protargol (H) (A–H, original; I–K, from Song [47]; L, M, from Hu et al. [19]). (A, B) Ventral view of two typical individuals. (C–E) Showing different body shapes. (F) Ventral view of anterior part, arrow showing the buccal area where the cytostome is located. (G) Showing the globular granules and biconcave crystals (arrows) in the cytoplasm. (H) Anterior part of ventral view of infraciliature showing the buccal area. (I) Side view, a typical individual. (J, K) Infraciliature of ventral and dorsal side, arrowhead in J indicates the posterior end of the scutica, while in K marks the small bald apex. (L) Right lateral view. (M) Slender body shape, left lateral view. Abbreviations: CC, caudal ; CV, contractile vacuole; Ma, macronucleus; M1, 2, 3, membranelles 1, 2 and 3; PM1, 2, the anterior and posterior parts of the paroral membrane. Scale bars: 20 μm.

The ML tree (Fig. 3) and the BI/MP trees (Fig. 4) showed a similar congeners. However, three Schizocalyptra species grouped together topology. Within the class , the subclass Peniculia with strong support (99% ML, 1.00 BI, 97% MP). (3) Thigmotrichida, branched basal, followed by an assemblage including the subclasses represented by B. subcylindrica, always grouped with Cyclidium Astomatia, Apostomatia, Hymenostomatia, Peritrichia, and the genus glaucoma (100% ML, 1.00 BI, 100% MP). (4) Loxocephalida, whose Dexiotrichides,andfinally the subclass Scuticociliatia (Figs. 3 and 4). The validity is still under discussion, was polyphyletic with Paratetrahymena differences between the ML tree and the BI/MP trees are mainly in the and Cardiostomatella consistently clustering together, and Dexiotrichides phylogenetic positions of some philasterid genera, especially the clade forming a clade with Apostomatia+Hymenostomatia+Peritrichia. of Paranophrys magna+Uronema elegans and Pseudocohnilembus. Within the subclass Scuticociliatia, there were four orders: (1) 4. Discussion Philasterida was represented by 11 out of 15 families [16] in the present study. All analyses (Figs. 3 and 4) recovered the order Philasterida as a 4.1. Species in Philasterides monophyletic group with strong support, whereas the relationships among these families remained unresolved. Of these 11 families, the Currently, three morphospecies have been reported in the genus family Philasteridae appeared to be a well supported monophyletic Philasterides: the type species Philasterides armata, P. dicentrarchi [49] clade (92% ML, 1.00 BI, 80% MP), in which P. armatalis formed a sister and the recently described P. armatalis (Song, 2000). P. dicentrarchi is a group to Philaster spp. (2) Pleuronematida included the genera histophagous parasite of sea bass (Dicentrarchus labrax)foundby Schizocalyptra, Pleuronema, Histiobalantium, Eurystomatella and Cycli- Dragesco et al. [49] in the Mediterranean Sea. Since Miamiensis dium.ThegeneraPleuronema and Cyclidium were not monophyletic. avidus exhibits macro- and microstome stages [50],Songand Especially species of the genus Cyclidium showed great genetic Wilbert [8] proposed P. dicentrarchi as a junior synonym of M. divergences, which placed C. glaucoma outside the order Pleuronema- avidus, based on their extremely similar morphological character tida and as a sister taxon to B. subcylindrica, instead close to its (e.g. body size, buccal apparatus, number of somatic kineties, 552 F. Gao et al. / Parasitology International 59 (2010) 549–555

Fig. 2. Photomicrographs of B. subcylindrica from live cells (A–E) and after staining (F–H) (A–H, original; I–L, from Xu and Song [48]), and C. glaucoma (M, from Song et al. [11]) for comparison. (A–C) View of three typical individuals, arrow marks the paroral membranelle (PM). (D) The posterior portion of cell, arrowhead indicates the contractile vacuole, arrow refers to the PM. (E) Detail, to show the serrate pellicle. (F) Oral apparatus. (G–H) Arrangement of the somatic kineties (SK), arrowhead marks the SK1. (I, J) Ventral and lateral view. (K, L) Para-ventral view and para-dorsal view of the same specimen. (M) Ventral view of C. glaucoma after staining. Abbreviations: