中国科技论文在线 http://www.paper.edu.cn Zoologica Scripta

Molecular evolution of Cinetochilum and Sathrophilus (, Ciliophora, ), two genera of with morphological affinities to

QIANQIAN ZHANG,MIAO MIAO,MICHAELA C. STRU¨ DER-KYPKE,KHALED A. S. AL-RASHEID, SALEH A. AL-FARRAJ &WEIBO SONG

Submitted: 6 October 2010 Zhang, Q., Miao, M., Stru¨der-Kypke, M. C., Al-Rasheid, K. A. S., Al-Farraj, S. A. & Accepted: 29 January 2011 Song, W. (2011). Molecular evolution of Cinetochilum and Sathrophilus (Protozoa, Cilio- doi:10.1111/j.1463-6409.2011.00473.x phora, Oligohymenophorea), two genera of ciliates with morphological affinities to scuti- cociliates. — Zoologica Scripta, 40, 317–325. The order Loxocephalida sensu Li et al. (2006) has been considered to be systemati- cally uncertain within the subclass Scuticociliatia. Loxocephalids display mixed morpholog- ical features and morphogenetic patterns that are found in two different oligohymenophorean subclasses: scuticociliates and . To reveal their phylo- genetic positions, molecular information on this group is urgently needed but still inade- quate. In the present study, we have sequenced the small subunit rRNA gene of two newly described loxocephalids, Cinetochilum ovale Gong & Song 2008; and Sathrophilus planus Fan et al. 2010; which have never been discussed based on molecular analysis. Results show: (i) all phylogenetic trees are nearly identical in placing Cinetochilum closest to the subclass Apostomatia and form a monophyletic group divergent from the typical scuticociliates, (ii) the genus Sathrophilus, together with Anoplophrya, a poorly known Astomatia, forms a peripheral branch separated from the scuticociliatian assemblage and (iii) the affiliation of the loxocephalid genera sensu Li et al. (2006) is not confirmed due to a dispersion in four deeply diverged clades. In addition, the polyphyly of the genus Cyclidium, shown in previ- ous studies, is confirmed by our phylogenetic analyses and supported by the approximately unbiased test based on the new database in this work. Corresponding author: M. Miao, Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, University of China, Qingdao 266003, China. E-mail: [email protected] Q. Zhang and M. Miao, Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China M. C. Stru¨der-Kypke, Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada K. A. S. Al-Rasheid, and Saleh A. Al-Farraj, Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia W. Song, Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean Univer- sity of China, Qingdao 266003, China

Introduction 2009a,b; Gao et al. 2010). In recent studies, the Loxoce- Lynn (2008) divided the ciliate class Oligohymenophorea phalida sensu Li et al. 2006, an order in the subclass Scuti- into six subclasses: Scuticociliatia, Peritrichia, Hymenos- cociliatia, has become the subject of great importance to tomatia, Peniculia, Apostomatia and Astomatia. Over the researchers interested in the phylogeny of class Oligohy- past 50 years, various studies have been carried out on the menophorea (Song et al. 2005; Li et al. 2006, 2010; Miao morphology, morphogenetic and phylogeny of this class, et al. 2010; Yi et al. 2010; Zhang et al. 2010). The order especially of the two main groups, scuticociliates and includes all taxa with obliquely oriented oral hymenostomes (Small 1967; Corliss 1979; Lynn & Small membranelles and -like features of somatic 2002; Fan et al. 2009, 2011; Miao et al. 2009b; Wang et al. ciliature (e.g. holotrichous ciliature, postoral kineties)

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Phylogeny of Cinetochilum and Sathrophilus d Q. Zhang et al.

(Song et al. 2005; Li et al. 2006). Previous morphogenetic cally as loxocephalids nor their association with the Hyme- and small subunit (SSU) rRNA gene sequence studies sug- nostomatia has been well supported (Zhang et al. 2010). In gested that some loxocephalids, (i.e. Dexiotrichides, Paratet- spite of their importance in the evolutionary hypothesis, rahymena, Cardiostomatella) may be phylogenetically placed the group is relatively controversial due to insufficient between typical scuticociliates and the subclass Hymenos- molecular and stomatogenetic evidence and also the ambi- tomatia (Song et al. 2005; Li et al. 2006; Fan et al. 2009; guity of morphological and ⁄ or morphogenetical features Yi et al. 2009). However, more recent molecular studies in key taxa, e.g. Paratetrahymena and Cardiostomatella (Yi including richer taxa and detailed analyses suggested that et al. 2009; Li et al. 2010; Zhang et al. 2010). the inter-related genera (including Sathrophilus, Paratetra- The psammophilic genus Cinetochilum was classified as a hymena, Cardiostomatella, Dexiotrichides), except for Dexio- member of the order (Corliss 1979; Lynn & trichides, are affiliated with the scuticociliates as a diverse Small 2002; Lynn 2008). This systematic position is group generally basal to the orders Philasterida and Pleu- questionable because Cinetochilum possesses an oral appa- ronematida (Li et al. 2010; Zhang et al. 2010). Neither the ratus with transversely positioned oral membranelles homogeneity of ciliates that can be classified morphologi- (Fig. 1A,B), which distinguishes it from other typical

