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Phylogenetic position of the marine ciliate, Cardiostomatella vermiforme (Kahl, 1928) Corliss, 1960 inferred from the complete SSrRNA gene sequence, with establishment of a new order Loxocephalida n. ord. (Ciliophora, Oligohymenophorea)
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 42 (2006) 107–114 www.elsevier.de/ejop
Phylogenetic position of the marine ciliate, Cardiostomatella vermiforme (Kahl, 1928) Corliss, 1960 inferred from the complete SSrRNA gene sequence, with establishment of a new order Loxocephalida n. ord. (Ciliophora, Oligohymenophorea) Lifang Lia, Weibo Songa,, Alan Warrenb, Yangang Wanga, Honggang Maa, Xiaozhong Hua, Zigui Chena a
Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, PR China Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
b
Received 14 November 2005; received in revised form 27 December 2005; accepted 27 January 2006
Abstract The small subunit rRNA (SSrRNA) gene was sequenced for Cardiostomatella vermiforme, a large marine ciliate the systematic position of which is uncertain but which has been regarded as a scuticociliate for about forty years. The present work indicates that this organism, together with a closely related species, Dexiotrichides pangi, always form a separate assemblage as a sister group to the scuticociliates sensu stricto. The fact that the clade comprising Cardiostomatella and Dexiotrichides branches between the typical scuticociliates and Hymenostomatia, and shares a series of morphological and morphogenetical characters with both, supports the conclusion that it belongs to an intermediate group between the two. We suggest that this group represents a new order, Loxocephalida n. ord. within the subclass Scuticociliatia, which possibly contains all taxa within the families Loxocephalidae and Cinetochilidae and with Loxocephalidae as the type family. r 2006 Elsevier GmbH. All rights reserved. Keywords: SSrRNA; Phylogenetic position; Cardiostomatella vermiforme; Loxocephalida n. ord.; Scuticociliates
Introduction Over the past four decades, numerous studies have been carried out on the taxonomy and cell development of the scuticociliates (Corliss 1979; Lynn 1979; Small 1967; Song 2000; Song and Wilbert 2000). Based mainly on morphological and morphogenetical data, Lynn and Small (1997) divided the scuticociliates into 3 orders: Philasterida, Pleuronematida and Thigmotrichida. Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2006.01.004
Comparatively few studies have been performed on the systematics of this group (Lynn and Small 1997), despite the fact that the morphology and morphogenesis of many species, especially those in the order Philasterida, have been investigated in detail using modern methods (Grolie`re 1980; Hu et al. 1996; Song 2000; Morade and Small 1994; Song and Wilbert 2000). Molecular methods, in particular the determination of small subunit rRNA (SSrRNA) sequences, have been commonly used to re-evaluate the phylogenetic relationships of many ciliate groups in recent years (Chen and Song 2001; Elwood et al. 1985; Ragan et al. 1996; Shang
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et al. 2002, 2003; Stoeck et al. 1998). Several of these studies provided different conclusions from those based on morphological and/or ontogenetic characters (Chen and Song 2002; Ragan et al. 1996; Stru¨der-Kypke et al. 2000). Nevertheless, sequence data for poorly known groups such as the Cardiostomatella–Dexiotrichidescomplex remain comparatively rare and incomplete. As a contribution to the analysis of ciliate phylogeny we have sequenced the SSrRNA gene from the marine ciliate, Cardiostomatella vermiforme, in order to ascertain its systematic position with molecular biological methods, particularly as the placement of the Cardiostomatella–Dexiotrichides complex within the true scuticociliates is now thought to be highly questionable (Song et al. 2005).
Material and methods Ciliate collection and identification Cardiostomatella vermiforme (Kahl 1928) Corliss 1960 was collected from the coast of Qingdao, China in November 2004. Clonal cultures were established and maintained at room temperature in autoclaved marine water with the appropriate prey. Observations on living cells were carried out using differential interference microscopy. Protargol (Wilbert 1975) and pyridinated silver carbonate (Ferna´ndez-Galiano 1976) impregnation techniques were applied to reveal the infraciliature.
