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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 and Related Taxa (Ciliophora, ) 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 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

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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 . 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

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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) a GU(A)UCAGUG versus CACUGG(A)GAUUCA-30 closed loop with four helices (helix I, II, III and IV); (except Cardiostomatella vermiformis and Pleur- (2) helix II is highly conserved containing a motif onema coronatum). This region contains 14 bp 50-GYGRUUGA versus UCYCYCRY-30 at its base; that are conserved in 77% of species studied. (3) helix III is the longest of the four and bears Frequencies of bases at each position and mutual three bulge loops; (4) helix I shows variation information on base-pair regions in Helices II and among the different taxa; (5) helix IV does not III among ‘‘typical’’ scuticociliates (except Cardi- always occur (Fig. 2). ostomatella vermiformis and Dexiotrichides pangi) The estimated thermodynamic energy of putative are shown in RNA structure logos (Fig. 5) secondary structures ranges from 49.60 kcal/mol Among the species included in the present (Uronema elegans)to74.77 kcal/mol (Cardiosto- study there are several compensatory base matella vermiformis)(Table 2). The four domains changes (CBCs) or hemi-CBCs within the helices: show distinct size classes. Usually Helix I, the most the three pairings of the bases of Helix II (Fig. 3) variable region of ITS2, is 4—7 bp long but shorter and the relatively conserved region of Helix III in in Uronema elegans (2 bp). Helix II varies from 7 bp Figure 4, marked with arrows where CBCs and (Parauronema longum)to11bp(Orchitophrya stel- hemi-CBCs occur. In fact, most of these changes larum, Pseudocohnilembus hargisi, Mesanophrys concern only one of the two paired positions. chesapeakensis and M. carcini), although it is Although less stable than the Watson—Crick extremely elongated in C. vermiformis(19bp).Helix complementarities, the presence of GU apposi- III ranges from 32 to 37 bp. Finally, Helix IV, which tions retain the RNA helical structure. is usually absent, is generally 3—5 bp long but Finally, for each taxon at least one of the sometimes is only 2 bp (Uronema elegans and MFOLD-generated structures included three or Metanophrys similis). four of the substructural features and subsequent The sequence domains are engaged in base manual adjustments easily yielded the remainder. pairing and are exposed as bulges or loops. There Therefore, sequences were resubmitted to MFOLD is a pyrimidine—pyrimidine bulge branching from using constraints for plausible base-pairing rela- helix II by a short base pairing consisting of 50- tions that would generate all consensus substruc- UU/YC-30 (50-UU/CC-30 in Dexiotrichides pangi, tural features. ARTICLE IN PRESS

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522 M. Miao et al.

Figure 1. The putative secondary structures of ITS2 in 13 scuticociliates and related species.

Alignment of Sequences and Construction results, provide consistent support for most of Phylogenetic Trees clades. The segregation between some genera within the scuticociliates is relatively deep and In order to assess the utility of ITS2 in phyloge- easy to distinguish. In all three analyses, the netic analyses, three datasets with different systematically questionable genera Dexiotrichides approaches were selected to recover the most and Cardiostomatella always cluster together in a robust clades (Fig. 6A—C). The first two topolo- clade that is basal to the scuticociliates s.s., while gies, carried out using maximum likelihood, Pleuronema branches from other typical urone- Bayesian inference and maximum parsimony matids at a deep level. The ‘‘true’’ philasterids are analyses, differed from each other on the basis recovered as a monophyletic group, though with of datasets: sequence data alone versus moderate to low bootstrap support. Within the sequence data combined with structural informa- philasterids sister relationships exist between tion (Fig. 6A, B). The third topology was based Orchitophrya and Mesanophrys, with high boot- only on putative secondary structure analysis strap support; Uronema showed a close relation- (Fig. 6C). The proposed phylogenies, though ship with the group including Paranophrys and differing between sequence- and structure-based Parauronema; moreover, the two species of ARTICLE IN PRESS

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ITS2 Structure of Scuticociliates and Related Species 523

