The Journal of Published by 中国科技论文在线 http://www.paper.edu.cnthe International Society of Eukaryotic Microbiology Protistologists J. Eukaryot. Microbiol., 58(1), 2011 pp. 11–21 r 2010 The Author(s) Journal of Eukaryotic Microbiology r 2010 International Society of Protistologists DOI: 10.1111/j.1550-7408.2010.00518.x Re-Evaluation on the Diversity of the Polyphyletic Genus Metaurostylopsis (Ciliophora, Hypotricha): Ontogenetic, Morphologic, and Molecular Data Suggest the Establishment of a New Genus Apourostylopsis n. g. WEIBO SONG,a NORBERT WILBERT,b LIQIONG LIa and QIANQIAN ZHANGa aLaboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China, and bZoologisches Institut, Universita¨t Bonn, 53115 Bonn, Germany ABSTRACT. The urostylid genus Metaurostylopsis Song et al., 2001 was considered to be a well-outlined taxon. Nevertheless, recent evidence, including morphological, ontogenetic, and molecular information, have consistently revealed conflicts among congeners, regarding their systematic relationships, ciliature patterns, and origins of ciliary organelles. In the present work, the morphogenetic and morphogenetic features were re-checked and compared, and the phylogeny of nominal species was analysed based on information inferred from the small subunit ribosomal RNA (SS rRNA) gene sequence. In addition, the binary divisional process in a new isolate of Meta- urostylopsis struederkypkeae Shao et al., 2008 is described. All results obtained reveal that the genus is a polyphyletic assemblage whose nominal congeners fall into three clades within the core Urostylida, based on SS rRNA gene sequences. These three clades not match the groups inferred from morphological/morphogenetical evidences. Some conflicting data from molecular and ontogenetic studies also indicate that single-gene information might not be consistently reliable in detecting the phylogenetic relationships among closely related groups and comprehensive multi-gene analyses are necessary to give a more exact evaluation for this divergent assemblage. According to our new understandings, five forms are confirmed to be true Metaurostylopsis. The morphotype Metaurostylopsis sinica Shao et al., 2008 should be excluded from the genus and represents a distinct type, and, thus, a new genus Apourostylopsis n. g. with it as the type specie, i.e. Apourostylopsis sinica (Shao et al., 2008) n. comb. Key Words. Hypotricha, Metaurostylopsis and Apourostylopsis, molecular phylogeny, new genus, ontogenesis. HE ciliate group Hypotricha (s.l.) contains approximately positions of taxa (Chen et al. 2010b; Wang et al. 2011; Yi et al. T 1,000 nominal species and all of them exhibit complicated 2008). morphogenesis whose features of physiological reorganization In the present work, we have compared the ciliature patterns and ontogenesis play important roles in defining the relationships and modes of cortical development of all known members so far among them (Berger 1999, 2006, 2008; Chen et al. 2010b; assigned to this genus, and observed the morphology and mor- Foissner 1996; Foissner et al. 2010; Hemberger 1982; Ku¨ppers phogenesis of a new isolate of Metaurostylopsis struederkypkeae and Claps 2010; Liu et al. 2010; Paiva and Silva-Neto 2009; Shao Shao et al., 2008, and we have analysed the systematic relation- et al. 2007a). The monophyletic origin of this assemblage is ba- ship of this species complex based on sequences of the SS rRNA sically supported by both small subunit ribosomal RNA (SS gene. We had three main aims: (1) to re-check the systematic rRNA) gene phylogenies (Bernhard, Stechmann, and Foissner arrangement of this assemblage, (2) to get further insight into the 2001; Huang et al. 2010; Jiang et al. 2010a, b; Paiva et al. 2009; diversity of their ontogenetic processes, and (3) to confirm the Schmidt et al. 2007), by the conservative ciliature patterns, and by distinction of congeners using more comprehensive data. features of stomatogenesis (Berger 1999, 2006, 2008; Berger and Foissner 1997; Borror 1979; Chen et al. 2010c; Li et al. 2010). On the other hand, hypotrichs display highly diverse somatic struc- MATERIALS AND METHODS tures and the origin of the ciliature as well as the modes of cortical Sample collection, morphological and morphogenetic stud- development divide them into many polyphyletic subgroups (Ber- ies. All isolates used in the present studies were either from the ger 2006; Foissner and Stoeck 2008; Foissner et al. 2004; Jiang cell bank of the Laboratory of Protozoology, Ocean University of and Song 2010; Song et al. 2009). Nevertheless, recent studies China, which were collected either from costal areas near Qingdao using molecular information have consistently questioned the (Tsingtao, 1201180E; 361040N) or from the