Extraction of genomic DNA and PCR amplification Ciliates were starved overnight, rinsed with sterile artificial sea water and then sedimented by centrifugation. 50 ml lysis buffer (Shang et al. 2003) was added to 20 ml of the concentrated cells and the mixture incubated at 56 1C for 1–2 h to extract DNA, and then at 94 1C for 15 min to denature protein. The PCR reaction steps were performed according to Medlin et al. (1988) and with the primers used by Chen and Song (2002).
and M13-20 primers, using the ABI Prism 377 Automated DNA Sequencer (Applied Biosystems Inc.).
Sequence availability The SSrRNA gene sequences of 33 other taxa were obtained from the GenBank/EMBL databases under the following accession numbers: Cyclidium glaucoma Z22879, Cyclidium plouneouri U27816, Entodiscus borealis AY541687, Entorhipidium pilatum AY541689, Entorhipidium tenue AY541688, Miamiensis avidus AY550080, Philasterides dicentrarchi AY642280, Plagiopyliella pacifica AY541685, Pleuronema coronatum AY103188, Schizocaryum dogieli AF527756, Thyrophylax vorax AY541686, Frontonia vernalis U97110, Lembadion bullinum AF255358, Epicarchesium abrae DQ190462, Zoothamnopsis sinica DQ190469, Zoothamnium arbuscula AF401523, Pseudovorticella punctata DQ190466, Vorticella fusca DQ190468, Anophryoides haemophila U51554, Cohnilembus verminus Z22878, Parauronema longum AY212807, Dexiotrichides pangi AY212805, Paranophrys magna AY103191, Metanophrys similis AY314803, Mesanophrys carcini AY103189; Uronema marinum Z22881, Pseudocohnilembus hargisi AY212806, Glaucoma chattoni X56533, Tetrahymena asiatica X56167, Tetrahymena hyperangularis X56173, Tetrahymena vorax AF364038, Lambornella sp. AF364043, Glaucoma scintillans AJ511861, Glaucomides bromelicola AJ810077, Bromeliophrya brasiliensis AJ810075, Colpidium campylum X56532, Ophryoglena catenula U17355, Ichthyophthirius multifiliis U17354, Pseudomicrothorax dubius X65151, Obertrumia georgiana X65149, Furgasonia blochmanni X65150, Colpoda inflata M97908, Sorogena stoianovitchae AF300285, Euplotes woodruffi AF492707, Paramecium bursaria AF100314, Paramecium tetraurelia X03772, Paramecium nephridiatum AF100317, Dysteria derouxi AY378112, Tokophrya quadripartita AY102174, Tetrahymena corlissi U17356, and a karyorelictid ciliate, Loxodes striatus L24248 was selected as the outgroup species.
Data analysis Cloning and sequencing of SSU rRNA gene The PCR products were extracted with UNIQ-5 DNA Cleaning Kit (Sangon Bio. Co., Canada) and cloned into a pUCm-T vector according to the manufacturer’s protocol. The plasmid DNA was extracted using the mini-prep spin column kit (Sangon Bio. Co., Canada), according to the manufacturer’s protocol. The SSUrDNA gene for Cardiostomatella vermiforme was double strand-sequenced with three forward and three reverse modified 18S sequencing primers (Elwood et al. 1985; Medlin et al. 1988) and the vector based primers, RV-M
The SSrRNA gene sequences of the various taxa were aligned using Clustal W, V.1.80 (Thompson et al. 1994), and then refined through consideration of the conserved primary structures. The computer program, MrBayes v3.0b4 (Huelsenbeck and Ronquist 2001) was used for the Markov chain Monte Carlo (MCMC) algorithm to construct a Bayesian tree. The chain length for our analysis was 10,000,000 generations with trees sampled every 50 generations. PHYLIP V.3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide
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Fig. 1. Morphological characters of Cardiostomatella vermiforme in vivo (A-H) and after staining by protargol or the silver carbonate method (I–M). A–E, To show variations in cell shape, arrow in B marks the endoplasmic granules, while arrow in D indicates the large contractile vacuole. F, Detail of the cortex showing the extrusomes (double-arrowheads) and the cilia (arrow). G, To show the endoplasmic granules and rice-grain-like granules (arrowheads) which make the cell appear black in colour. H, View of pellicle, note that the cell surface appears to have a ‘‘relief sculpture’’ pattern. Arrow marks a food vacuole beneath the pellicle. I, Nuclear apparatus, arrows mark the macronuclear nodules, while the arrowhead indicates the micronucleus. J, Ventral view, to show the oral apparatus, arrow marks the paroral membrane, arrowheads depict the membranelles. K, General view, arrowheads mark the macronuclear nodules, arrow indicates the oral apparatus. L, Apical–lateral view, to show the anterior suture (arrows). M, Caudal portion, arrows mark the prominent basal bodies of the caudal cilia. Scale bars: 100 mm.