Ma, macronucleus; Mi, micronucleus; M1, 2, 3, membranelles 1, 2 and 3; PM paroral membrane; SK1 somatic kinety 1. Scale bars: 30 μm. position of contractile vacuole pore and life style, etc.). Jung et al. on patterns of the stomatogenetic process, the data of apomorphic– [51] also concluded that M. avidus and P. dicentrarchi are synony- plesiomorphic characters, as well as the morphological features, Ma et mous, based not only on morphological but also on 18 S rRNA gene al. [20] assigned Philaster and Philasterides to two different families, sequence data. In the present work, we have sequenced the 18 S the Philasteridae and the Uronematidae, respectively. However, based rRNA gene sequence of P. armatalis, and detected 95 site differences on their similar morphology, especially the “similar” structure of the between P. armatalis and P. dicentrarchi (sequence identity, 94.5%), buccal apparatus (e.g. the dominant membranelles 1 and 2), these while only 3 site differences were detected between P. dicentrarchi two genera were placed in the same family Philasteridae by some and M. avidus (AY550080) (sequence identity, 99.8%). Our phyloge- other investigators [12,14–16]. Notably, misled by the wrong species netic trees inferred from the 18 S rRNA gene sequences also indicate name of AY642280 (first named as Philasterides dicentrarchi, and that P. dicentrarchi (AY642280) and M. avidus (AY550080) always recently corrected to M. avidus) in GenBank, Miao et al. [35] concluded cluster in a fully supported (100% ML, 1.00 BI, 100% MP) clade that Philaster and Philasterides were conspicuously separated in the divergent from the newly sequenced P. armatalis (Figs. 3 and 4). topology, and suggested removing Philasterides from the family Therefore, we confirm that P. dicentrarchi is a junior synonym of M. Philasteridae. By adding the sequence of P. armatalis, a typical species avidus and that there are only two species in this genus, P. armata of this genus, our phylogenetic trees indicate that Philasterides and and P. armatalis. Meanwhile, the name of P. dicentrarchi has been Philaster group together in a well supported clade with strong support corrected as M. avidus in GenBank by its submitter (personal (1.00 BI, 93% ML, 80% MP) (Figs. 3 and 4), which suggests that they communication with Sung Mi Kim). have a reliable close relationship. Therefore, we suggest retaining both Philasterides and Philaster in the family Philasteridae.

4.2. Systematic position of Philasterides and related genera 4.3. Systematic position of Boveria and the order Thigmotrichida The assignment of different genera to the families within the Scuticociliatia has been generally controversial, because no widely According to Lynn [16], the order Thigmotrichida includes six families: acceptable criteria have been presented for them [12,14–16,20]. Based Ancistridae, Hemispeiridae, Hysterocinetidae, Protanoplophryidae, F. Gao et al. / Parasitology International 59 (2010) 549–555 553

Fig. 3. Maximum likelihood tree inferred from 18 S rRNA gene sequences. Numbers at nodes represent the bootstrap values of ML out of 1000 replicates. Hyphen (-) indicates disagreement between Bayesian and ML. *, first reported at the name of Philasterides dicentrarchi in Kim et al. [54]. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. Newly sequenced species in this work are highlighted in bold.

Paraptychostomidae, and Nucleocorbulidae. B. subcylindrica, the only which is inconsistent with their morphological similarity (the site species with a 18 S rRNA gene sequence being sequenced (in the present changes detected among these three species are 222–275 bp, and the work), belongs to the family Hemispeiridae. Based on the morphology, sequence identities among them are 84.0%–87.2%). Moreover, the especiallythepresenceofa‘scutica-field’ [48,52], Thigmotrichida should present phylogenetic trees (Figs. 3 and 4) depicted C. glaucoma and C. undoubtedly be assigned to the Scuticociliatia. However, their systematic porcatum as very divergent from other pleuronematids. There are position remains uncertain because neither