AB E

CD

F

Fig. 1 Infraciliature of Cinetochilum ovale and Sathrophilus planus from life (A, C) and after protargol (B, D), showing their specific morphological features. —A, B. Cinetochilum ovale (after Gong & Song 2008); —C, D. Sathrophilus planus (after Fan et al. 2010). M1–3, membranelle 1–3; PF, postoral kinetofragments; PK, postoral kinety; PM, paroral membrane; Sc, scutica; SK1, n, somatic kinety1, n. Scale bars in A, B = 10 lm; in C = 30 lm; in D = 40 lm. —E. Maximum likelihood phylogeny of small subunit (SSU) rRNA genes. —F. Maximum parsimony phylogeny of SSU rRNA genes. Numbers on branches are values generated from 1000 bootstrap replicates. Full support numbers are symbolized by a black dot. Species sequenced in the present study are shown in bold type, while Loxocephalida sensu Li et al. (2006) are highlighted in grey. Systematic classification follows Lynn (2008).

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Q. Zhang et al. d Phylogeny of Cinetochilum and Sathrophilus

philasterids (Song et al. 2005). However, the phylogenetic extension for 10 min). The purified PCR product of the placement of the genus Cinetochilum has never been dis- expected size (1.8 kb) was cloned into the pUCm-T vec- cussed because neither ontogenetic nor molecular informa- tor (Sangon, Shanghai, China) and cultured in Escherichia tion was available. coli DH5a cells. Cultures transformed overnight were Similarly, the genus Sathrophilus is also a candidate for detected by PCR amplifications using the M13F and the order Loxocephalida and is one of the loxocephalid M13R primers (Miao et al. 2009a). Sequencing reactions genera originally placed in the scuticociliate order Philas- were performed (Sangon sequencing facility, Shanghai, terida (Fig. 1C,D). The phylogenetic position of this China) using M13F and M13R primers. genus has primarily been discussed based on the molecular data of its species, S. holtae (Zhang et al. 2010). It diverges Phylogenetic analyses based on sequences of SSU rRNA at a deep level from all scuticociliates, sister to the com- gene plex comprised of other two loxocephalids — Paratetra- Sequences for all other ciliates used in this analysis were hymena and Cardiostomatella. Nevertheless, these three from the GenBank ⁄ EMBL database. Sequences were species did not show close relationships to another loxo- aligned with CLUSTAL W V. 1.83 (Thompson et al. 1994) cephalid genus, Dexiotrichides. Therefore, the phylogenetic and the alignment was refined manually to excise highly position of this genus is still considered as controversial variable regions using BIOEDIT (Hall 1999). The final and more samples of taxa from this genus as well as from alignment comprised 90 taxa and 1676 nucleotide posi- the loxocephalid group are required. tions. A Bayesian inference (BI) approach as imple- Indeed, taxon sampling plays an important role in phy- mented in MRBAYES V. 3.1.2 (Ronquist & Huelsenbeck logenetic analyses, and here we provide sequences of two 2003) was used to infer phylogenetic relationships using scuticociliates with transversely positioned oral membran- the GTR+I+G evolutionary model selected under the elles, Sathrophilus planus and Cinetochilum ovale, with the AIC criterion by MRMODELTEST V. 2 (Nylander 2004). aim to assess their phylogenetic positions as well as the Markov chain Monte Carlo simulations were run for systematic assignment of the order Loxocephalida. 2 000 000 generations, with two parallel runs and each run with four simultaneous chains, using a sample fre- Materials and methods quency of 100 and a burn-in of 5000 trees. A maximum Sample collection, observation and identification likelihood (ML) tree was constructed with PHYML V. Cinetochilum ovale Gong & Song 2008 was isolated on 13 2.4.4 (Guindon & Gascuel 2003), using the GTR+I+G November 2008 from Jiao Zhou Bay, Qingdao, China model selected by MODELTEST (Posada & Crandall 1998) (3604¢N; 12018¢E), from a water sample with a tempera- under the AIC criterion, with the proportion of invari- ture of 18 C, pH 8.0 and salinity 31&. Microscopy able sites (I) = 0.32 and gamma distribution shape observations, impregnation and terminology were accord- parameter (G) = 0.56. A maximum-parsimony (MP) tree ing to Wilbert (1975) and Pan et al. (2010), and identifica- was calculated by a heuristic search with all characters tion was based on Gong & Song (2008). Sathrophilus coded as unordered in PAUP* V. 4.0b10 (Swofford 2002); planus Fan et al. 2010, was sampled from the upper 0– 732 characters were parsimony informative and used to 4 cm sand layer from the coast of the Second Bathing find the most parsimonious tree using the heuristic Beach (3603¢N; 12021¢E) of Qingdao in April 2009. search option. Parsimony data were bootstrap-resampled Specimens were collected, processed and identified as 1000 times. Eight species of the classes Colpodea, Plagi- described by Fan et al. (2010). Terminology and systematics opylea and Prostomatea were selected as outgroup in are according to Lynn (2008). the analyses. The support of the data set for competing phylogenetic DNA extraction, PCR amplification and sequencing hypotheses was evaluated using approximately unbiased Genomic DNA was extracted from the washed cells using (AU) tests (Shimodaira 2002) to test the monophyly of the a REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, order Loxocephalida. The ML tree was generated with a MO, USA) according to the manufacturer’s instructions constraint block in PAUP (Swofford 2002), under a (Yi et al. 2009). Primers for SSU rRNA gene amplification GTR+G+I model, which was identical to the one used for ciliates were EukA (5¢-AACCTGGTTGATCCTGC when estimating the global ML tree. The same model was CAGT-3¢) and EukB (5¢-TGATCCTTCTGCAGGTT used to estimate the site likelihoods for those trees prior to CACCTAC-3¢) (Medlin et al. 1988). PCR conditions were doing the AU test. Scores of each constraint tree were com- as follows: 30 cycles with denaturation at 94 C for 30 s pared with the unconstrained ML result using the AU test (initial denaturation for 5 min), primer annealing at 52 C option implemented in CONSEL (Shimodaira & Hasegawa for 1 min and primer extension at 72 C for 1 min (final 2001).

