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Phylogenetic position of the marine ciliate, Certesia quadrinucleata (Ciliophora; Hypotrichia; Hypotrichida) inferred from the complete small subunit ribosomal RNA gene sequence
ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 42 (2006) 55–61 www.elsevier.de/ejop
Phylogenetic position of the marine ciliate, Certesia quadrinucleata (Ciliophora; Hypotrichia; Hypotrichida) inferred from the complete small subunit ribosomal RNA gene sequence Lifang Li, Weibo Song Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, China Received 29 June 2005; received in revised form 15 September 2005; accepted 29 September 2005
Abstract The complete small subunit rRNA (SSrRNA) gene sequence of the rare marine hypotrich, Certesia quadrinucleata Fabre-Domergue, 1885, was determined, and found to be 1752 nucleotides long. The phylogenetic position of this species was deduced using distance matrix, maximum parsimony and maximum likelihood methods. Certesia was consistently demonstrated to be a member of the Aspidisca–Euplotes group and clearly exhibits a very close relationship to the well-known genus Euplotes (99% Bay, 99% LS, 99% NJ, 99% MP). The phylogenetic trees further suggest that: (1) Uronychia and Diophrys, traditionally placed in the family Uronychiidae, branch earlier and share a closer relationship to each other than to other hypotrichs; (2) taxa in Gastrocirrhidae, represented by Euplotidium arenarium, might be an ‘‘ancestral’’ group among ‘‘traditional’’ hypotrichs. r 2005 Elsevier GmbH. All rights reserved. Keywords: SSrRNA; Phylogenetic position; Certesia quadrinucleata; Hypotrich ciliate
Introduction The hypotrich (e.g., euplotid) and stichotrich (e.g., oxytrichid) ciliates are among the most easily recognizable ciliates. Morphological attributes, features of the life cycle and physiological properties are used to deduce relationships among the families within the order Euplotida (Borror 1972; Borror and Hill 1995; Curds and Wu 1983; Song 1995). However, euplotid phylogeny still remains confusing as regards the evolutionary relationships and systematic positions of many wellknown groups. This is due to the high diversity of their morphology, the difficulty in recognizing which similarities are due to convergent evolution, and the loss of Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2005.09.005
intermediate forms during the long period of time euplotids have existed. 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, 2002b; Elwood et al. 1985; Greenwood et al. 1991; Ragan et al. 1996; Shang et al. 2002, 2003; Shin et al. 2000). These studies provided many results inconsistent with those from traditional morphological and ontogenetic characters (Chen et al. 2000, 2003; Chen and Song 2002a; Ragan et al. 1996; Stru¨derKypke et al. 2000). As part of a comprehensive analysis of ciliate phylogeny being carried out in the authors’ group, we have recently sequenced the SSrRNA gene of the rare marine hypotrich Certesia quadrinucleata for the first
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L. Li, W. Song / European Journal of Protistology 42 (2006) 55–61
time, in order to determine its systematic position with molecular biological methods and provide more information on phylogenetic relationships within the class Spirotrichea. The results are supplied and discussed here.
Material and methods Ciliate collection and identification C. quadrinucleata Fabre-Domergue, 1885 was found in samples collected from the open water of a scallopfarm off Qingdao (Tsingtao, 361080 N; 1201430 E), China, in December 2004. After isolation, specimens were maintained in Petri dishes in the laboratory for about 4 days as raw cultures. From these clonal cultures were established and maintained in boiled seawater at room temperature with rice grains to enrich natural bacteria as food for the ciliates. Observations on living cells were carried out with bright field and differential interference contrast microscopy. The protargol silver staining method according to Wilbert (1975) was used to reveal the infraciliature. Our result showed that this isolate was morphologically identical to that described recently (Lin and Song 2004) (Fig. 1). General systematic arrangement and terminology are mainly according to Lynn and Small (2002).
Extraction of genomic DNA and PCR amplification Ciliates were starved overnight, rinsed with sterile artificial sea water and then sedimented by centrifugation. Fifty microliters of lysis buffer (Shang et al. 2002) 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 (2002a).