sequences using the Kimura two-parameter model (Kimura 1980). Distance-matrix trees were constructed using the Fitch and Margoliash (1967) least-squares
(LS) method and the neighbor-joining (NJ) method (Saitou and Nei 1987) and the distance data bootstrapped (1000 replicates). Heuristic searches with a
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parsimony ratchet were used to generate trees for the maximum-parsimony (MP) analysis in PAUP (V. 4.0b10) (Swofford 2002). Bootstrap values were also generated in PAUP (V. 4.0b10). For maximum parsimony analysis, data were bootstrap resampled 1000 times.
Results Identification of the Qingdao population (Fig. 1) Cardiostomatella vermiforme was recently redescribed by Al-Rasheid (2001). The isolate used in the present study corresponds exactly with that reported by Al-Rasheid (2001) thus confirming the identity of our organism (Fig. 1).
Sequences and comparisons The complete SSrRNA gene sequence was determined for Cardiostomatella vermiforme (1769 nucleotides, GenBank/EMBL accession number AY881632). The GC content (43.82%) is in the same range as that of other ciliates (Chen and Song 2002; Elwood et al. 1985; Schlegel et al. 1991; Shang et al. 2003; Sogin and Elwood 1986). The sequence of Cardiostomatella vermiforme differed in 123 nucleotides from the sequence of Dexiotrichides pangi (structural similarity 93%). 159 sites are different from that in Anophryoides haemophila (structural similarity 91%), while 300 sites differ from Tetrahymena asiatica (structural similarity 83%).
Bayesian and distance matrix analysis To determine the systematic position of C. vermiforme, we constructed phylogenetic trees using multiple algorithms. As shown in Fig. 2, there is strong support for the monophyly of each of the four subclasses of Oligohymenophora included within this study, viz. Scuticociliatia (100% Bay, 92% LS, 96% NJ), Peritrichia (100% Bay, 100% LS, 100% NJ), Hymenostomatia (100% Bay, 100% LS, 100% NJ) and Peniculia (100% Bay, 99% LS, 99% NJ). The Peniculia branches at a very deep level within the oligohymenophorean clade and forms a sister group to the Peritrichia and Hymenostomatia. The Cardiostomatella–Dexiotrichides complex forms a well-defined group within the subclass Scuticociliatia that branches at a deep level from, and forms an outgroup to, the other scuticociliates (Fig. 2).
Maximum parsimony analysis The major aspects of the topology of the maximum parsimony tree (Fig. 3) are generally very similar to those of the Bayesian tree (Fig. 2) and can be summarized as follows: (1) the monophyly of each of the four subclasses of Oligohymenophorea included within this study, viz. Scuticociliatia, Peritrichia, Hymenostomatia and Peniculia; (2) C. vermiforme and D. pangi, branch at a deep level within the Scuticociliatia clade that shows a close relationship with Hymenostomatia (Fig. 3).