ontogenetic nor molecular possibly two reasons: 1) considering that misidentification of dataareavailablesofar. Cyclidium species is a potential problem [47,53], their 18 S rRNA In our phylogenetic trees, B. subcylindrica clusters with C. gene sequences are very possibly in question; and 2) the genus glaucoma, a well-known form in the order Pleurostomatida, which Cyclidium itself is not a monophyletic assemblage, possibly based on a is acceptable considering the morphological features: both have the fast evolutionary divergence rate of these Cyclidium-like species, and extremely long paroral membrane (PM) which surrounds the whole the topology shown in the present work exhibits only the gene trees buccal field and simple membranelles (Fig. 2K–M). The major based on few species (under sampling). Thus further studies are difference is that the buccal field in B. subcylindrica moves to the needed. posterior end of the cell with clearly differentiated three membra- Since only a few representative pleuronematid and one thigmo- nelles (Fig. 2K, L) (vs. the buccal field in Cyclidium/C. glaucoma is on trichid sequences are available so far, it is too early to draw a final the ventral cell side with membranelles more or less reduced or conclusion about the exact relationship among them. However, we incompletely differentiated, Fig. 2M). We suppose that the morpho- are confident that, based on both morphological and molecular data, logical divergence might be explained by their different ways of living the order Thigmotrichida falls into the subclass Scuticociliatia, and has (parasitic vs. free living). Therefore, the order Thigmotrichida, a close genetic relationship with the order Pleuronematida. represented by Boveria, might have a sister relationship with Cyclidium-related pleuronematids, which is consistent with the Acknowledgments morphological data. Unexpectedly, the three Cyclidium species do not cluster together, This work was supported by the National Science Foundation of and C. porcatum even branches outside the order Pleuronematida, China (Project nos. 30870264, 40976099). Many thanks are also due 554 F. Gao et al. / Parasitology International 59 (2010) 549–555

[14] Lynn DH, Small EB: Phylum Ciliophora Doflein, 1901; in Lee JJ, Leedale GF, Bradbury P (eds): An illustrated guide to the Protozoa, Allen Press Inc, 2002. [15] Puytorac P. Phylum Ciliophora Doflein, 1901. In: de Puytorac P, editor. Traité de zoologie, infusoires ciliés. Paris: Masson; 1994. [16] Lynn DH. The ciliated Protozoa: characterization, classification and guide to the literature. Dordrecht: Springer Verlag; 2008. [17] Grolière CA. Morphologie et stomatogenèse chez deux ciliés Scuticociliatida des genres Philasterides Kahl, 1926 et Cyclidium O. F. Müller, 1786. Acta Protozool 1980;19:195–206. [18] Song WB, Wilbert N. Taxonomische untersuchungen an aufwuchsciliaten (Protozoa, Ciliophora) im Poppelsdorfer Weiher, Bonn. Lauterbornia 1989;3: 2–221. [19] Hu XZ, Warren A, Song WB. Stomatogenesis and morphological re-description of the marine ciliate, Philasterides armatalis (Protozoa: Ciliophora: Scuticociliatida). J Mar Bio Ass UK 2008;88:29–34. [20] Ma HW, Song WB, Ma HG. On phylogenetic relationships of Scuticociliatida (Protozoa, Ciliophora) mainly based on stomatogenetic and morphological data. Acta Zootax Sin 2005;30:684–91. [21] Chatton E, Lwoff A. Recherches sur les ciliés thigmotriches. I. Arch Zool Exp Gén 1949;86:169–253. [22] Chatton E, Lwoff A. Recherches sur les ciliés thigmotriches. II. Arch Zool Exp Gén 1950;86:393–485. [23] Fenchel T. Ciliates from Scandinavian molluscs. Ophelia 1965;2:71–174. [24] Raabe Z. Ordo Thigmotricha (Ciliata–Holotricha). II. Familiae Hemispeiridae. Acta Protozool 1970;7:117–80. [25] Raabe Z. Ordo Thigmotricha (Ciliata–Holotricha). III. Familiae Ancistrocomidae et Sphenophryidae. Acta Protozool 1970;7:385–463. [26] Raabe Z. Ordo Thigmotricha (Ciliata–Holotricha). IV. Familiae Thigmophryidae. Acta Protozool 1971;9:121–70. [27] Raabe Z. The morphogenetic principles of Sewertzoff, their extension and application to Protozoa. Acta Protozool 1971;9:1–22. [28] Raabe