Cloning and sequencing of the SSrRNA gene The amplified products were extracted with UNIQ-5 DNA Cleaning Kit (Sangon Bio. Co., Canada) and inserted into a pUCm-T vector. The plasmid mini-prep spin column kit (Sangon Bio. Co., Canada) was used to harvest and purify plasmid DNA. DNA sequencing for C. quadrinucleata was accomplished using the ABI Prism 377 Automated DNA Sequencer (Applied Biosystems Inc.) with three forward and three reverse modified 18S sequencing primers (Elwood et al. 1985; Medlin et al. 1988) and the RV-M and M13-20 primers. All sequences were confirmed from both strands.
Fig. 1. Morphology and infraciliature of Certesia quadrinucleata, from life (A–B) and after protargol impregnation (C–D). Scale bar: 20 mm. Reproduced with permission from the detailed account by Lin and Song (2004).
The GenBank/EMBL accession number for C. quadrinucleata is DQ059581.
Sequence availability The nucleotide sequences used in the present paper are available from the GenBank/EMBL databases under the following accession numbers: Aspidisca steini AF305625, Diophrys appendiculata AY004773, Euplotes charon AF492705, Euplotes vannus AY004772, Euplotidium arenarium Y19116, Uronychia transfuga AF260120, Strombidium inclinatum AJ488911, Strombidium purpureum U97112, Protocruzia adherens AY217727, Protocruzia sp. X65153, Phacodinium metchnikoffi AJ277877, Tetmemena pustulata X03947, Oxytricha nova X03948, Styxophrya quadricornuta X53485, Gastrostyla steinii AF164133, Stylonychia mytilus AJ310498, Anteholisticha multistylata AJ277876, Strobilidium caudatum AY143573, Tintinnopsis dadayi AY143562 and a heterotrichous form, Blepharisma americanum M97909 was selected as the outgroup species.
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Phylogenetic analyses The sequence was aligned with other SSrRNA gene sequences using a computer-assisted procedure, Clustal W, ver. 1.80 (Thompson et al. 1994), and refined by considering the conservation of 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 under the GTR model of substitution (Lanave et al. 1984; Rodriguez et al. 1990; Tavare 1986) and considering a gamma-shaped distribution of the rates of substitution among sites. The chain length for our analysis was 100,000 generations with trees sampled every 100 generations. The PHYLIP package, version 3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide sequences using the Kimura (1980) two-parameter model. Distance-matrix trees were then constructed using the Fitch and Margoliash (1967) leastsquares (LS) method and the neighbor-joining (NJ) method (Saitou and Nei 1987). Then, the distance data were bootstrap resampled 1000 times. For the max-
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imum-parsimony (MP) analysis, a consensus tree was generated by heuristic tree searches using the parsimony ratchet in the PAUP (v. 4.0b10) (Swofford 2002) program. Bootstrap values were also generated in PAUP (v. 4.0b8). For maximum parsimony analysis, data were bootstrap resampled 1000 times.
Results Sequences and comparisons The complete SSrRNA gene sequence 1752 nucleotides in length was determined for C. quadrinucleata. The GC content (45.21%) is in the same range as in other ciliates. The sequence of C. quadrinucleata differs in 244 nucleotides from the sequence of Euplotes vannus (structural similarity 86.09%). A total of 230 sites are different from that in Aspidisca steini (structural similarity 86.87%), while 233 sites differ from Euplotidium arenarium (structural similarity 86.86%).
Fig. 2. A Bayesian tree inferred from the nucleotide sequences of complete small subunit rRNA (SSrRNA) of spirotrichous ciliated protozoa. Numbers at nodes represent bootstrap values (in %) out of 1000 replicates: the first number is the Bayesian credibility value using the MrBayes algorithm, the second number is from the distance-matrix-based LS method and the third number is derived from the distance matrix-based on the NJ method. Blepharisma americanum (Order Heterotrichida) was selected as the outgroup taxon. Asterisks indicate bootstrap values less than 50%. 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. The new sequence is highlighted in boldface.