Daya Bay, Southern current systematic arrangement regarding the relationships and China (1141320E; 221420N). Material identification and cultiva- assignments of many families and orders which were traditionally tion were carried out according to Song et al. (2001). Morpho- believed to be monophyletic, such as many taxa in the orders logical and morphogenetic studies on M. struederkypkeae were Urostylida, Sporadotrichida, and Stichotrichida (Hu et al. 2009b; performed after Wilbert (1975) and Li et al. (2009b). To illustrate Lynn 2008; Yi et al. 2009a). This bewildering situation makes it the changes occurring during morphogenesis, old (i.e. parental) difficult to describe their evolutionary relationships using only a cirri are depicted with contour whereas new cirri are shaded black. single line of evidence (i.e. morphological/morphogenetical or Terminology and systematics are mainly according to Song et al. molecular) (Hu et al. 2009a). (2001) and Berger (2006). The urostylid genus Metaurostylopsis currently contains eight DNA extraction, polymerase chain reaction (PCR) amplifi- morphotypes (Table 1) (Chen, Huang, and Song 2010a; Lei et al. cation, and sequencing. Two populations of Metaurostylopsis 2005; Shao et al. 2008a, c; Song and Wilbert 2002; Song, Petz, cheni and one population of Metaurostylopsis flavicans were and Warren 2001; Wang et al. 2011), and morphogenesis has been treated in the present work. Genomic DNA extraction, PCR am- reported for four of them (Chen et al. 2010a; Shao et al. 2008c; plification, and sequencing of the SS rRNA gene were performed Song et al. 2001). Recent reports indicate that there is greater according to Gong et al. (2009). The SS rRNA gene was amplified diversity within the genus, leading to confusion regarding the by PCR with primers Euk A (50-AACCTGGTTGATCCTGC identification of species, genus definition, and the systematic CAGT-30) and Euk B (50-TGATCCTTCTGCAGGTTCACC TAC-30). Polymerase chain reaction amplification and sequenc- Corresponding Author: W. Song, Laboratory of Protozoology, Insti- ing were according to Miao et al. (2009). tute of Evolution & Marine Biodiversity, Ocean University of China, Phylogenetic analyses. The new SS rRNA gene sequences, Minxing Building B, Qingdao 266003, China—e-mail: wsong@ouc. together with sequences of 52 hypotrichous taxa (s.l.) downloaded edu.cn from GenBank database (see Fig. 37 for GenBank accession 11 转载 中国科技论文在线 http://www.paper.edu.cn 12 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011 numbers), were aligned using CLUSTAL W implemented in Bio- buccal cavity. Subsequently, seven morphotypes have been edit 7.0 (Hall 1999). Phacodinium metchnikoffi was selected as the assigned to this genus (Chen et al. 2010a; Lei et al. 2005; Shao out-group species. Ambiguously aligned regions and gaps were et al. 2008a, c; Song and Wilbert 2002; Wang et al. 2011) (Table 1). excluded before phylogenetic analyses. Bayesian inference (BI), Characteristically, all known forms are found in marine or maximum likelihood (ML), and maximum parsimony (MP) were brackish waters and all of them seem to have remarkable cortical used to construct phylogenetic trees. Model selection and phylo- granules, which often render the cell coloured (e.g. yellow-brown- genetic analyses were according to Yi et al. (2009c). ish, reddish, or yellow-greenish) (Fig. 4–6). Three morphotypes The AU tests were used to test for competing phylogenetic even have two types of granules, that is, larger and smaller ones hypotheses of monophyly of the genus (Shimodaira 2002), with (Table 1). The larger granules are mostly grouped and located the constraint tree generated by PAUP. The MP analysis was per- along/near the ciliary organelles while the smaller ones are usu- formed with the software package PAUPÃ 4.0b10, and support for ally sparsely distributed on both sides of the cell. the internal branches was estimated using the bootstrap method Three morphological groups can be recognized according to the with 1,000 replicates (Yi et al. 2010). TREEVIEW v1.6.6 and ciliature patterns. Group I consists of five members, making the MEGA 4.0 were used to visualize tree topologies (Tamura et al. ‘‘core group,’’ with a pattern typical of the type species (Fig. 3) 2007). (Chen et al. 2010a; Lei et al. 2005; Shao et al. 2008a, c; Song et al. 2001; Song and Wilbert 2002). In contrast, groups II and III have only one or two species: Metaurostylopsis sinica (group II; Fig. 1), RESULTS Metaurostylopsis songi and M. flavicans (group III; Fig. 2). Morphology and infraciliature of nominal species in Meta- Among them, M. sinica differs from the group I in (1) having urostylopsis. The genus Metaurostylopsis was established by only two frontoterminal cirri that are generated