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Phylogeny of Cinetochilum and Sathrophilus d Q. Zhang et al.

Results Gene sequence and distance analyses Deposition and comparison of sequences Trees constructed by all three methods had similar topolo- The complete SSrRNA gene sequences are as follows: gies but differed in some details (Fig. 1E,F and 2). In all Cinetochilum ovale (accession no. FJ870103) 1752 nucleo- trees, C. ovale clustered with the subclass Apostomatia in a tides in length and GC content of 44.75%; Sathrophilus well-supported clade (1.00 BI, 97% ML, 89% MP) but planus (accession no. FJ868186) 1762 nucleotides in length was unresolved relative to nearby clades. It was a sister and GC content of 43.76%. These values are in the same clade to the typical scuticociliates in the orders Philasteri- range as those reported for related ciliates (Yi et al. 2009; da and and a clade containing the genera Li et al. 2010; Zhang et al. 2010). However, the sequence Sathrophilus and Anoplophrya in the BI tree with low pos- of S. planus differs from S. holtae by 83 nucleotides repre- terior probability (0.54); was grouped with clades contain- senting a divergence of 5% (published in Zhang et al. ing Cardiostomatella and Paratetrahymena, Dexiotrichides and 2010), which suggests a new species in Sathrophilus (Fan Urocentum and Cyclidium porcatum in the ML tree with et al. 2010). similarly low bootstrap support (22%); and was completely

Fig. 2 Phylogenetic tree of small subunit rRNA gene sequences showing the positions of Cinetochilum and Sathrophilus, by Bayesian inferences applying the GTR+G+I model. Species sequenced in the present study are shown in bold type, while Loxocephalida sensu Li et al. (2006) are highlighted in grey and noted with arrows. Numbers near branches are posterior probability values. Full support numbers are symbolized by a black dot. Asterisks (*) mark three nominal Cyclidium species, which fall in three different positions. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. Systematic classification follows Lynn (2008).

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Q. Zhang et al. d Phylogeny of Cinetochilum and Sathrophilus