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Bayesian and distance-matrix analysis To determine the systematic position of C. quadrinucleata, we constructed phylogenetic trees using multiple algorithms. As shown in Fig. 2, C. quadrinucleata was placed within the cluster of the subclass Hypotrichia sensu Lynn and Small 2002, forming a sister clade to the genus Euplotes with high bootstrap values (99% Bay, 99% LS, 99% NJ). Bayesian and distance-matrix based analyses produced identical trees and provide strong bootstrap support for the monophyly of the class Spirotrichea sensu Lynn and Small 2002 (see Fig. 2). The monophyly of four of its six subclasses: Protocruziidia (100% Bay, 100% LS, 100% NJ), Choreotrichia (99% Bay, 100% LS, 100% NJ), Stichotrichia (98% Bay, 100% LS, 100% NJ) and Oligotrichia (98% Bay, 100% LS, 100% NJ) are also confirmed as expected. In all these trees, Protocruziidia branches firstly from the spirotrichean clade at a very deep level (100% Bay, 100% LS, 100% NJ), while the branch comprising Choreotrichia and Oligotrichia forms a sister group of Stichotrichia (97% Bay, 56% LS, 75% NJ) (Fig. 2). As shown in Fig. 2, in the assemblage of Hypotrichia, Diophrys and Uronychia branch first from the hypotrich clade at a deep level and represent a monophyletic clade as a sister group to all other hypotrichous taxa though
some bootstrap values are not very high. Euplotidium, Aspidisca, Certesia and Euplotes form another branching lineage. Certesia is placed in the Aspidisca–Euplotes group and clearly exhibits a very close relationship to the well-known genus Euplotes, which agrees with results derived from both the morphological and morphogenetical descriptions (Corliss 1979; Lin and Song 2004).
Maximum parsimony analysis As shown in Fig. 3, the major aspects of the topology of the maximum parsimony tree are generally very similar to those of the Bayesian tree (Fig. 2). Fig. 3 exhibits the following features: (1) the monophyly of class Spirotrichea is clear; (2) the family Gastrocirrhidae, represented by Euplotidium arenarium, appears as an ancestral group among traditional hypotrichs; (3) Certesia clearly has a very close relationship to the wellknown genus Euplotes. However, the result suggests that the subclass Hypotrichia is not a monophyletic assemblage, in that Diophrys and Uronychia, traditionally regarded as hypotrichs, group with stichotrich and oligotrich taxa, although the bootstrap value is very low (36% MP).
Fig. 3. A maximum parsimony (MP) tree of the spirotrichous ciliates constructed from complete small subunit ribosomal RNA (SSrRNA) sequences indicating the systematic position of Certesia quadrinucleata and phylogenetic relationships among the spirotrichs whose sequences are available. The numbers at the forks indicate the percentage of times that specific branch pattern occurred in 1000 trees. No significance is placed on branch lengths connecting the species. The new sequence is highlighted in boldface.
