ARTICLE IN PRESS 中国科技论文在线 http://www.paper.edu.cn Protist, Vol. 159, 519—533, October 2008 http://www.elsevier.de/protis Published online date 30 July 2008 ORIGINAL PAPER Analysis of the Internal Transcribed Spacer 2 (ITS2) Region of Scuticociliates and Related Taxa (Ciliophora, Oligohymenophorea) to Infer their Evolution and Phylogeny Miao Miaoa, Alan Warrenb,1, Weibo Songa, Shi Wangc, Huimin Shanga, and Zigui Chena aLaboratory of Protozoology, Ocean University of China, Qingdao 266003, China bDepartment of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK cLaboratory of Molecular Genetics and Breeding of Molluscs, Ocean University of China, Qingdao 266003, China Submitted November 25, 2007; Accepted May 31, 2008 Monitoring Editor: Michael Melkonian The ITS2 (ITS — internal transcribed spacer) region of the rDNA in 11 representative scuticociliates and two ambiguously related genera was analyzed. In common with other eukaryotes, the putative ITS2 folding pattern consists of a closed loop with four helices supported by minimum free energy and compensatory base changes (CBCs), although two of these helices are variable and sometimes absent. Three topologies were obtained on the basis of traditional primary sequence analysis, ‘‘string’’ strategy of secondary structure and analysis of the combined data. It was found that the secondary structure information could help to improve alignment and utilize appropriately phylogenetic strategies. The proposed phylogenies, though differing between sequence- and structure-based results, provide consistent support for high-level clades: the systematically questionable genera Dexiotrichides and Cardiostomatella always cluster together in a clade basal to the scuticociliates s.s., whereas Pleuronema branches from other uronematids at a deep level, and is hence a divergent taxon. Within the well-supported monophyletic philasterids, a sister relationship exists between Orchitophrya and Mesanophrys, while Uronema shows a close relationship with the group including Paranophrys and Parauronema. The positions of Metanophrys, Pseudocohnilembus and Ano- phryoides among the philasterids remain poorly resolved. Our findings firmly support the proposed evolutionary scenario inferred previously both from morphological and molecular data. & 2008 Elsevier GmbH. All rights reserved. Key words: ITS2 secondary structure; molecular phylogeny; scuticociliates. Introduction The internal transcribed spacer (ITS) regions, transcripts, are interspersed among the rRNA which can excise themselves during the matura- genes (Maroteaux et al. 1985). The ITS regions tion of the precursor of ribosomal RNA (rRNA) can be further subdivided into ITS1, which is located between small subunit (SSU) and 5.8S 1 Corresponding author. rRNA genes, and ITS2, which separates the 5.8S e-mail [email protected] (A. Warren). and large subunit (LSU) rRNA genes. Owing to & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2008.05.002 转载 ARTICLE IN PRESS 中国科技论文在线 http://www.paper.edu.cn 520 M. Miao et al. their relatively high divergence, ITS2 sequences provided support for the recognition of the are widely used for phylogenetic reconstructions scuticociliates as a distinct, high ranking group at both genus and species levels (Alverez and within the class Oligohymenophorea (Li et al. Wendel 2003; Park et al. 2003). However, ITS2 has 2006; Ragan et al. 1996; Shang et al. 2006). not been considered appropriate for molecular Molecular data derived from random amplified phylogenetics at high taxonomic ranks, mostly polymorphic DNA-fingerprinting (RAPD), and due to excessive INDELs (insertions—deletions), restriction fragment length polymorphism (RFLP) saturation, and/or intragenomic variation (Fabry have also been used to reconstruct the phyloge- et al. 1999; Vollmer and Palumbi 2004). It has netic relationships both within the Scuticociliatia recently been reported that ITS2 has a conserved and with related taxa (Shang and Song 2002, 2005; core structure that comprises four helices, the Shang et al. 2003; Stoeck et al. 1998). However, third being the longest (Coleman 2007; Schultz et comparisons between different studies reveal that al. 2005; Wolf et al. 2005a). More than 150,000 there is much confusion over phylogeny and ITS2 sequences have been deposited in GenBank evolution of taxa within the Scuticociliatia. (NCBI), ca. 86,000 of which follow a homology In the present study, we examined the folding modeling of structures (via ITS2 database http:// patterns of the ITS2 segment and the conserva- its2.bioapps.biozentrum.uni-wuerzburg.de) tion in its putative secondary structure for a range (Schultz et al. 2006; Selig et al. 2008). The ITS2 of scuticociliates and related species. The inclu- appears to evolve at a