specific instances where the topologies disagreed could be traced to differences in bootstrap support (Fig. 6A, B). The genera Orchitophrya and Mesanophrys occupied a relatively basal position within the philasterids in the trees inferred from ITS2 data combined with primary sequences and putative secondary structure (Fig. 6B), rather than clustering with Anophryoides as in the tree inferred from ITS2 primary sequences alone (Fig. 6A). Furthermore, in the ITS2 primary sequence ana- lysis without adjustment, the well-characterized peniculine genus Paramecium clustered with Pleuronema rather than Tetrahymena (Fig. 6A). The topology based only on the predicted secondary structure of the ITS2 region resolved most relationships among the species. The ana- lyses based on secondary structural characters, was performed using the ‘‘string’’ strategy (Fig. 6C). In this strategy, the whole secondary structure is presented as a string in bracket notation bonded with each other, thus it is unnecessary to deconstruct the putative second- ary structure into homologous substructural com- ponents. The neighbor-joining tree based on ITS2 secondary structure alone showed that in the order Philasterida, Metanophrys clustered in a group with Uronema, Paranophrys and Parauro- nema whereas this affiliation is somewhat different from those based on other analyses (Fig. 6A—C). The maximum-likelihood phylogram for the SSU rRNA gene sequence data is shown in Figure 6D. The phylogeny is typical of previous SSU rRNA analyses of the Oligohymenophorea, with high bootstrap support for all the established groups. Figure 2. The putative secondary structure model Trees based on Bayesian inference and maximum of the ITS2 transcript in scuticociliates and related parsimony analyses have similar topologies with the species, supported by CBCs and hemi-CBCs that scuticociliates segregating into two monophyletic preserve the helix pairing. The four domains, each with a stem—loop, are labeled I—IV, Note the assemblages (93%ML, 1.00BI, 79%MP): a periphe- characteristic pyrimidine—pyrimidine bulge in Helix ral clade comprising Cardiostomatella and Dexio- II. In Helix III, there are three small bulges — B1, B2 trichides with moderate bootstrap support (55%ML, and B3. Helix IV occurs in some species but not all 0.94BI, 66%MP), and a second clade, representing and is indicated by the dashed line. Helices II and III the typical scuticociliates, with high bootstrap sup- and part of the closed loop are in bold because of port (97%ML, 1.00BI, 61%MP). The latter clade is the relatively well-conserved nucleotide positions. further subdivided into two lineages: (1) the order Pleuronematida including Pleuronema, Cyclidium and Schizocalyptra as a well-supported basal line- Pseudocohnilembus and the two Mesanophrys age (99%ML, 1.00BI, 98%MP); (2) the order Phila- spp., grouped together respectively. sterida which is considered to be a monophyletic Alignment of the ITS2 primary sequences was assemblage (96%ML, 1.00BI, 95%MP). Compared considerably improved based on adjustment of with the trees based on ITS2 data, Pseudocohni- the secondary structure (Fig. 6A, B). Conserved lembus clusters with Metanophrys (77%ML, 0.98BI, stems identified in the secondary structural 55%MP); Parauronema is sister to Uronema and domain provided consistent bases for correcting Paranophrys with strong support (67%ML, 0.72BI, the alignment of variable loop regions for the 95%MP); Anophryoides and Mesanophrys cluster phylogenetic analyses (data not shown). The with each other (98%ML, 1.00BI, 88%MP). ARTICLE IN PRESS

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Table 2. Numerical and statistical values of the putative secondary structures (ITS2) determined in this study. Species Length (nt) in each helix Unpaired DG (25 1C, bases in kcal/mol) total Dexiotrichides pangi 10 20 66 — 55 57.0 Cardiostomatella vermiformis 10 38 72 8 79 74.77 Anophryoides haemophila 14 18 72 — 48 56.61 Orchitophrya stellarum 14 22 72 — 63 60.52 Pseudocohnilembus hargisi 12 22 72 8 64 69.16 Pseudocohnilembus persalinus 10 18 72 6 61 62.35 Metanophrys similis 10 20 66 4 56 56.90 Mesanophrys chesapeakensis 14 22 74 — 57 60.84 Mesanophrys carcini 14 22 74 — 56 60.84 Paranophrys magna 8 20701078 61.51 Parauronema longum 81472873 55.94 Uronema elegans 41670468 49.60 Pleuronema coronatum 82064—66 59.65 The species sequenced by the senior author’s laboratory (OUC) are highlighted in boldface.

Figure 3. Structure of Helix II in ITS2 of representative scuticociliates and related species. There is a small pyrimidine—pyrimidine bulge branching from the base of the helix by an unpairing of two bases consisting of 50-UU/YC-30. The motif with 50-GYGRUUGA versus UCYCYCRY-30 at its base is boxed. Arrows indicate nucleotide sites of compensating base changes (CBCs), hollow arrows indicate those of semi-CBCs.