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Discussion As demonstrated in the present work and many other papers (e.g. Lynn and Small 1997, 2002), the class Spirotrichea is strongly confirmed as a monophyletic clade with its six major subclasses so far represented by SSrRNA sequences. Based on the information of morphology, stomatogenesis, ultrastructure, cyst structure, and behaviour, Borror and Hill (1995) divided the order Euplotida (s. str., ¼ Hypotrichida s. l.) into five families: Gastrocirrhidae, Certesiidae, Uronychiidae, Aspidiscidae and Euplotidae. Considering some morphological and ontogenetic characters involved in early differentiation in some taxa (e.g. short and stubby dorsal cilia, stable number of five developmental streaks in the frontoventral cirral field, lower ratio of cell length to width), they postulated phylogenetic relationships among these families. Our current work supports their hypothesis basically in: (1) Uronychia and Diophrys, placed in the family Uronychiidae by Borror and Hill (1995), branch early from the remaining hypotrichs and exhibit a closer relationship to each other than to other euplotids; (2) Gastrocirrhidae, represented by Euplotidium, always branches at a deep level and is clearly isolated from other typical euplotid genera, which indicates that it might indeed be an ‘‘ancestral’’ group among hypotrichs. The genus Certesia, which has a short row of left marginal cirri, no caudal cirri and a macronucleus in
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parts, was created by Fabre-Domergue in 1885. It was originally placed (uncertainly) between the oxytrichids and euplotids (Fabre-Domergue, 1885). Bu¨tschli (1889) and Sauerbrey (1928) considered it as a subgenus of Euplotes. Later, it was assigned to the family Euplotidae (Corliss, 1979; Curds and Wu, 1983; Kahl, 1932). Then, Borror and Hill (1995) revised the euplotids and erected a new family Certesiidae to include this taxon (Lynn and Small 2002). Based on the ciliary pattern, especially the frontoventral cirri, and the number of macro-nuclear parts, Lin and Song (2004) concluded that Certesia should be placed in the Aspidisca–Euplotes group very close to Euplotes. The present work has confirmed this. In Figs. 2 and 3, C. quadrinucleata clustered in the subclass Hypotrichia sensu Lynn and Small 2002, forming a sister clade to the genus Euplotes with high bootstrap values. However, this result is not completely in agreement with some previous conclusions based on morphogenetic and morphological studies. Using data obtained from morphological examination (protargol or Chatton-Lwoff staining methods) in interphase and from the binary fission, Song (1995) discussed the possible phylogenetic relationships among the 11 genera of the family Euplotidae s.l. with the methods of fuzzy cluster and cladistic analysis (apomorph-plesiomorph). The results suggested that the 11 genera could be divided into seven clades: Gastrocirrhus; Swedmarkia-Discocephalus; Uronychia; Diophrys; Euplotidium-Certesia; Cytharoides-Euplotes; Euplotaspis-Aspidisca (see Fig. 4). On the basis of variability of ciliation
Fig. 4. Tentative evolutionary tree showing the possible phylogenetic relationship of 11 genera of the family Euplotidae deduced by Song (1995) (modified from Song 1995).
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Medicine, New York, for kind suggestions for data treatment. We are grateful to Dr. Xiaofeng Lin, Laboratory of Protozoology, OUC, for identifying the material. Many thanks are also due to Mr. Hongan Long, graduate student of the Laboratory of Protozoology, OUC, for his help in sample collection.
References
Fig. 5. Diagram of possible relationships among families and genera of the order Euplotida. Upper diagrams illustrate typical morphologies of ventral surfaces of ciliates of each of the five families of the order Euplotida, and diagrams A–D represent possible hypothetical ancestors (from Borror and Hill 1995).
and nuclear apparatus, Borror and Hill (1995) concluded that Certesia is more closely related to Cytharoides, Euplotidium, and Gastrocirrhus than to Euplotes or Aspidisca (see Fig. 5), which is again different from the inference of SSrRNA gene sequence analyses. There are two principal types of explanation about why different conclusions emerge: (a) that the morphological and morphogenetic characters do not necessarily reflect the evolutionary relationships for some unknown reason, e.g. phenetic adaptation; (b) that the information from SSrRNA gene sequence analysis does not always accurately reflect the actual phylogeny, as noticed in some studies by other researchers (Bernhard et al. 1995; Hirt et al. 1995; Shang et al. 2003; Stru¨derKypke et al. 2000; Wright and Lynn 1995). Since molecular data from several critical genera (e.g. Cytharoides, Gastrocirrhus) are still lacking, it is too early to suggest where in the phylogenetic tree these highly specialized taxa may belong. We eagerly await the presentation of sequence data on other genera of hypotrichs to strengthen hypotheses about phylogenetic relationships among these ciliates.