relatively moderate rate sion of thirteen ITS2 sequences representing and its value has been described for eukaryote eleven genera and six families allowed us to evolution (Coleman 2003; Schlo¨ tterer et al. 1994). acquire a broad perspective of rRNA spacer The secondary structure of the ITS region has evolution and phylogeny for the scuticociliates been increasingly taken into account in the and related species. The main aim of this paper sequence alignments in order to compare homo- was to demonstrate the value of the ITS2 putative logous characteristics (Gottschling et al. 2005). secondary structure for a group of marine ciliates. Furthermore, several strategies based on struc- To this end, two hypotheses were tested as tural parameters have been developed to improve follows: (1) there is a conserved core structure in the reconstruction of phylogenies (Billoud et al. the ITS2 of scuticociliates as in other eukaryotes; 2000; Subbotin et al. 2007; Wang et al. 2007). (2) ITS2 genealogy is congruent with SSU rRNA These improvements provide a well-supported sequence phylogeny, moreover ITS2 putative background for the phylogenetic application of secondary structure can improve and enhance ITS2. phylogenetic inference among scuticociliates and Classifications of the ciliates within the subclass related species. Scuticociliatia reflect significant variations regard- ing their morphologies, life styles, and behaviors (Corliss 1979; Foissner 1996; Lynn and Small Results 2002). Small (1967) established this taxon as an independent lineage of hymenostomes character- Primary Sequence Comparisons ized by their mode of stomatogenesis and the possession of the scutica or scuticovestige, which The ITS2 sequences showed considerable varia- is a group of basal bodies or kinetosomes that tion among the species studied with substitutions, typically arise posterior of, or parallel to, the insertions, and deletions as presented in Table 1. paroral membrane. It is generally accepted that The lengths of the ITS2 sequences of 13 species the subclass Scuticociliatia comprises three ranged from 168 nt (Pleuronema coronatum)to orders: Philasterida, Pleuronematida, and Thig- 217 nt (Cardiostomatella vermiformis) with a mean motrichida (Lynn and Small 2002). Li et al. (2006) of 179 nt. The average G+C content of the ITS2 added a fourth order, Loxocephalida, although the sequence was 39.6%, with the highest G+C taxonomic rank of this group remains in question content detected in C. vermiformis (47.9%) and (Yi and Song, personal communication). Although the lowest in Uronema elegans (31.4%). morphological and morphogenetic attributes are routinely used to identify species and to deduce Putative Secondary Structure Tracts in the relationships among them, the systematic Related Scuticociliates positions of certain taxa remain ambiguous (Morade and Small 1994; Song and Wilbert Putative secondary structures of the ITS2 tran- 2000). In recent years, ribosomal RNA data have script of the 13 species are shown in Figure 1. ARTICLE IN PRESS 中国科技论文在线 http://www.paper.edu.cn ITS2 Structure of Scuticociliates and Related Species 521 Table 1. Details of ITS regions from scuticociliates and related species. Species Genbank Reference or data source ITS2 ITS2 GC accession no. (persons who sequenced the Length Content (%) gene) (nt) Dexiotrichides pangi AY513758 Present work (Shang) 171 36.26 Cardiostomatella vermiformis EU262621 Present work (Miao) 217 47.93 Anophryoides haemophila AF107779 Goggin and Murphy (2000) 174 35.63 Orchitophrya stellarum AF107773 Goggin and Murphy (2000) 177 37.29 Pseudocohnilembus hargisi AY513753 Present work (Shang) 178 39.89 Pseudocohnilembus EU262622 Present work (Miao) 169 43.20 persalinus Metanophrys similis AY513757 Present work (Shang) 174 36.78 Mesanophrys chesapeakensis AF107778 Goggin and Murphy (2000) 174 39.66 Mesanophrys carcini AY513756 Present work (Shang) 173 39.88 Paranophrys magna AY513755 Present work (Shang) 182 36.81 Parauronema longum AY513759 Present work (Shang) 175 36.00 Uronema elegans AY513760 Present work (Shang) 172 31.40 Pleuronema coronatum AY513754 Present work (Shang) 168 47.02 The species sequenced in the senior author’s laboratory (OUC) are highlighted in boldface. In spite of distinct sequence variation, these 50-UU/UC-30 in others) (Fig. 3). In Helix III we found taxa shared a very similar pattern of secondary a highly conserved region in all species studied structure with homologous sequence segments comprising bulge B1 (highlighted gray in Fig. 4) having homologous locations. Therefore a gen- and two small bulges (B2 and B3), positioned next erally putative secondary structure model could to the region presenting the motif 50-UGAAUC- be developed, the main features of which are: (1)