中国科技论文在线 http://www.paper.edu.cn Zoologica Scripta Molecular evolution of Cinetochilum and Sathrophilus (Protozoa, Ciliophora, Oligohymenophorea), two genera of ciliates with morphological affinities to scuticociliates 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 ciliate 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 hymenostomes. 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, Ocean 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 scuticociliate taxa with obliquely oriented oral hymenostomes (Small 1967; Corliss 1979; Lynn & Small membranelles and hymenostome-like features of somatic 2002; Fan et al. 2009, 2011; Miao et al. 2009b; Wang et al. ciliature (e.g. holotrichous ciliature, postoral kineties) ª 2011 The Authors d Zoologica Scripta ª 2011 The Norwegian Academy of Science and Letters, 40, 3, May 2011, pp 317–325 317 转载 中国科技论文在线 http://www.paper.edu.cn 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 Philasterida (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). 318 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 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