Acknowledgements This work was supported by the ‘‘Nature Science Foundation of China’’ (Project Nos. 30430090; 40376045). Many thanks are due to Dr. Zigui Chen, Cancer Research Center, Albert Einstein College of
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PROTISTOLOGY European Journal of Protistology 42 (2006) 55–61 www.elsevier.de/ejop
Phylogenetic position of the marine ciliate, Certesia quadrinucleata (Ciliophora; Hypotrichia; Hypotrichida) inferred from the complete small subunit ribosomal RNA gene sequence Lifang Li, Weibo Song Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, China Received 29 June 2005; received in revised form 15 September 2005; accepted 29 September 2005
Abstract The complete small subunit rRNA (SSrRNA) gene sequence of the rare marine hypotrich, Certesia quadrinucleata Fabre-Domergue, 1885, was determined, and found to be 1752 nucleotides long. The phylogenetic position of this species was deduced using distance matrix, maximum parsimony and maximum likelihood methods. Certesia was consistently demonstrated to be a member of the Aspidisca–Euplotes group and clearly exhibits a very close relationship to the well-known genus Euplotes (99% Bay, 99% LS, 99% NJ, 99% MP). The phylogenetic trees further suggest that: (1) Uronychia and Diophrys, traditionally placed in the family Uronychiidae, branch earlier and share a closer relationship to each other than to other hypotrichs; (2) taxa in Gastrocirrhidae, represented by Euplotidium arenarium, might be an ‘‘ancestral’’ group among ‘‘traditional’’ hypotrichs. r 2005 Elsevier GmbH. All rights reserved. Keywords: SSrRNA; Phylogenetic position; Certesia quadrinucleata; Hypotrich ciliate
Introduction The hypotrich (e.g., euplotid) and stichotrich (e.g., oxytrichid) ciliates are among the most easily recognizable ciliates. Morphological attributes, features of the life cycle and physiological properties are used to deduce relationships among the families within the order Euplotida (Borror 1972; Borror and Hill 1995; Curds and Wu 1983; Song 1995). However, euplotid phylogeny still remains confusing as regards the evolutionary relationships and systematic positions of many wellknown groups. This is due to the high diversity of their morphology, the difficulty in recognizing which similarities are due to convergent evolution, and the loss of Corresponding author.
E-mail address: [email protected] (W. Song). 0932-4739/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2005.09.005
intermediate forms during the long period of time euplotids have existed. 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, 2002b; Elwood et al. 1985; Greenwood et al. 1991; Ragan et al. 1996; Shang et al. 2002, 2003; Shin et al. 2000). These studies provided many results inconsistent with those from traditional morphological and ontogenetic characters (Chen et al. 2000, 2003; Chen and Song 2002a; Ragan et al. 1996; Stru¨derKypke et al. 2000). As part of a comprehensive analysis of ciliate phylogeny being carried out in the authors’ group, we have recently sequenced the SSrRNA gene of the rare marine hypotrich Certesia quadrinucleata for the first
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time, in order to determine its systematic position with molecular biological methods and provide more information on phylogenetic relationships within the class Spirotrichea. The results are supplied and discussed here.
Material and methods Ciliate collection and identification C. quadrinucleata Fabre-Domergue, 1885 was found in samples collected from the open water of a scallopfarm off Qingdao (Tsingtao, 361080 N; 1201430 E), China, in December 2004. After isolation, specimens were maintained in Petri dishes in the laboratory for about 4 days as raw cultures. From these clonal cultures were established and maintained in boiled seawater at room temperature with rice grains to enrich natural bacteria as food for the ciliates. Observations on living cells were carried out with bright field and differential interference contrast microscopy. The protargol silver staining method according to Wilbert (1975) was used to reveal the infraciliature. Our result showed that this isolate was morphologically identical to that described recently (Lin and Song 2004) (Fig. 1). General systematic arrangement and terminology are mainly according to Lynn and Small (2002).
Extraction of genomic DNA and PCR amplification Ciliates were starved overnight, rinsed with sterile artificial sea water and then sedimented by centrifugation. Fifty microliters of lysis buffer (Shang et al. 2002) 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 (2002a).