Discussion Based on analyses of the SSrRNA sequence data presented here, our work supports the contention that the subclasses Scuticociliatia, Peniculia, Peritrichia and Hymenostomatia are invariably separate taxa within the class Oligohymenophorea. This is consistent with previous findings based on molecular (Stru¨der-Kypke et al. 2000) and morphological data, particularly the radially oriented transverse ribbons of the somatic kinetids (Bardele 1981; Beran 1990; Lynn 1979, 1981). The genus Cardiostomatella, which was moved from Hymenostomatia to Scuticociliatia by Corliss (1960), possesses a similar ciliary pattern to that of Dexiotrichides, i.e. a Tetrahymena-like oral apparatus with three transversely orientated membranelles, a postoral intercalary kinety and an anterior suture but without a scutica (Al-Rasheid 2001; Fenchel et al. 1995). Morphologically, Cardiostomatella bears a closer resemblance to the tetrahymenids than to the ‘‘true’’ scuticociliates, especially with respect to its infraciliature and silverline system, e.g. the absence of the apical plate and scutica and the presence of the preoral suture and postoral intercalary kineties. However its stomatogenesis, a feature of particular significance in evaluating the systematic position of ciliates (Foissner 1996; Grolie`re 1980), has yet to be described. The systematic position of the taxa Cardiostomatella and Dexiotrichides remained uncertain until recent research suggested that the genus Dexiotrichides should occupy an intermediate position between true scuticociliates and Hymenostomatia, based on morphological, morphogenetic and molecular studies of Dexiotrichides pangi (Song et al. 2003, 2005). The present study shows that the ‘‘true’’ scuticociliates (represented by Paranophrys magna and Parauronema longum) together form a monophyletic clade with strong posterior probability in Bayesian tree and high or moderate bootstrap support in distance matrix, maximum parsimony tree construction methods (98% BAY, 82% LS, 97% NJ, 52% MP). The species within Hymenostomatia form a conspicuous monophyly
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Fig. 2. A Bayesian tree inferred from the nucleotide sequences of complete small subunit rRNA (SSrRNA) of Oligohymenophorean ciliated protozoa. A karyorelictid ciliate, Loxodes striatus was set as the outgroup taxon. Asterisks indicate bootstrap values less than 50%. ‘‘N’’ reflects disagreement between a method and the reference Bayesian tree at a given node. Evolutionary distance is represented by the branch length to separate the species in the figure. The scale bar corresponds to five substitutions per 100 nucleotide positions. Numbers at nodes represent: first No. ¼ Bayesian posterior probability (in %), second No. ¼ LS bootstrap values (in %) out of 1000 replicates, third No. ¼ NJ bootstrap values (in %) out of 1000 replicates. The taxa in bold typeface were sequenced at the Laboratory of Protozoology, OUC.
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Fig. 3. A Maximum Parsimony (MP) tree constructed from complete small subunit ribosomal RNA (SSrRNA) sequences indicating the systematic position of Cardiostomatella vermiforme. The numbers at the forks indicate the percentage of times that specific branch pattern occurred in 1000 trees. Asterisks indicate percentages less than 50%. No significance is placed on branch lengths connecting the species. The taxa in bold typeface were sequenced at the Laboratory of Protozoology, Ocean University of China.
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with high posterior probability in Bayesian tree and strong bootstrap support in other methods (100% BAY, 100% LS, 100% NJ, 100% MP), while in each analysis Cardiostomatella and Dexiotrichides form a separate clade that occupies an intermediate position between the typical scuticociliates and Hymenostomatia. This finding is consistent with that of Song et al. (2005) based on morphogenetic and molecular studies of D. pangi. In conclusion, we suggest that Cardiostomatella and Dexiotrichides should be placed in a separate order, Loxocephalida n. ord. with the type family Loxocephalidae, which is characterized by possessing a Dexiotrichida-pattern of infraciliature as described by Song et al. (2005), e.g. with obliquely oriented Tetrahymena-like membranelles; a deeply excavate buccal cavity; an evenly curved paroral membrane; one to several postoral intercalary kineties (in most cases); a short fragment with densely packed dikinetids at the anterior end of SK1. This new order might include the following taxa which are placed in the families Loxocephalidae and Cinetochilidae: Dexiotricha, Dexiotrichides, Platynematum, Cardiostomatella, Cinetochilum, Balanonema, Pseudoplatynematum, Cristigera, Paratetrahymena, Pseudocinetochilum, Loxocephalus, Paraloxocephalus, Sphenostomella ( ¼ Sathrophilus) and Paradexiotricha.
Acknowledgements This work was supported by the ‘‘Nature Science Foundation of China’’ (Project number 30430090) and the Darwin Initiative Programme (Project no. 14-015). We are grateful to Mr. H. Long, postgraduate of the Laboratory of Protozoology, OUC, for sampling.