unresolved relative to all of these other taxa in the MP ments (PF), a structure very similar to the postoral kinety tree. The newly sequenced species S. planus clustered with (PK) of other loxocephalids to some extent, making it S. holtae with maximum support in every tree. Sathrophilus unique compared with other scuticociliates (no PF or PK clustered with the astomatian Anoplophrya marylandensis, for almost all known Philasterida and Pleuronematida with moderate to low support in BI and ML trees (0.70 taxa). This feature is obviously present in the infraciliature BI, 37% ML) but was unresolved relative to all nearby of C. ovale (Fig. 1A,B; Gong & Song 2008). taxa in the MP tree. In turn, the Sathrophilus–Anoplophrya In our study based on the SSU rRNA gene sequence clade was sister to the typical scuticociliates and the Cine- information, C. ovale is most closely related to taxa of the tochilum–Apostomatia clade in the BI tree with low poster- subclass Apostomatia (Fig. 1E,F and 2). The widely ior probability (0.54) but was sister to the typical accepted placement of Cinetochilum in the subclass Scutico- scuticociliates and a diverse assemblage of taxa comprised ciliatia (Lynn 2008) is not supported. The Cinetochilum– of apostomes, loxocephalids, Urocentrum and Cyclidium in Apostomatia clade is clearly separated from other typical the ML tree with moderate bootstrap support (70% ML). scuticociliates and groups with several clades of loxocepha- Other loxocephalid taxa sensu Li et al. (2006) (Paratetra- lid genera (i.e. Cardiostomatella–Paratetrahymena, Dexiotric- hymena, Cardiostomatella, Dexiotrichides) and the putative lo- hides–Urocentum and Sathrophilus). The assignment of xocephalid C. porcatum (Zhang et al. 2010) were basal to C. ovale to the order Loxocephalida, as suggested by Li and divergent from typical scuticociliates (orders Philaste- et al. (2006), is not supported by our trees, as it never rida and Pleuronematida) in all trees but also very diver- grouped individually with any loxocephalid groups. gent from each other (Fig. 1E,F and 2). Cardiostomatella In terms of the result of the AU test (Table 1), the and Paratetrahymena were closely related in a moderately topology was rejected (AU < 0.05) for trees in which to well-supported clade (1.00 BI, 72% ML, 73% MP) in all trees, but the position of Dexiotrichides was not stable. Table 1 Approximately unbiased (AU) results comparing trees Dexiotrichides and Urocentrum, currently classified as peni- representative of alternative hypotheses about the phylogenetic culid, were basal to hymenostomes and peritrichia in the associations of focal groups, results in which P < 0.05 are shaded BI tree with strong posterior probability (0.99); however Dexiotrichides was unresolved relative to other taxa and Hypotheses tested -ln L AU

Urocentrum was associated with hymenostomes and peri- Loxocephalida 31537.7036 0.015 trichs with only weak bootstrap support (57%) in the MP Loxocephalida without Cinetochilum ovale 31481.10941 0.203 tree. By contrast, Dexiotrichides associated with Urocentrum Cinetochilum ovale + Apostomatia + other 31490.91108 0.259 in a weakly supported clade (70% bootstrap value), which Loxocephalida Loxocephalida + typical Scuticociliatia 31551.12306 0.012 in turn formed a poorly supported sister group (31%) to Loxocephalida without Cinetochilum 31475.07045 0.406 the loxocephalid Cardiostomatella–Paratetrahymena clade in ovale + typical Scuticociliatia the ML tree. C. porcatum was in an unresolved position Loxocephalida + Hymenostomatia 31632.58846 <0.001 near the Cardiostomatella–Paratetrahymena clade in the BI Loxocephalida without Cinetochilum ovale 31577.33891 0.003 tree and basal to the same clade in the ML tree. It was in + Hymenostomatia Cinetochilum ovale + Sathrophilus spp. 31540.78622 0.007 the large assemblage of unresolved scuticociliate, loxo- Sathrophilus spp. + Cinetochilum ovale and 31493.75608 0.18 cephalid, apostomatian, and astomatian taxa in the MP Apostomatia tree. Two other species of Cyclidium fell within the clade Dexiotrichides pangi + Paratetrahymena 31471.65241 0.372 of pleuronematid scuticociliates with maximal support but spp. + Cardiostomatella vermiformis in divergent positions. Cinetochilum ovale + typical Scuticociliatia 31526.19803 0.034 Cinetochilum ovale + Apostomatia + typical 31475.0015 0.373 Scuticociliatia Discussion Cinetochilum ovale + Hymenostomatia 31568.53449 0.008 The systematic position of Cinetochilum: are there Cinetochilum ovale + Apostomatia + 31535.77699 0.01 relationships between Cinetochilum and the subclass Hymenostomatia Apostomatia Sathrophilus + typical Scuticociliatia 31498.73068 0.150 Sathrophilus spp. + Hymenostomatia 31593.22963 <0.001 Morphologically, C. ovale resembles most typical scutico- Cyclidium spp. 31740.07138 <0.001 ciliates by having a scutica and an apical plate (Gong & C. porcatum + loxocephalida 31526.50606 0.035 Song 2008); however, its transversely positioned oral C. porcatum + loxocephalida + Apostomatia 31476.36793 0.791 membranelles differ from the expansive membranelles of C. porcatum + typical Scuticociliatia 31489.71428 0.346 many scuticociliates and are more like those of loxocepha- Cyclidium spp. + other pleuronematid spp. 31510.85298 0.005 lids (e.g. Paratetrahymena). In addition, some species like Loxocephalida: Cinetochilum ovale + Sathrophilus spp. + Dexiotrichides C. ovale and C. australiense possess postoral kinetofrag- pangi + Paratetrahymena spp. + Cardiostomatella vermiformis

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Phylogeny of Cinetochilum and Sathrophilus d Q. Zhang et al.