Z. Ordo Thigmotricha (Ciliata–Holotricha). V. Familiae Hysterocinetidae et Fig. 4. Bayesian tree inferred from 18 S rRNA gene sequences. Numbers at nodes Protoanoplophryidae. Acta Protozool 1972;10:115–84. represent posterior probability of Bayesian analysis and the bootstrap percentages for [29] Miao M, Warren A, Song WB, Wang S, Shang HM, Chen ZG. Analysis of the internal maximum parsimony (1000 replicates). Hyphen (-) indicates disagreement between transcribed spacer 2 (ITS2) region of scuticociliates and related taxa (Ciliophora, Bayesian and MP. *, first reported at the name of Philasterides dicentrarchi in Kim et al. Oligohymenophorea) to infer their evolution and phylogeny. Protist 2008;159: [54]. The scale bar corresponds to 5 substitutions per 100 nucleotide positions. Newly 519–33. sequenced species in this work are highlighted in bold. [30] Shang HM, Song WB. Separation and relationship of ten marine scuticociliates (Protozoa, Ciliophora) using RAPD fingerprinting method. Acta Oceanol Sin 2005;24:78–85. to Dr. Hongwei Ma, Gulf Coast Research Laboratory, USM, and Prof. [31] Yi ZZ, Song WB, Gong J, Warren A, Al-Rasheid KAS, Al-Farraj SA, et al. Phylogeny of Xiaozhong Hu, Dr. Chen Shao, Ms. Qianqian Zhang, Laboratory of six oligohymenophoreans (Protozoa, Ciliophora) inferred from small subunit rRNA gene sequences. Zool Scr 2009;38:323–31. Protozoology, OUC, for their kind help in experiment operation and [32] Gao S, Chen ZG, Shao C, Long HA, Al-Rasheid KAS, Song WB. Reconsideration of the comments on the manuscript. phylogenetic position of Frontonia-related Peniculia (Ciliophora, Protozoa) inferred from the small subunit ribosomal RNA gene sequences. Acta Protozool 2008;47:47–54. [33] Wang YG, Miao M, Zhang QQ, Gao S, Song WB, Al-Rasheid KAS, et al. Three marine interstitial scuticociliates, Schizocalyptra similis sp. n., S. sinica sp. n. and References Hippocomos salinus Small and Lynn, 1985 (Ciliophora: Scuticociliatida), isolated from Chinese coastal waters. Acta Protozool 2008;47:377–87. [1] Coats DW, Small EB. Stomatogenesis in the philasterine scuticociliate Philaster [34] Fan XP, Miao M, Al-Rasheid KAS, Song WB. A new marine scuticociliate (Protozoa, hiatti Thompson, 1969. Trans Am Microsc Soc 1976;95:718–25. Ciliophora) from northern China, with a brief note on its phylogenetic position [2] Grolière CA. Étude comparée de la stomatogenèse chez quelques ciliés inferred from small subunit rDNA sequence data. J Euk Microbiol 2009;56:577–82. des genres Paralembus Kahl, 1933 Philaster Fabre-Domergue, [35] Miao M, Wang YG, Li LQ, Al-Rasheid KAS, Song WB. Molecular phylogeny of the 1885 Parauronema Thompson, 1967, Tetrahymena Furgasson, 1940. Protistologica scuticociliate Philaster (Protozoa, Ciliophora) based on SSU rRNA gene sequences 1974;10:319–31. information, with description of a new species P. apodigitiformis sp. n. Syst [3] Thompson JC. A description of Cohnilembus verminus from Eniwetok Atoll. J Biodivers 2009;7:381–8. Protozool 1968;15:396–9. [36] Wilbert N. Eine verbesserte technik der protargolimprä gnation für ciliaten. [4] Wilbert N, Kahan D. Ciliates of Solar Lake on the Red Sea shore. Arch Protistenkd Mikrokosmos 1975;64:171–9. 1981;124:70–95. [37] Yi ZZ, Song WB, Shao C, Warren A, Al-Rasheid KAS, Roberts DM, et al. Phylogeny of [5] Aescht E, Foissner W. Biology of a high-rate activated sludge plant of a some systematically uncertain urostyloids — Apokeronopsis, Metaurostylopsis, pharmaceutical company. Arch Hydrobiol 1992;90(Suppl):207–51. Thigmokeronopsis (Ciliophora, Stichotrichia) estimated with small subunit rRNA [6] Fernandez-Leborans G, Novillo A. Three new species of ciliate in the genera gene sequence information: discrepancies and agreements with morphological Pseudocohnilembus, Pleuronema, and Urotricha (Ciliophora). Proc Biol Soc Wash data. Eur J Protistol 2008;44:254–62. 