Phylogenetic Consideration of Two Scuticociliate Genera, Philasterides and Boveria (Protozoa, Ciliophora) Based on 18 S Rrna Gene Sequences
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Parasitology International 59 (2010) 549–555 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Phylogenetic consideration of two scuticociliate genera, Philasterides and Boveria (Protozoa, Ciliophora) based on 18 S rRNA gene sequences Feng Gao a, Xinpeng Fan a, Zhenzhen Yi a,b, Michaela Strüder-Kypke c, Weibo Song a,⁎ a Laboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China b Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, South China Normal University, Guangzhou 510631, China c Department of Integrative Biology, University of Guelph, Guelph, ON, Canada NIG 2W1 article info abstract Article history: Many scuticociliates are facultative parasites of aquatic organisms and are among the most problematic Received 22 January 2010 ciliate taxa regarding their systematic relationships. The main reason is that most species, especially taxa in Received in revised form 14 May 2010 the order Thigmotrichida have similar morphology and have not been studied yet using molecular methods. Accepted 9 July 2010 In the present work, two scuticociliate genera, represented by two rare parasitic species, Philasterides Available online 15 July 2010 armatalis (order Philasterida) and Boveria subcylindrica (order Thigmotrichida), were studied, and phylogenetic trees concerning these two genera were constructed based on their 18 S rRNA gene sequences. Keywords: fi Ciliophora The results indicate that: 1) Philasterides forms a sister group with Philaster, supporting the classi cation that Phylogeny these two genera belong to the family Philasteridae; 2) it is confirmed that the nominal species, Philasterides Morphology dicentrarchi Dragesco et al., 1995 should be a junior synonym of Miamiensis avidus as revealed by both Philasterida previous investigations and the data revealed in the present work; and 3) the poorly known form B. Thigmotrichida subcylindrica, the only member in the order Thigmotrichida, of which molecular data are available so far, 18 S rRNA gene always clusters with Cyclidium glaucoma, a highly specialized scuticociliate, indicating a sister relationship between the orders Thigmotrichida and Pleuronematida. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Chatton and Lwoff [21,22], Fenchel [23], and Raabe [24–28], and no molecular information is available for any species of this order. Scuticociliates are found in a variety of habitats with great Phylogenetic analyses based on molecular data (e.g., 18 S rRNA and biodiversity regarding their life styles, structure, behaviour and RFLP) have been successfully used to identify and separate scutico- many other biological characters [1–11]. Although scuticociliates ciliate species, however, only few representative pleuronematids, and were separated into different groups by different taxonomists [12– no thigmotrichid species have been examined by molecular techni- 15], three scuticociliate orders, namely Philasterida, Pleuronematida ques so far [29–35]. Thus, there are still large gaps in our knowledge and Thigmotrichida, are generally well recognized [14,16]. about these taxa. The genus Philasterides is characterized by a Tetrahymena-like As a new contribution, we isolated and sequenced 18 S rRNA gene silverline system, the bipartite paroral membrane, and three well- of Philasterides armatalis, a philasterid species, and of Boveria defined oral membranelles, which are arranged in a Paranophrys-like subcylindrica, a thigmotrichid species. The main goal is to provide pattern [17,18]. This genus was originally assigned to the family molecular evidence for the phylogenetic assignments of the genus Philasteridae based on its morphological and morphogenetic features Philasterides and the order Thigmotrichida. The present study also [17–19], but recently transferred to the family Uronematidae provides photomicrographs and a brief redescription of the morphol- according to its stomatogenesis pattern [20]. However, the phyloge- ogy of both species. netic placement of Philasterides, especially its relationships to other morphologically similar genera, such as Philaster, has never been 2. Materials and methods assessed using molecular data. The order Thigmotrichida, characterized by a highly differentiated 2.1. Ciliate collection and identification thigmotactic somatic ciliature and a subequatorial oral region [16], has received relatively little attention since the monographic works of B. subcylindrica was isolated from the mantle cavity of the marine mollusk, Pinna pectinata on March 3 rd, 2009 from Qingdao (36° 04′ N; 120° 23′ E), China, by flushing the gills of hosts with seawater. P. ⁎ Corresponding author. Tel./fax: +86 532 8203 2283. armatalis was collected on June 26th, 2008 from the coastal area, the E-mail address: [email protected] (W. Song). same place as above. Microscopical observations and silver 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.07.002 550 F. Gao et al. / Parasitology International 59 (2010) 549–555 impregnations are according to Wang et al. [9] and Wilbert [36]. replicates was constructed using PAUP* v. 4.0b10 [46] by means of Terminology and systematics are mainly following Lynn [16]. a heuristic search with all characters coded as unordered. 