C. ovale was individually included in the clades of Loxoce- reans. Bradbury (1989) suggested that similarities in the phalida, typical Scuticociliatia or Hymenostomatia. Fur- life cycles and the somatic ciliary ultrastructure of hyme- thermore, the grouping of the C. ovale–Apostomatia nostomes and apostomes justified a placement of the branch together with the Hymenostomatia was rejected apostome ciliates as a suborder within the order Hyme- (AU < 0.05). However, a branching of the clade C. ovale– nostomatida. Phylogenetic analyses inferred from SSU Apostomatia together with other species of the order Lo- rRNA gene sequence data supported the monophyly of xocephalida or with the typical Scuticociliatia cannot be apostomates and revealed sufficient genetic divergence to rejected. Based on this result, we could hypothesize that other subclasses to maintain this group as a separate the subclasses Scuticociliatia and Apostomatia are closely subclass of the Oligohymenophorea (Clamp et al. 2008). related, with the genus Cinetochilum being an evolutionary Our phylogenetic analyses support these results: the link between them. apostome clade is distinctly monophyletic and shows a According to Lynn (2008), species in the subclass deep divergence (1.00 BI, 100% ML, 100% MP). In Apostomatia are ciliates with a polymorphic life cycle; addition, we also find 20 nucleotide positions that are usually epibionts of marine . The tomites of uniquely shared among apostomes (Fig. 3). Among these species have somatic monokinetids with an ultra- these, 10 sites, located in the stem regions of the sec- structural pattern that resembles other oligohymenopho- ondary structure of SSU rRNA gene (Peer de et al.

A

B

C

Fig. 3 Twenty-seven parsimony-informative positions of the alignment of nuclear small subunit (SSU) rRNA gene sequences of scuticociliate, apostome, loxocephalid, and hymenostome genera from which the trees in Figs. 1E,F and 2 were derived. Positions of homologous nucleotides in the alignment are given at the top of each column. Homologous groups of nucleotides between Cinetochilum ovale and species of subclass Apostomatia are shown in light grey boxes. Semi conserved positions and homologous groups among apostomes are indicated by grey and dark grey boxes. Species in subclass Apostomatia are shown within a box in the list of taxa. Other groups are noted with brackets: —A. typical species of the subclass Scuticociliatia; —B. order Loxocephalida; —C. subclass Hymenostomatia. *Nucleotides that locate on the stems of the secondary structure according to the SSU rRNA secondary structure model of canadensis (Peer de et al. 2000).

322 ª 2011 The Authors d Zoologica Scripta ª 2011 The Norwegian Academy of Science and Letters, 40, 3, May 2011, pp 317–325 中国科技论文在线 http://www.paper.edu.cn