1994;107:221–38. [38] Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically [7] Foissner W, Adam H, Foissner I. Morphologie, infraciliatur und silberliniensystem amplified eukaryotic 16 S-like rRNA-coding regions. Gene 1988;71:491–9. einiger wenig bekannter Scuticociliatida (Protozoa, Ciliophora). Zool Jb Syst [39] Wuyts J, Van de Peer Y, Winkelmans T, De Wachter R. The European database on 1982;109:443–68. small subunit ribosomal RNA. Nucleic Acids Res 2002;30:183–5. [8] Song WB, Wilbert N. Redefinition and redescription of some marine scuticociliates [40] Eddy SR. HMMER: profile hidden Markov models for biological sequence analysis; from China, with report of a new species, Metanophrys sinensis nov. spec. 2001. Available at http://hmmer.janelia.org/(Accessed on 3 December 2009). (Ciliophora, Scuticociliatida). Zool Anz 2000;239:45–74. [41] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis [9] Wang YG, Hu XZ, Long HA, Al-Rasheid KAS, Al-Farraj SA, Song WB. Morphological program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999;41:95–8. studies indicate that Pleuronema grolierei nov. spec. and P. coronatum Kent, 1881 [42] Yi ZZ, Song WB, Warren A, Roberts DM, Al-Rasheid KA, Chen ZG, et al. A molecular represent different sections of the genus Pleuronema (Ciliophora: Scuticociliatida). phylogenetic investigation of Pseudoamphisiella and Parabirojimia (Protozoa, Eur J Protistol 2008;44:131–40. Ciliophora, Spirotrichea), two genera with ambiguous systematic positions. Eur J [10] Wang YG, Song WB, Warren A, Al-Rasheid KAS, Al-Quraishy SA, Al-Farraj SA, et al. Protistol 2008;44:45–53. Descriptions of two new marine scuticociliates, Pleuronema sinica n. sp. and P. [43] Nylander JA. MrModeltest v2. Uppsala University, 2004. wilberti n. sp. (Ciliophora: Scuticociliatida), from the Yellow Sea, China. Eur J [44] Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under Protistol 2009;45:29–37. mixed models. Bioinformatics 2003;19:1572–4. [11] Song WB, Warren A, Hu XZ. Free-living ciliates in the Bohai and Yellow Seas, China. [45] Guindon S, Gascuel O. A simple, fast and accurate algorithm to estimate large Beijing: Science Press; 2009. phylogenies by maximum likelihood. Syst Biol 2003;52:696–704. [12] Corliss JO. The ciliated Protozoa: characterization, classification and guide to the [46] Swofford DL. PAUP*. Phylogenetic analysis using parsimony (*and other literature. Oxford: Pergamon Press; 1979. methods). Version 4: Sunderland, MA, 2002. [13] Jankowski AW. Conspectus of a new system of the phylum Ciliophora. Tr [47] Song WB. Morphological and taxonomical studies on some marine scuticociliates Zoologiche Ins Leningrad 1980;94:103–21. from China Sea, with description of two new species, Philasterides armatalis sp. n. F. Gao et al. / Parasitology International 59 (2010) 549–555 555

and Cyclidium varibonneti sp. n. (Protozoa: Ciliophora: Scuticociliatida). Acta [51] Jung SJ, Kitamura SI, Song JY, Oh MJ. Miamiensis avidus (Ciliophora: Scuticocilia- Protozool 2000;39:295–322. tida) causes systemic infection of olive flounder Paralichthys olivaceus and is a [48] Xu KD, Song WB. Studies on pathogenetic ciliates from marine molluscs III senior synonym of Philasterides dicentrarchi. Dis Aquat Organ 2007;73:227–34. Thigmotrichine ciliates (Protozoa, Ciliophora, Scuticociliatida). J Ocean Univ [52] Long HA, Song WB, Chen JX, Gong J, Ji DD, Hu XZ, et al. Studies on an endoparasitic Qingdao 2000;30:230–6. ciliate Boveria labialis (Protozoa, Ciliophora) from the sea cucumber, Apostichopus [49] Dragesco A, Dragesco J, Coste F, Gasc C, Romestand B, Raymond J, et al. Philasterides japonicus. J Mar Bio Ass UK 2006;86:823–8. dicentrarchi, n. sp., (Ciliophora, Scuticociliatida), a histophagous opportunistic [53] Fenchel T, Finlay BJ. The diversity of microbes: resurgence of the phenotype. Philos parasite of Dicentrarchus labrax (Linnaeus, 1758), a reared marine fish. Eur J Trans R Soc Lond B Biol Sci 2006;361:1965–73. Protistol 1995;31:327–40. [54] Kim SM, Cho JB, Kim SK, Nam YK, Kim KH. Occurrence of in olive [50] Gomez-Saladin E, Small EB. Oral morphogenesis of the microstome to macrostome flounder Paralichthys olivaceus by Philasterides dicentrarchi (Ciliophora: Scutico- transformation in Miamiensis avidus strain Ma/2. J Euk Microbiol 1993;40:363–70. ciliatida). Dis Aquat Organ 2004;62:233–8.