Cloning and sequencing of the SSrRNA gene The amplified products were extracted with UNIQ-5 DNA Cleaning Kit (Sangon Bio. Co., Canada) and inserted into a pUCm-T vector. The plasmid mini-prep spin column kit (Sangon Bio. Co., Canada) was used to harvest and purify plasmid DNA. DNA sequencing for C. quadrinucleata was accomplished using the ABI Prism 377 Automated DNA Sequencer (Applied Biosystems Inc.) with three forward and three reverse modified 18S sequencing primers (Elwood et al. 1985; Medlin et al. 1988) and the RV-M and M13-20 primers. All sequences were confirmed from both strands.
Fig. 1. Morphology and infraciliature of Certesia quadrinucleata, from life (A–B) and after protargol impregnation (C–D). Scale bar: 20 mm. Reproduced with permission from the detailed account by Lin and Song (2004).
The GenBank/EMBL accession number for C. quadrinucleata is DQ059581.
Sequence availability The nucleotide sequences used in the present paper are available from the GenBank/EMBL databases under the following accession numbers: Aspidisca steini AF305625, Diophrys appendiculata AY004773, Euplotes charon AF492705, Euplotes vannus AY004772, Euplotidium arenarium Y19116, Uronychia transfuga AF260120, Strombidium inclinatum AJ488911, Strombidium purpureum U97112, Protocruzia adherens AY217727, Protocruzia sp. X65153, Phacodinium metchnikoffi AJ277877, Tetmemena pustulata X03947, Oxytricha nova X03948, Styxophrya quadricornuta X53485, Gastrostyla steinii AF164133, Stylonychia mytilus AJ310498, Anteholisticha multistylata AJ277876, Strobilidium caudatum AY143573, Tintinnopsis dadayi AY143562 and a heterotrichous form, Blepharisma americanum M97909 was selected as the outgroup species.
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Phylogenetic analyses The sequence was aligned with other SSrRNA gene sequences using a computer-assisted procedure, Clustal W, ver. 1.80 (Thompson et al. 1994), and refined by considering the conservation of 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 under the GTR model of substitution (Lanave et al. 1984; Rodriguez et al. 1990; Tavare 1986) and considering a gamma-shaped distribution of the rates of substitution among sites. The chain length for our analysis was 100,000 generations with trees sampled every 100 generations. The PHYLIP package, version 3.57c (Felsenstein 1995) was used to calculate the sequence similarity and evolutionary distances between pairs of nucleotide sequences using the Kimura (1980) two-parameter model. Distance-matrix trees were then constructed using the Fitch and Margoliash (1967) leastsquares (LS) method and the neighbor-joining (NJ) method (Saitou and Nei 1987). Then, the distance data were bootstrap resampled 1000 times. For the max-
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imum-parsimony (MP) analysis, a consensus tree was generated by heuristic tree searches using the parsimony ratchet in the PAUP (v. 4.0b10) (Swofford 2002) program. Bootstrap values were also generated in PAUP (v. 4.0b8). For maximum parsimony analysis, data were bootstrap resampled 1000 times.
Results Sequences and comparisons The complete SSrRNA gene sequence 1752 nucleotides in length was determined for C. quadrinucleata. The GC content (45.21%) is in the same range as in other ciliates. The sequence of C. quadrinucleata differs in 244 nucleotides from the sequence of Euplotes vannus (structural similarity 86.09%). A total of 230 sites are different from that in Aspidisca steini (structural similarity 86.87%), while 233 sites differ from Euplotidium arenarium (structural similarity 86.86%).
Fig. 2. A Bayesian tree inferred from the nucleotide sequences of complete small subunit rRNA (SSrRNA) of spirotrichous ciliated protozoa. Numbers at nodes represent bootstrap values (in %) out of 1000 replicates: the first number is the Bayesian credibility value using the MrBayes algorithm, the second number is from the distance-matrix-based LS method and the third number is derived from the distance matrix-based on the NJ method. Blepharisma americanum (Order Heterotrichida) was selected as the outgroup taxon. Asterisks indicate bootstrap values less than 50%. 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. The new sequence is highlighted in boldface.
