中国科技论文在线 http://www.paper.edu.cn Zoologica Scripta Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus (Alveolata, Ciliophora) SHAN GAO,MICHAELA C. STRU¨ DER-KYPKE,KHALED A. S. AL-RASHEID,XIAOFENG LIN &WEIBO SONG Submitted: 12 May 2009 Gao, S., Stru¨der-Kypke, M.C., Al-Rasheid, K.A.S., Lin, X. & Song, W. (2010). Molecular Accepted: 31 October 2009 phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus doi:10.1111/j.1463-6409.2010.00416.x (Alveolata, Ciliophora).—Zoologica Scripta, 39, 305–313. Very few molecular studies on the phylogeny of the karyorelictean ciliates have been car- ried out because data of this highly ambiguous group are extremely scarce. In the present study, we sequenced the small subunit ribosomal RNA genes of three morphospecies rep- resenting two karyorelictean genera, Kentrophoros, Trachelolophos, and one haptorid, Trache- lotractus, isolated from the South and East China Seas. The phylogenetic trees constructed using Bayesian inference, maximum likelihood, maximum parsimony and neighbor-joining methods yielded essentially similar topologies. The class Karyorelictea is depicted as a monophyletic clade, closely related to the class Heterotrichea. The generic concept of the family Trachelocercidae is confirmed by the clustering of Trachelolophos and Tracheloraphis with high bootstrap support; nevertheless, the order Loxodida is paraphyletic. The transfer of the morphotype Trachelocerca entzi Kahl, 1927 to the class Litostomatea and into the new haptorid genus Trachelotractus, as suggested by previous researchers based on morpho- logical studies, is consistently supported by our molecular analyses. In addition, the poorly known species Parduczia orbis occupies a well-supported position basal to the Geleia clade, justifying the separation of these genera from one another. Corresponding author: Weibo Song, Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China. Tel: +86 532 8203 2283; E-mail: [email protected] Shan Gao & Weibo Song, Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China. E-mail: [email protected], [email protected] Michaela C. Stru¨der-Kypke, Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: [email protected] Khaled A. S. Al-Rasheid, Zoology Department, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia. E-mail: [email protected] Xiaofeng Lin, Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou, 510631, China. E-mail: [email protected] Introduction Dragesco (1996a,b) and Foissner & AL-Rasheid (1999a,b) Karyorelictean ciliates play an important role in under- obtained excellent preparations from all main groups of standing nuclear dimorphism and phylogeny of ciliates karyorelicteans and revealed a world of new details using a because their diploid macronuclei are non-dividing and ‘strong’ fixative and Wilbert’s protargol technique (Wil- originate from micronuclei during and after each cell divi- bert 1975). sion (Corliss 1974; Lynn & Small 1997). However, mor- Until now, numerous conflicting classifications based on phological analyses of the karyorelicteans were limited for morphological, developmental or ultrastructural features a long time by their extreme fragility, although many basic have been proposed for karyorelicteans (Corliss 1979; features (e.g. body shape, nuclear structure and somatic Lynn & Corliss 1991; de Puytorac et al. 1993; Foissner cortical ultrastructure) were explored in the pioneering 1998). Emphasizing oral structures, the classification studies by Dragesco (1960), Dragesco & Dragesco-Kerne´is scheme of Corliss (1979) united the orders Karyorelictida, (1986), Raikov et al. (1975) and Wilbert (1986). More Primociliatida, Prostomatida, Haptorida and Pleurostom- recently, Foissner (1995a,b,c, 1996, 1997a,b), Foissner & atida into the subclass Gymnostomata and deemed ª 2010 The Authors. Journal compilation ª 2010 The Norwegian Academy of Science and Letters d Zoologica Scripta, 39, 3, May 2010, pp 305–313 305 转载 中国科技论文在线 http://www.paper.edu.cn Molecular phylogeny of three ciliate genera d S. Gao et al. karyorelictids to be the most primitive ciliates. However, morphological characters were combined to achieve a new this hypothesis was challenged by ultrastructural data that evaluation of the phylogenetic relationships of the karyor- suggested a close relationship between karyorelictids and elictean ciliates. heterotrichs, which both possess overlapping postciliary microtubular ribbons forming the conspicuous postcili- Materials and methods odesmata to the right side of the kinety (Raikov et al. Ciliate collection and identification 1975). The schemes proposed by later authors (Foissner Kentrophoros fasciolatum and Kentrophoros sp.