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PROTISTOLOGY European Journal of Protistology 42 (2006) 107–114 www.elsevier.de/ejop
Phylogenetic position of the marine ciliate, Cardiostomatella vermiforme (Kahl, 1928) Corliss, 1960 inferred from the complete SSrRNA gene sequence, with establishment of a new order Loxocephalida n. ord. (Ciliophora, Oligohymenophorea) Lifang Lia, Weibo Songa,, Alan Warrenb, Yangang Wanga, Honggang Maa, Xiaozhong Hua, Zigui Chena a
Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, PR China Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK
b
Received 14 November 2005; received in revised form 27 December 2005; accepted 27 January 2006
Abstract The small subunit rRNA (SSrRNA) gene was sequenced for Cardiostomatella vermiforme, a large marine ciliate the systematic position of which is uncertain but which has been regarded as a scuticociliate for about forty years. The present work indicates that this organism, together with a closely related species, Dexiotrichides pangi, always form a separate assemblage as a sister group to the scuticociliates sensu stricto. The fact that the clade comprising Cardiostomatella and Dexiotrichides branches between the typical scuticociliates and Hymenostomatia, and shares a series of morphological and morphogenetical characters with both, supports the conclusion that it belongs to an intermediate group between the two. We suggest that this group represents a new order, Loxocephalida n. ord. within the subclass Scuticociliatia, which possibly contains all taxa within the families Loxocephalidae and Cinetochilidae and with Loxocephalidae as the type family. r 2006 Elsevier GmbH. All rights reserved. Keywords: SSrRNA; Phylogenetic position; Cardiostomatella vermiforme; Loxocephalida n. ord.; Scuticociliates
Introduction Over the past four decades, numerous studies have been carried out on the taxonomy and cell development of the scuticociliates (Corliss 1979; Lynn 1979; Small 1967; Song 2000; Song and Wilbert 2000). Based mainly on morphological and morphogenetical data, Lynn and Small (1997) divided the scuticociliates into 3 orders: Philasterida, Pleuronematida and Thigmotrichida. Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2006.01.004
Comparatively few studies have been performed on the systematics of this group (Lynn and Small 1997), despite the fact that the morphology and morphogenesis of many species, especially those in the order Philasterida, have been investigated in detail using modern methods (Grolie`re 1980; Hu et al. 1996; Song 2000; Morade and Small 1994; Song and Wilbert 2000). Molecular methods, in particular the determination of small subunit rRNA (SSrRNA) sequences, have been commonly used to re-evaluate the phylogenetic relationships of many ciliate groups in recent years (Chen and Song 2001; Elwood et al. 1985; Ragan et al. 1996; Shang
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et al. 2002, 2003; Stoeck et al. 1998). Several of these studies provided different conclusions from those based on morphological and/or ontogenetic characters (Chen and Song 2002; Ragan et al. 1996; Stru¨der-Kypke et al. 2000). Nevertheless, sequence data for poorly known groups such as the Cardiostomatella–Dexiotrichidescomplex remain comparatively rare and incomplete. As a contribution to the analysis of ciliate phylogeny we have sequenced the SSrRNA gene from the marine ciliate, Cardiostomatella vermiforme, in order to ascertain its systematic position with molecular biological methods, particularly as the placement of the Cardiostomatella–Dexiotrichides complex within the true scuticociliates is now thought to be highly questionable (Song et al. 2005).
Material and methods Ciliate collection and identification Cardiostomatella vermiforme (Kahl 1928) Corliss 1960 was collected from the coast of Qingdao, China in November 2004. Clonal cultures were established and maintained at room temperature in autoclaved marine water with the appropriate prey. Observations on living cells were carried out using differential interference microscopy. Protargol (Wilbert 1975) and pyridinated silver carbonate (Ferna´ndez-Galiano 1976) impregnation techniques were applied to reveal the infraciliature.