Q. Zhang et al. d Phylogeny of Cinetochilum and Sathrophilus

2000), have important effect on the branching topology data set (e.g. Loxocephalus, Dexiotricha, Platynematum and of the tree. Cristigera), this cluster is very likely artifactual. An increase Moreover, the present work strongly argues a hypothesis of species include in future analyses might provide a better that Apostomatia may have close relationship to the loxo- degree of resolution. The association between Sathrophilus cephalid species C. ovale. Apostomes tomites have three spp. and the typical scuticociliates is not rejected kineties (kineties x, y, z) in essentially a postoral position, (AU > 0.1) by the AU test (Table 1), whereas a relationship among which the anterior part of the x kinety was identi- between Sathrophilus spp. and the hymenostomes is rejected fied as the primordium for the oral organelle during the (AU < 0.001); this corresponds well to the observations of stomatogenetic. In the morphogenetic descriptions of dif- Zhang et al. (2010) that Sathrophilus is more closely related ferent species of apostomes (Chatton & Lwoff 1935), the to scuticociliates than to hymenostomes. x, y, z, kineties of some taxa show remarkable similarity to the three PF of Cinetochilum in terms of their position and Phylogenetic position of the order Loxocephalida and its appearance. We could assume that the PF of Cinetochilum branching pattern may be homologues of the x, y, z kineties of apostomates. The AU test results show that the hypothesis of a mono- However, stomatogenetic data of the Cinetochilum are phyly of the order Loxocephalida is rejected with this data needed to give further indication on this point. set (AU < 0.05; Table 1). However, the monophyly of The apostome species are highly divergent compared loxocephalids without Cinetochilum or the assemblage of with other groups (Fig. 3). To avoid the possibility of loxocephalids and apostomes cannot be rejected. Long-Branch Artifact effect from the out-group (classes Lynn (2008) placed the genera Sathrophilus and Cineto- Colpodea, Plagiopylea and Prostomatea), we performed a chilum together in the family Cinetochilidae, and Paratet- second subset of analyses including only the scuticociliate, rahymena, Cardiostomatella and Dexiotrichides in the family hymenostome, apostome and species; the topology Loxocephalidae. This familial assignment was not for the Apostomatia and Cinetochilum clade did not differ supported by our molecular analyses. Cinetochilum and in the resulting trees (data not shown). Additionally, Cine- subclass Apostomatia formed a distinct well-supported tochilum and the apostome species share four signature nu- clade, clearly separated from the genus Sathrophilus. The cleotides and they also share five nucleotides that exist cluster of Paratetrahymena and Cardiostomatella supports otherwise only in certain loxocephalid species (Fig. 3). the concept of the family Loxocephalidae, with the The results of AU tests also indicate the rejection of any exception of Dexiotrichides, which groups with Urocentrum. hypothesis that separates Cinetochilum and the apostomes The AU test performed also does not reject a cluster of (Table 1). This and the high support values for the node Paratetrahymena, Cardiostomatella and Dexiotrichides. How- (1.00 BI, 97% ML, 89% MP) strengthen the hypothesis of ever, an assemblage of Sathrophilus and Cinetochilum is an association between Cinetochilum and the apostomes as rejected, whereas the hypothesis of a cluster for Cinetochi- suggested by the SSU rRNA gene analyses. lum, Sathrophilus and the apostomes is not rejected (Table 1). Phylogenetic position of Sathrophilus All rejections of constraint topologies among the loxo- The newly sequenced S. planus groups well with its conge- cephalids seem to be based on the inclusion of the genus ner S. holtae, corresponding to the high resemblance of the Cinetochilum, suggesting that it is dissimilar from other lo- somatic ciliary pattern (Fan et al. 2010). This clade xocephalids. Considering the molecular phylogeny of loxo- branches off as one of the peripheral clades of subclass Scu- cephalids presented so far, it is still at a comparatively ticociliatia in all our trees, which questions the placement early stage (only 5 of 13 proposed genera), consisting of a of Sathrophilus as a member of the order Philasterida (Lynn modest number of sequences of only one gene. At this 2008). Our results partly agree with a previous study based point, we cannot draw any conclusions with regard to the on the morphology, ontogeny and SSU rRNA gene order Loxocephalida — neither on its monophyly nor on sequence of its other species S. holtae (Long et al. 2007; the genera included in it. More data clearly need to be Zhang et al. 2010). The genus clusters as a distinguished added. form, separated from the typical scuticociliates (Zhang et al. 2010). Sathrophilus does not show close relationship Tests with new database for the genus Cyclidium with any other clade in the phylogenetic analyses, as its The positions of three Cyclidium species in our trees were clustering with the astome species A. marylandensis is not markedly divergent (Fig. 2), corresponding to the most supported (0.70 BI, 37% ML). As the subclass Astomatia is recent molecular analyses (Zhang et al. 2010) and also sup- represented by only one species, and some major taxa of porting the previous morphologic data, which have the order Loxocephalida are still missing in the current revealed a polyphyletic nature of the genus Cyclidium (Lynn

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Phylogeny of Cinetochilum and Sathrophilus d Q. Zhang et al.