2.2. DNA extraction, PCR amplification and sequencing 3. Results Total genomic DNA was extracted from cells using REDExtract-N-Amp 3.1. Morphological redescription of P. armatalis Song, 2000 (Fig. 1) Tissue PCR Kit (Sigma, St. Louis, USA) as described by Yi et al. [37].ThePCR amplifications of 18 S rRNA genes were performed with primers Euk A Considering the morphology and infraciliature, the current popula- (5′-AAC CTG GTT GAT CCT GCC AGT-3′)andEukB(5′-TGA TCC TTC TGC tion corresponds well to the populations described by Song [47] AGG TTC ACC TAC-3′) [38]. PCR amplification, cloning and sequencing (Fig. 1 I–K) and Hu et al. [19] (Fig. 1L, M). Therefore, our description were done according to Yi et al. [31]. concentrates only on those features differing from the previous report. Cells approximately 60–70×20–25 μminvivo(Fig. 1A, B), generally thicker than the populations reported previously with 2.3. Sequence availability and phylogenetic analyses asymmetric body shape when viewed from ventral. Cytoplasm mostly full of large globular granules (about 3 μm in diameter), which Besides the newly sequenced 18 S rRNA genes, all other sequences indicates that the cells might be newly left from the host (Fig. 1A–G). used in the present analysis were obtained from the NCBI GenBank Always several to many sparsely distributed biconcave crystals database (Table 1). Three Colpodea species were selected as the detected (Fig. 1G). Contractile vacuole terminally located at posterior outgroup for all analyses. All available 18 S rRNA gene sequences of end of cell, formed slowly and then contracted rapidly. Locomotion the subclass Scuticociliatia were included in our phylogenetic generally hastily and swimming helically ahead or motionless for a analyses. However, Schizocaryum sp. EC13, Cyclidium citrullus and long while when feeding on surface of debris. Infraciliature and buccal Entorhipidium pilatum were excluded due to their limited sequence apparatus (Fig. 1H) as described by Song [47] and Hu et al. [19]. length (441 bp, 836 bp and 1376 bp, respectively). The secondary structure-based 18 S rRNA gene sequence alignment of ciliates downloaded from the European Ribosomal 3.2. Morphological redescription of B. subcylindrica Stevens, 1901 Database [39] wasusedasthe‘“seed” alignment to build a profile (Fig. 2) hidden Markov model (HMM) using Hmmer Package version 2.3.2 [40];theHMMprofile obtained was then used to create an Our isolation resembles the original redescription very well alignment of the 56 sequences we assembled using Hmmalign [24,48], thus only some features are emphasized/documented here. – – μ within the package. The masked regions that could not be aligned Body usually 40 60×20 25 m in vivo with both ends conspicu- unambiguously were removed from the initial alignment using ously truncated. Cytoplasm colourless and hyaline, often with many to – Bioedit 7.0.0 [41].Thefinal alignment of 1704 characters was used numerous food granules/vacuoles (Fig. 2A D). Somatic cilia about μ μ to construct phylogenetic trees according to Yi et al. [42].The 10 m in length, while cilia in buccal area about 30 minlength(Fig. 2A, program MrModeltest v.2 [43] selected the GTR+I (=0.3430)+G D, arrow). Contractile vacuole located near the posterior cell end, about μ (=0.4870) as the best model with AIC criterion, which was then 10 mindiameter(Fig. 2D, arrowhead). Locomotion by rotating around used for both Bayesian inference (BI) and maximum likelihood long axis of body. Isolated cells can be maintained in Petri dish for about (ML). The MrBayes 3.1.2 program [44] was used for Bayesian 1 h. Infraciliature (Fig. 2F, K, L) corresponds well with previous reports analyses, and four simultaneous chains were run for 1,500,000 [24,48]. Number of somatic kineties (SK) about 27. generations sampling every 100 generations. The first 4000 trees were discarded as burn-in. All trees remaining after the discarding 3.3. Phylogenetic analyses were used to calculate posterior probabilities (PP) using a majority rule consensus. A ML tree was constructed with the PhyML V2.4.4 The two new 18 S rRNA gene sequences