Extraction of genomic DNA and PCR amplification Ciliates were starved overnight, rinsed with sterile artificial sea water and then sedimented by centrifugation. 50 ml lysis buffer (Shang et al. 2003) was added to 20 ml of the concentrated cells and the mixture incubated at 56 1C for 1–2 h to extract DNA, and then at 94 1C for 15 min to denature protein. The PCR reaction steps were performed according to Medlin et al. (1988) and with the primers used by Chen and Song (2002).
and M13-20 primers, using the ABI Prism 377 Automated DNA Sequencer (Applied Biosystems Inc.).
Sequence availability The SSrRNA gene sequences of 33 other taxa were obtained from the GenBank/EMBL databases under the following accession numbers: Cyclidium glaucoma Z22879, Cyclidium plouneouri U27816, Entodiscus borealis AY541687, Entorhipidium pilatum AY541689, Entorhipidium tenue AY541688, Miamiensis avidus AY550080, Philasterides dicentrarchi AY642280, Plagiopyliella pacifica AY541685, Pleuronema coronatum AY103188, Schizocaryum dogieli AF527756, Thyrophylax vorax AY541686, Frontonia vernalis U97110, Lembadion bullinum AF255358, Epicarchesium abrae DQ190462, Zoothamnopsis sinica DQ190469, Zoothamnium arbuscula AF401523, Pseudovorticella punctata DQ190466, Vorticella fusca DQ190468, Anophryoides haemophila U51554, Cohnilembus verminus Z22878, Parauronema longum AY212807, Dexiotrichides pangi AY212805, Paranophrys magna AY103191, Metanophrys similis AY314803, Mesanophrys carcini AY103189; Uronema marinum Z22881, Pseudocohnilembus hargisi AY212806, Glaucoma chattoni X56533, Tetrahymena asiatica X56167, Tetrahymena hyperangularis X56173, Tetrahymena vorax AF364038, Lambornella sp. AF364043, Glaucoma scintillans AJ511861, Glaucomides bromelicola AJ810077, Bromeliophrya brasiliensis AJ810075, Colpidium campylum X56532, Ophryoglena catenula U17355, Ichthyophthirius multifiliis U17354, Pseudomicrothorax dubius X65151, Obertrumia georgiana X65149, Furgasonia blochmanni X65150, Colpoda inflata M97908, Sorogena stoianovitchae AF300285, Euplotes woodruffi AF492707, Paramecium bursaria AF100314, Paramecium tetraurelia X03772, Paramecium nephridiatum AF100317, Dysteria derouxi AY378112, Tokophrya quadripartita AY102174, Tetrahymena corlissi U17356, and a karyorelictid ciliate, Loxodes striatus L24248 was selected as the outgroup species.
Data analysis Cloning and sequencing of SSU rRNA gene The PCR products were extracted with UNIQ-5 DNA Cleaning Kit (Sangon Bio. Co., Canada) and cloned into a pUCm-T vector according to the manufacturer’s protocol. The plasmid DNA was extracted using the mini-prep spin column kit (Sangon Bio. Co., Canada), according to the manufacturer’s protocol. The SSUrDNA gene for Cardiostomatella vermiforme was double strand-sequenced with three forward and three reverse modified 18S sequencing primers (Elwood et al. 1985; Medlin et al. 1988) and the vector based primers, RV-M
The SSrRNA gene sequences of the various taxa were aligned using Clustal W, V.1.80 (Thompson et al. 1994), and then refined through consideration of the conserved primary structures. The computer program, MrBayes v3.0b4 (Huelsenbeck and Ronquist 2001) was used for the Markov chain Monte Carlo (MCMC) algorithm to construct a Bayesian tree. The chain length for our analysis was 10,000,000 generations with trees sampled every 50 generations. PHYLIP V.3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide
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Fig. 1. Morphological characters of Cardiostomatella vermiforme in vivo (A-H) and after staining by protargol or the silver carbonate method (I–M). A–E, To show variations in cell shape, arrow in B marks the endoplasmic granules, while arrow in D indicates the large contractile vacuole. F, Detail of the cortex showing the extrusomes (double-arrowheads) and the cilia (arrow). G, To show the endoplasmic granules and rice-grain-like granules (arrowheads) which make the cell appear black in colour. H, View of pellicle, note that the cell surface appears to have a ‘‘relief sculpture’’ pattern. Arrow marks a food vacuole beneath the pellicle. I, Nuclear apparatus, arrows mark the macronuclear nodules, while the arrowhead indicates the micronucleus. J, Ventral view, to show the oral apparatus, arrow marks the paroral membrane, arrowheads depict the membranelles. K, General view, arrowheads mark the macronuclear nodules, arrow indicates the oral apparatus. L, Apical–lateral view, to show the anterior suture (arrows). M, Caudal portion, arrows mark the prominent basal bodies of the caudal cilia. Scale bars: 100 mm.