& Stru¨der-Kypke 2005). We have tested the hypotheses for and Boveria (Protozoa, Ciliophora) based on the phylogeny of the genus Cyclidium based on our new 18S rRNA gene sequences. Parasitology International, 59, 549– database with the addition of C. ovale and S. planus. The 555. Gong, J. & Song, W. (2008). Morphology and infraciliature of a hypothesis that Cyclidium spp. are monophyletic is rejected new marine ciliate, Cinetochilum ovale n. sp. (Ciliophora: by the AU test (AU < 0.001; Table 1). The association Oligohymenophorea). Zootaxa, 1939, 51–57. of all three Cyclidium spp. with the order Pleuronematida is Guindon, S. & Gascuel, O. (2003). A simple, fast and accurate also rejected (AU < 0.05; Table 1), mainly due to the algorithm to estimate large phylogenies by maximum inclusion of C. porcatum. On the contrary, an association of likelihood. Systematic Biology, 52, 696–704. C. porcatum with either the putative loxocephalids (includ- Hall, T. A. (1999). BioEdit: a user-friendly biological sequence ing Apostomatia) or typical scuticociliates (Philasterida and alignment editor and analysis program for Windows 95 ⁄ 98 ⁄ NT. Nucleic Acids Symposium Series, 41, 95–98. Pleuronematida) is not rejected (Table 1). Li, L., Song, W., Warren, A., Wang, Y., Ma, H., Hu, X. & Chen, Z. (2006). Phylogenetic position of the marine ciliate, Acknowledgements Cardiostomatella vermiforme (Kahl, 1928) Corliss, 1960 inferred This work was supported by the National Natural Science from the complete SSrRNA gene sequence, with establishment Foundation of China (project nos 31030059 and of a new order Loxocephalida n. ord (Ciliophora, 40976075) and the Centre of Excellence in Biodiversity, Oligohymenophorea). European Journal of Protistology, 42, 107– King Saud University. Many thanks are due to Mr Hon- 114. Li, J., Lin, X., Yi, Z., Liu, W. & Al-Rasheid, K. A. S. (2010). gbo Pan and Ms Jie Huang, graduate students of the Lab- Molecules or morphogenesis: how to determine the oratory of Protozoology, OUC, for the help in material phylogenetic assignment of Paratetrahymena (Protozoa, identification and gene sequencing. We also thank the Ciliophora, Oligohymenophorea)? Zoologica Scripta, 39, 499– anonymous reviewers for their constructive criticism on 510. the first version of this manuscript. Long, H., Song, W., Warren, A., Al-Rasheid, K., Gong, J. & Chen, X. (2007). Two new ciliates from the North China Seas, References Schizocalyptra aeschtae nov. spec. and Sathrophilus holtae nov. Bradbury, P. C. (1989). Evidence for hymenostome affinities in an spec., with new definition of the genus Sathrophilus (Ciliophora, apostome ciliate. Journal of Protozoology, 36, 95–103. Oligohymenophora). Acta Protozoologica, 46, 229–245. Chatton, E. & Lwoff, A. (1935). Les cilie´s apostomes I. Aperc¸u Lynn, D. H. (2008). The Ciliated Protozoa: Characterization, historique et ge´ne´ral; e´tude monographique des genres et des Classification and Guide to the Literature. Dordrecht: Springer. espe`ces. Archives de Zoologie Expe´rimentale et Ge´ne´rale, 77,1– Lynn, D. H. & Small, E. B. (2002). Phylum Ciliophora Doflein, 453. 1901. In J. J. Lee, G. F. Leedale & P. Bradbury (Eds) An Clamp, J. C., Bradbury, P., Struder-Kypke, M. & Lynn, D. Illustrated Guide to the Protozoa (pp. 371–656). Lawrence, KS: (2008). Phylogenetic position of the apostome ciliates (phylum Society of Protozoologists. Ciliophora, subclass Apostomatia) tested using small subunit Lynn, D. H. & Stru¨der-Kypke, M. (2005). Scuticociliate rRNA gene sequences. Denisia, 23, 395–402. endosymbionts of echinoids (phylum Echinodermata): Corliss, J. O. (1979). The Ciliated Protozoa: Characterization, phylogenetic relationships among species in the genera Classification and Guide to the Literature. Oxford, New York: Entodiscus, Plagiopyliella, Thyrophylax and Entorhipidium (phylum Pergamon Press. Ciliophora). Journal of Parasitology, 91, 1190–1199. Fan, X., Miao, M., Al-Rasheid, K. A. S. & Song, W. (2009). A Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. (1988). The new genus of marine scuticociliate (Protozoa, Ciliophora) from characterization of enzymatically amplified eukaryotic 16S-like Northern China, with a brief note on its phylogenetic position rRNA-coding regions. Gene, 71, 491–499. inferred from small subunit ribosomal DNA sequence data. Miao, M., Song, W., Clamp, J. C., Al-Resehid, K. A. S., Journal of Eukaryotic Microbiology, 56, 577–582. Al-Khedhairy, A. A. & Al-Arifi, S. (2009a). Further Fan, X., Chen, X., Al-Rasheid, K. A. S., Warren, A. & Song, W. consideration of the phylogeny of some ‘traditional’ (2010). Two new ciliates from coastal water of north China, heterotrichs (Protista, Ciliophora) of uncertain affinities, based Sathrophilus planus n. sp. and Pseudoplatynematum willii n. sp., on new sequences of the small subunit rRNA gene. Journal of with new definition of the genus Pseudoplatynematum Eukaryotic Microbiology, 56, 244–250. (Ciliophora, Oligohymenophora). European Journal of Miao, M., Wang, Y., Li, L., Al-Rasheid, K. A. S. & Song, W. Protistology, 46, 212–220. (2009b). Molecular phylogeny of the scuticociliate Philaster Fan, X., Chen, X., Song, W., Al-Rasheid, K. A. S. & Warren, A. (Protozoa, Ciliophora) based on SSU rRNA gene sequences (2011). Two new marine Frontonia species, F. mengi spec. nov. information with description of a new species P. apodigitiformis and F. magna spec. nov (Protozoa; Ciliophora), with notes on sp. n. Systematics and Biodiversity, 7, 381–388. their phylogeny based on SSU rRNA gene sequence data. Miao, M., Wang, Y., Song, W., Clamp, J. C. & Al-Rasheid, K. A. International Journal of Systematic and Evolutionary Microbiology, S. (2010). Description of Eurystomatella sinica n. gen., n. sp., In press. with establishment of a new family Eurystomatellidae n. fam Gao, F., Fan, X., Yi, Z., Stru¨der-Kypke, M. & Song, W. (2010). (Protista, Ciliophora, Scuticociliatia) and analyses of its Phylogenetic consideration of two scuticociliate genera, phylogeny inferred from sequences of the small-subunit rRNA