-QD-06112601 1998; Lynn & Small 2002; Lynn 2008) generally sup- were collected from the coast of Qingdao, northern China ported this relationship but they differ with respect to the (36°08¢N 120°43¢E). Trachelotractus entzi (Kahl, 1927) taxonomic structure within the class Karyorelictea Foissner 1997a,b and T. gigas were sampled from Daya (Table 1). Bay (22°42¢N 114°32¢E), southern China. Isolation and Compared with the large number of morphospecies morphological investigations of species carried out accord- described and new taxa recognized to date, the number of ing to Foissner (1991) and Foissner & Dragesco (1996a). karyorelictean species with SS rRNA gene sequence infor- mation available from GenBank is low (over 130 described DNA extraction and polymerase chain reaction (PCR) morphospecies and just eight sequenced). Some key taxa amplification are still needed in analyses to achieve better understanding Isolation of cells and extraction of genomic DNA were and interpretation of the phylogenetic relationships of performed according to Yi et al. (2008b). In brief, cells these organisms. We have identified three karyorelictean were starved in sterilized seawater at room temperature species, i.e. two Kentrophoros species and Trachelolophos overnight to minimize contents of food vacuoles and con- gigas, as well as Trachelotractus entzi, which was formerly taminants, and then DNA was extracted using an REDEx- classified as Trachelocerca within the class Karyorelictea, tract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, but has been transferred to the class Litostomatea by USA) according to the manufacturer’s protocol, with the Foissner (1997a). None of these sequences has so far been slight modification that only 1 ⁄ 10 of the volume suggested analysed in phylogenetic studies. Gene sequences and for each reagent solution was used (Gong et al. 2007). DNA samples were stored at )20 °C. The universal eukaryotic forward primer Euk A (5¢- AACCTGGTTGATCCTGCCAGT-3¢) and reverse Euk Table 1 Four taxonomic schemes for the classification of B(5¢-TGATCCTTCTGCAGGTTCACCTAC-3¢) were karyorelictean ciliates. used to amplify the SS rRNA gene (Medlin et al. 1988) using the PCR protocol of Gao (2008). Corliss (1979) Foissner (1998) Lynn & Small (2002) Lynn (2008) et al. C. Kinetofragminophora C. Karyorelictea C. Karyorelictea C. Karyorelictea Cloning and sequencing of SS rRNA gene S.C. Gymnostomata S.C. Trachelocercia O. Karyorelictida O. Loxodida O. Protostomatida O. Protostomatida Polymerase chain reaction products were purified using an F. Trachelocercidae F. Kentrophoridae F. Kentrophoridae F. Kentrophoridae H.Q & Q Gel Extraction Kit II (U-gene, Jixi, Anhui, Trachelocerca Kentrophoros Kentrophoros Kentrophoros China) and then inserted into pUCm-T vectors (Sangon, Trachelonema F. Cryptopharyngidae Prototrachelocerca F. Trachelocercidae Tracheloraphis Apocrypharynx Trachelolophos Kovalevaia Toronto, ON, Canada). Plasmids were harvested using the F. Loxodidae Cryptopharynx Trachelonema Prototrachelocerca Mini-prep Spin Column kit (Sangon) and were sequenced Kentrophoros F. Loxodidae Tracheloraphis Sultanophrys by the Invitrogen sequencing facilities in Shanghai and Loxodes Loxodes F. Trachelocercidae Trachelocerca Remanella Remanella Trachelocerca Trachelolophos Guangzhou, China. Subsequent sequencing was performed F. Geleiidae O. Trachelocercida O. Loxodida Tracheloraphis in both directions by primer walking (Yi et al. 2008a). Avelia F. Trachelocercidae F. Cryptopharyngidae O. Loxodida Geleia Trachelocerca Apocrypharynx F. Cryptopharyngidae O. Primociliatida Trachelophos Cryptopharynx Apocrypharynx Phylogenetic analyses O. Prostomatida Tracheloraphis F. Loxodidae Cryptopharynx Other sequences used in this study were obtained from O. Haptorida F. Prototrachelocercidae Loxodes F. Loxodidae the NCBI ⁄ GenBank database (Table 2). Sequences were O. Pleurostomatida Prototrachelocerca Remanella Loxodes S.C. Protoheterotrichia O. Protoheterotrichida Remanella aligned using CLUSTAL W 1.83 (Thompson et al. 1994). O. Protoheterotrichida F. Geleiidae O. Protoheterotrichida Ends were trimmed and the ambiguously aligned sites F. Geleiidae Avelia F. Geleiidae were Gblocks v.0.91b (Castresana, 2000), yielding an Avelia Geleia Avelia Geleia Gellertia Geleia alignment of 1387 characters. Gellertia The program MRMODELTEST v.2 (Nylander 2004) Parduczia selected GTR + I (= 0.3474) + G (= 0.6060) under AIC Our new sequences are underlined. criterion as the best model, which was then used in the 306 Zoologica Scripta, 39, 3, May 2010, pp 305–313 d ª 2010 The Authors. Journal compilation ª 2010 The Norwegian Academy of Science and Letters 中国科技论文在线 http://www.paper.edu.cn