sequences using the Kimura two-parameter model (Kimura 1980). Distance-matrix trees were constructed using the Fitch and Margoliash (1967) least-squares
(LS) method and the neighbor-joining (NJ) method (Saitou and Nei 1987) and the distance data bootstrapped (1000 replicates). Heuristic searches with a
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parsimony ratchet were used to generate trees for the maximum-parsimony (MP) analysis in PAUP (V. 4.0b10) (Swofford 2002). Bootstrap values were also generated in PAUP (V. 4.0b10). For maximum parsimony analysis, data were bootstrap resampled 1000 times.
Results Identification of the Qingdao population (Fig. 1) Cardiostomatella vermiforme was recently redescribed by Al-Rasheid (2001). The isolate used in the present study corresponds exactly with that reported by Al-Rasheid (2001) thus confirming the identity of our organism (Fig. 1).
Sequences and comparisons The complete SSrRNA gene sequence was determined for Cardiostomatella vermiforme (1769 nucleotides, GenBank/EMBL accession number AY881632). The GC content (43.82%) is in the same range as that of other ciliates (Chen and Song 2002; Elwood et al. 1985; Schlegel et al. 1991; Shang et al. 2003; Sogin and Elwood 1986). The sequence of Cardiostomatella vermiforme differed in 123 nucleotides from the sequence of Dexiotrichides pangi (structural similarity 93%). 159 sites are different from that in Anophryoides haemophila (structural similarity 91%), while 300 sites differ from Tetrahymena asiatica (structural similarity 83%).
Bayesian and distance matrix analysis To determine the systematic position of C. vermiforme, we constructed phylogenetic trees using multiple algorithms. As shown in Fig. 2, there is strong support for the monophyly of each of the four subclasses of Oligohymenophora included within this study, viz. Scuticociliatia (100% Bay, 92% LS, 96% NJ), Peritrichia (100% Bay, 100% LS, 100% NJ), Hymenostomatia (100% Bay, 100% LS, 100% NJ) and Peniculia (100% Bay, 99% LS, 99% NJ). The Peniculia branches at a very deep level within the oligohymenophorean clade and forms a sister group to the Peritrichia and Hymenostomatia. The Cardiostomatella–Dexiotrichides complex forms a well-defined group within the subclass Scuticociliatia that branches at a deep level from, and forms an outgroup to, the other scuticociliates (Fig. 2).
Maximum parsimony analysis The major aspects of the topology of the maximum parsimony tree (Fig. 3) are generally very similar to those of the Bayesian tree (Fig. 2) and can be summarized as follows: (1) the monophyly of each of the four subclasses of Oligohymenophorea included within this study, viz. Scuticociliatia, Peritrichia, Hymenostomatia and Peniculia; (2) C. vermiforme and D. pangi, branch at a deep level within the Scuticociliatia clade that shows a close relationship with Hymenostomatia (Fig. 3).