324 ª 2011 The Authors d Zoologica Scripta ª 2011 The Norwegian Academy of Science and Letters, 40, 3, May 2011, pp 317–325 中国科技论文在线 http://www.paper.edu.cn

Q. Zhang et al. d Phylogeny of Cinetochilum and Sathrophilus

gene. International Journal of Systematic and Evolutionary Thompson, J. D., Higgins, D. G. & Gilbson, T. J. (1994). Microbiology, 60, 460–468. CLUSTAL W: improving the sensitivity of progressive Nylander, J. A. (2004). MrModeltest v2. Uppsala: Uppsala multiple sequence alignment through sequence weighting, University. positions-specific gap penalties and weight matrix choice. Pan, H., Huang, J., Hu, X., Fan, X., Al-Rasheid, K. A. S. & Nucleic Acids Research, 22, 4673–4680. Song, W. (2010). Morphology and SSU rRNA gene sequences Wang, Y., Miao, M., Zhang, Q., Gao, S., Song, W., Al-Rasheid, of three marine ciliates from Yellow Sea, China, including one K., Warren, A. & Ma, H. (2009a). Three marine interstitial new species, heteromarinum nov. spec (Ciliophora, scuticociliates, Schizocalyptra similis sp. n., S. sinica sp. n. and Scuticociliatida). Acta Protozoologica, 49, 45–59. Hippocomos salinus Small and Lynn, 1985 (Ciliophora: Peer de, Y. V., Rijk, P. D., Wuyts, J., Winkelmans, T. & Scuticociliatida), isolated from Chinese coastal waters. Acta Wachter, R. D. (2000). The European small subunit ribosomal Protozoologica, 47, 377–387. RNA database. Nucleic Acids Research, 28, 175–176. Wang, Y., Song, W., Warren, A., Al-Rasheid, K., Al-Quraishy, S. Posada, D. & Crandall, K. A. (1998). Model Test: testing the A., Al-Farraj, S. A., Hu, X. & Pan, H. (2009b). Descriptions of model of DNA substitution. Bioinformatics, 14, 817–818. two new marine scuticociliates, Pleuronema sinica n. sp. and P. Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian wilberti n. sp (Ciliophora: Scuticociliatida), from the Yellow phylogenetic inference under mixed models. Bioinformatics, 19, Sea, China. European Journal of Protistology, 45, 29–37. 1572–1574. Wilbert, N. (1975). Eine verbesserte Technik der Shimodaira, H. (2002). An approximately unbiased test of Protargolimpragnation fu¨r Ciliaten. Mikrokosmos, 64, 171–179. phylogenetic tree selection. Systematic Biology, 51, 492–508. Yi, Z., Song, W., Gong, J., Warren, A., Al-Rasheid, K., Al-Farraj, Shimodaira, H. & Hasegawa, M. (2001). Consel: for assessing the S. A. & Al-Khedhairy, A. (2009). Phylogeny of six confidence of phylogenetic tree selection. Bioinformatics, 17, oligohymenophoreans (Protozoa, Ciliophora) inferred from 1246–1247. small subunit rRNA gene sequences. Zoologica Scripta, 38, 323– Small, E. B. (1967). The Scuticociliatida, a new order of the class 331. Ciliatea (phylum Protozoa, subphylum Ciliophora). Transactions Yi, Z., Wang, Y., Lin, X., Al-Rasheid, K. A. S. & Song, W. of the American Microscopical Society, 86, 345–370. (2010). Analysis of the phylogeny of the subclass Scuticociliatia Song, W., Warren, A., Roberts, D., Ma, H., Shang, H., Wilbert, (Protozoa, Ciliophora) using combined data inferred from N., Miao, M. & Yi, Z. (2005). Re-evaluation of the genetic, morphological and morphogenetic evidence. Chinese phylogenetic position of the genus Dexiotrichides (Protozoa, Journal of Oceanology and Limnology, 28, 778–784. Ciliophora, Scuticociliatida) inferred from stomatogenetic and Zhang, Q., Fan, X., Clamp, J. C., Yi, Z., Al-Rasheid, K. A. S. & molecular information for Dexiotrichides pangi. Progress Natural Song, W. (2010). Description of Paratetrahymena parawassi n. sp. Science, 15, 700–707. using morphological and molecular evidence and a phylogenetic Swofford, D. L. (2002). PAUP*. Phylogenetic Analysis Using analysis of the taxonomically ambiguous genera Sathrophilus and Parsimony (*and Other Methods). Version 4. Sunderland, MA: Paratetrahymena (Ciliophora, Oligohymenophorea. Journal of Sinauer Associates. Eukaryotic Microbiology, 57, 483–493.

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