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Bayesian and distance-matrix analysis To determine the systematic position of C. quadrinucleata, we constructed phylogenetic trees using multiple algorithms. As shown in Fig. 2, C. quadrinucleata was placed within the cluster of the subclass Hypotrichia sensu Lynn and Small 2002, forming a sister clade to the genus Euplotes with high bootstrap values (99% Bay, 99% LS, 99% NJ). Bayesian and distance-matrix based analyses produced identical trees and provide strong bootstrap support for the monophyly of the class Spirotrichea sensu Lynn and Small 2002 (see Fig. 2). The monophyly of four of its six subclasses: Protocruziidia (100% Bay, 100% LS, 100% NJ), Choreotrichia (99% Bay, 100% LS, 100% NJ), Stichotrichia (98% Bay, 100% LS, 100% NJ) and Oligotrichia (98% Bay, 100% LS, 100% NJ) are also confirmed as expected. In all these trees, Protocruziidia branches firstly from the spirotrichean clade at a very deep level (100% Bay, 100% LS, 100% NJ), while the branch comprising Choreotrichia and Oligotrichia forms a sister group of Stichotrichia (97% Bay, 56% LS, 75% NJ) (Fig. 2). As shown in Fig. 2, in the assemblage of Hypotrichia, Diophrys and Uronychia branch first from the hypotrich clade at a deep level and represent a monophyletic clade as a sister group to all other hypotrichous taxa though
some bootstrap values are not very high. Euplotidium, Aspidisca, Certesia and Euplotes form another branching lineage. Certesia is placed in the Aspidisca–Euplotes group and clearly exhibits a very close relationship to the well-known genus Euplotes, which agrees with results derived from both the morphological and morphogenetical descriptions (Corliss 1979; Lin and Song 2004).
Maximum parsimony analysis As shown in Fig. 3, the major aspects of the topology of the maximum parsimony tree are generally very similar to those of the Bayesian tree (Fig. 2). Fig. 3 exhibits the following features: (1) the monophyly of class Spirotrichea is clear; (2) the family Gastrocirrhidae, represented by Euplotidium arenarium, appears as an ancestral group among traditional hypotrichs; (3) Certesia clearly has a very close relationship to the wellknown genus Euplotes. However, the result suggests that the subclass Hypotrichia is not a monophyletic assemblage, in that Diophrys and Uronychia, traditionally regarded as hypotrichs, group with stichotrich and oligotrich taxa, although the bootstrap value is very low (36% MP).
Fig. 3. A maximum parsimony (MP) tree of the spirotrichous ciliates constructed from complete small subunit ribosomal RNA (SSrRNA) sequences indicating the systematic position of Certesia quadrinucleata and phylogenetic relationships among the spirotrichs whose sequences are available. The numbers at the forks indicate the percentage of times that specific branch pattern occurred in 1000 trees. No significance is placed on branch lengths connecting the species. The new sequence is highlighted in boldface.