Discussion Based on analyses of the SSrRNA sequence data presented here, our work supports the contention that the subclasses Scuticociliatia, Peniculia, Peritrichia and Hymenostomatia are invariably separate taxa within the class Oligohymenophorea. This is consistent with previous findings based on molecular (Stru¨der-Kypke et al. 2000) and morphological data, particularly the radially oriented transverse ribbons of the somatic kinetids (Bardele 1981; Beran 1990; Lynn 1979, 1981). The genus Cardiostomatella, which was moved from Hymenostomatia to Scuticociliatia by Corliss (1960), possesses a similar ciliary pattern to that of Dexiotrichides, i.e. a Tetrahymena-like oral apparatus with three transversely orientated membranelles, a postoral intercalary kinety and an anterior suture but without a scutica (Al-Rasheid 2001; Fenchel et al. 1995). Morphologically, Cardiostomatella bears a closer resemblance to the tetrahymenids than to the ‘‘true’’ scuticociliates, especially with respect to its infraciliature and silverline system, e.g. the absence of the apical plate and scutica and the presence of the preoral suture and postoral intercalary kineties. However its stomatogenesis, a feature of particular significance in evaluating the systematic position of ciliates (Foissner 1996; Grolie`re 1980), has yet to be described. The systematic position of the taxa Cardiostomatella and Dexiotrichides remained uncertain until recent research suggested that the genus Dexiotrichides should occupy an intermediate position between true scuticociliates and Hymenostomatia, based on morphological, morphogenetic and molecular studies of Dexiotrichides pangi (Song et al. 2003, 2005). The present study shows that the ‘‘true’’ scuticociliates (represented by Paranophrys magna and Parauronema longum) together form a monophyletic clade with strong posterior probability in Bayesian tree and high or moderate bootstrap support in distance matrix, maximum parsimony tree construction methods (98% BAY, 82% LS, 97% NJ, 52% MP). The species within Hymenostomatia form a conspicuous monophyly
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Fig. 2. A Bayesian tree inferred from the nucleotide sequences of complete small subunit rRNA (SSrRNA) of Oligohymenophorean ciliated protozoa. A karyorelictid ciliate, Loxodes striatus was set as the outgroup taxon. Asterisks indicate bootstrap values less than 50%. ‘‘N’’ reflects disagreement between a method and the reference Bayesian tree at a given node. Evolutionary distance is represented by the branch length to separate the species in the figure. The scale bar corresponds to five substitutions per 100 nucleotide positions. Numbers at nodes represent: first No. ¼ Bayesian posterior probability (in %), second No. ¼ LS bootstrap values (in %) out of 1000 replicates, third No. ¼ NJ bootstrap values (in %) out of 1000 replicates. The taxa in bold typeface were sequenced at the Laboratory of Protozoology, OUC.
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Fig. 3. A Maximum Parsimony (MP) tree constructed from complete small subunit ribosomal RNA (SSrRNA) sequences indicating the systematic position of Cardiostomatella vermiforme. The numbers at the forks indicate the percentage of times that specific branch pattern occurred in 1000 trees. Asterisks indicate percentages less than 50%. No significance is placed on branch lengths connecting the species. The taxa in bold typeface were sequenced at the Laboratory of Protozoology, Ocean University of China.
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with high posterior probability in Bayesian tree and strong bootstrap support in other methods (100% BAY, 100% LS, 100% NJ, 100% MP), while in each analysis Cardiostomatella and Dexiotrichides form a separate clade that occupies an intermediate position between the typical scuticociliates and Hymenostomatia. This finding is consistent with that of Song et al. (2005) based on morphogenetic and molecular studies of D. pangi. In conclusion, we suggest that Cardiostomatella and Dexiotrichides should be placed in a separate order, Loxocephalida n. ord. with the type family Loxocephalidae, which is characterized by possessing a Dexiotrichida-pattern of infraciliature as described by Song et al. (2005), e.g. with obliquely oriented Tetrahymena-like membranelles; a deeply excavate buccal cavity; an evenly curved paroral membrane; one to several postoral intercalary kineties (in most cases); a short fragment with densely packed dikinetids at the anterior end of SK1. This new order might include the following taxa which are placed in the families Loxocephalidae and Cinetochilidae: Dexiotricha, Dexiotrichides, Platynematum, Cardiostomatella, Cinetochilum, Balanonema, Pseudoplatynematum, Cristigera, Paratetrahymena, Pseudocinetochilum, Loxocephalus, Paraloxocephalus, Sphenostomella ( ¼ Sathrophilus) and Paradexiotricha.
Acknowledgements This work was supported by the ‘‘Nature Science Foundation of China’’ (Project number 30430090) and the Darwin Initiative Programme (Project no. 14-015). We are grateful to Mr. H. Long, postgraduate of the Laboratory of Protozoology, OUC, for sampling.
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