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Discussion As demonstrated in the present work and many other papers (e.g. Lynn and Small 1997, 2002), the class Spirotrichea is strongly confirmed as a monophyletic clade with its six major subclasses so far represented by SSrRNA sequences. Based on the information of morphology, stomatogenesis, ultrastructure, cyst structure, and behaviour, Borror and Hill (1995) divided the order Euplotida (s. str., ¼ Hypotrichida s. l.) into five families: Gastrocirrhidae, Certesiidae, Uronychiidae, Aspidiscidae and Euplotidae. Considering some morphological and ontogenetic characters involved in early differentiation in some taxa (e.g. short and stubby dorsal cilia, stable number of five developmental streaks in the frontoventral cirral field, lower ratio of cell length to width), they postulated phylogenetic relationships among these families. Our current work supports their hypothesis basically in: (1) Uronychia and Diophrys, placed in the family Uronychiidae by Borror and Hill (1995), branch early from the remaining hypotrichs and exhibit a closer relationship to each other than to other euplotids; (2) Gastrocirrhidae, represented by Euplotidium, always branches at a deep level and is clearly isolated from other typical euplotid genera, which indicates that it might indeed be an ‘‘ancestral’’ group among hypotrichs. The genus Certesia, which has a short row of left marginal cirri, no caudal cirri and a macronucleus in
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parts, was created by Fabre-Domergue in 1885. It was originally placed (uncertainly) between the oxytrichids and euplotids (Fabre-Domergue, 1885). Bu¨tschli (1889) and Sauerbrey (1928) considered it as a subgenus of Euplotes. Later, it was assigned to the family Euplotidae (Corliss, 1979; Curds and Wu, 1983; Kahl, 1932). Then, Borror and Hill (1995) revised the euplotids and erected a new family Certesiidae to include this taxon (Lynn and Small 2002). Based on the ciliary pattern, especially the frontoventral cirri, and the number of macro-nuclear parts, Lin and Song (2004) concluded that Certesia should be placed in the Aspidisca–Euplotes group very close to Euplotes. The present work has confirmed this. In Figs. 2 and 3, C. quadrinucleata clustered in the subclass Hypotrichia sensu Lynn and Small 2002, forming a sister clade to the genus Euplotes with high bootstrap values. However, this result is not completely in agreement with some previous conclusions based on morphogenetic and morphological studies. Using data obtained from morphological examination (protargol or Chatton-Lwoff staining methods) in interphase and from the binary fission, Song (1995) discussed the possible phylogenetic relationships among the 11 genera of the family Euplotidae s.l. with the methods of fuzzy cluster and cladistic analysis (apomorph-plesiomorph). The results suggested that the 11 genera could be divided into seven clades: Gastrocirrhus; Swedmarkia-Discocephalus; Uronychia; Diophrys; Euplotidium-Certesia; Cytharoides-Euplotes; Euplotaspis-Aspidisca (see Fig. 4). On the basis of variability of ciliation
Fig. 4. Tentative evolutionary tree showing the possible phylogenetic relationship of 11 genera of the family Euplotidae deduced by Song (1995) (modified from Song 1995).
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Medicine, New York, for kind suggestions for data treatment. We are grateful to Dr. Xiaofeng Lin, Laboratory of Protozoology, OUC, for identifying the material. Many thanks are also due to Mr. Hongan Long, graduate student of the Laboratory of Protozoology, OUC, for his help in sample collection.
References
Fig. 5. Diagram of possible relationships among families and genera of the order Euplotida. Upper diagrams illustrate typical morphologies of ventral surfaces of ciliates of each of the five families of the order Euplotida, and diagrams A–D represent possible hypothetical ancestors (from Borror and Hill 1995).
and nuclear apparatus, Borror and Hill (1995) concluded that Certesia is more closely related to Cytharoides, Euplotidium, and Gastrocirrhus than to Euplotes or Aspidisca (see Fig. 5), which is again different from the inference of SSrRNA gene sequence analyses. There are two principal types of explanation about why different conclusions emerge: (a) that the morphological and morphogenetic characters do not necessarily reflect the evolutionary relationships for some unknown reason, e.g. phenetic adaptation; (b) that the information from SSrRNA gene sequence analysis does not always accurately reflect the actual phylogeny, as noticed in some studies by other researchers (Bernhard et al. 1995; Hirt et al. 1995; Shang et al. 2003; Stru¨derKypke et al. 2000; Wright and Lynn 1995). Since molecular data from several critical genera (e.g. Cytharoides, Gastrocirrhus) are still lacking, it is too early to suggest where in the phylogenetic tree these highly specialized taxa may belong. We eagerly await the presentation of sequence data on other genera of hypotrichs to strengthen hypotheses about phylogenetic relationships among these ciliates.
Acknowledgements This work was supported by the ‘‘Nature Science Foundation of China’’ (Project Nos. 30430090; 40376045). Many thanks are due to Dr. Zigui Chen, Cancer Research Center, Albert Einstein College of
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