Discussion Joseph et al. 1999; Mai and Coleman 1997). As indicated previously, two trends can be found Primary Sequence Conservation during the evolution of eukaryotic ITS regions — and Divergence increase in length and higher G+C content (Gold- man et al. 1983). The ITS2 regions of ciliates, dinoflagellates and While the ITS2 of scuticociliates presents a yeasts are often short and contain a relatively low dramatic range of length variation compared to other percentage of G+C (Gottschling and Plo¨ tner 2004; eukaryotes, its size remains relatively homogeneous ARTICLE IN PRESS

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ITS2 Structure of Scuticociliates and Related Species 525

Figure 4. Structure of Helix III in ITS2 of representative scuticociliates and related species. A highly conserved region in all species studied is indicated by the shaded areas while another conserved region in eleven species (except Cardiostomatella vermiformis and Dexiotrichides pangi) is indicated by the boxes. Arrows locate nucleotide sites of compensating base changes (CBCs), hollow arrows indicate those of semi-CBCs.

with major eukaryotic groups. The insertions and Sequence Structural Evolution of ITS2 in deletions that account for a large proportion of Scuticociliatia and Implications for its variability in the ITS2 in the scuticociliates and Function related species do not seem to impede the formation of conserved structural elements. The ITS2 has In the present investigation it was found that the apparently evolved mostly by length variation of the 13 species shared the same ITS2 putative nucleotide sequence, which causes changes in secondary structure model, viz. a four-fingered the stems of the secondary structures. Perhaps the hand, which is similar to that in algae, plants, small size of these spacers (as compared with those mice, insects, protozoa and vertebrates (Coleman in vertebrates, for example) obviates the need for 2003, 2005; Hoef-Emden 2007; Joseph et al. G+C-rich DNA, or the high A+T content might favor a 1999; Mai and Coleman 1997; Michot et al. 1999; structure analogous to that favored by high G+C Oliverio et al. 2002). Among the four helices content. nucleotide sequences in scuticociliates appear ARTICLE IN PRESS

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526 M. Miao et al.

Figure 5. ITS2 structure logo of Helix II (A) and Helix III (B) in ITS2 of typical scuticociliates (except Cardiostomatella vermiformis and Dexiotrichides pangi). The height of a base in each column is proportional to its frequency in multiple sequence alignment. The relative entropy method was used where the frequency of bases in each column is compared to the background frequency of each base. A prior nucleotide distribution is set to A:C:G:U ¼ 1:1:1:1. Inversed sequence characters indicate a less-than-background frequency. Mutual information in pairs of columns is indicated by the letter M.

to have evolved most rapidly in Helix IV, and next (Lott et al. 1998). According to our present work, it most rapidly in Helix I, as in plants and green algae is clear that the conservation of the structural (Mai and Coleman 1997). Helix II is more stable domains (i.e. Helices II and III) found in all and characteristically has a small bulge, similar scuticociliates investigated to date is constrained to that found in the neogastropods (Oliverio et al. and probably plays a significant functional role in 2002). Helix II in Cardiostomatella vermiformis is the folding of the secondary structure of ITS2 conspicuously longer than ‘‘typical’’ scuticocili- during rRNA primary transcript processing. ates, suggesting that Cardiostomatella is an Helices I and IV, which are close to the ITS2 ends isolated genus close to the scuticociliates. Helix and are highly variable in length and form, could III usually contains several bulges and conserva- be a part of their stop signals. However it is tive regions. unknown whether other factors, such as the There are at least two strands of evidence that closed-loop-like palm, are involved. Furthermore support the accuracy of the models of ITS2 it is also unknown how genetic diversity can structure suggested here. Firstly, CBCs or hemi- be generated and whether there are additional CBCs have been found as defined previously helices in other scuticociliates. (Gutell et al. 1994). Many of the changes in the Two alternative models have recently been primary sequence are silent in terms of RNA reported for Saccharomyces cerevisiae ITS2: the structure, which suggests that at least some parts ‘‘hairpin model’’ and the ‘‘ring model’’ (Coˆ te´ et al. of the predicted structures have functional impor- 2002). A functional genetic assay was carried out tance because they are conserved. Heredity is and both of the models were identified as being guaranteed by faithful DNA replication, whereas important in efficient processing. Notably, the evolution depends upon errors accompanying sequences examined thus far can DNA replication. This suggests mechanisms of adopt the ring structure but appear unable to form individual nucleotide replacements, deletions, and a structure analogous to the hairpin model. It is, recombination events. And secondly, the identifi- however, difficult to draw any conclusions based cation of homologous alignment is somewhat on these findings because taxon sampling is still ambiguous based on the primary sequences, not sufficiently representative among the scutico- which supports the existence of the helices. ciliates. Although in Figure 1 Helix I is shown Our analysis also supports the assertion that the adjacent to the 50 end, it should be noted that this overall size of ITS2 is not critical for correct feature is variable and differs from species to transcription and, as a result, we agree that there species. Furthermore, Helix IV occurs near the may be a ‘‘minimum-nucleotide formula’’ for ITS2, 30 end in some species (e.g. Cardiostomatella i.e. a minimum number of necessary nucleotides vermiformis, Metanophrys similis, Parauronema ARTICLE IN PRESS

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ITS2 Structure of Scuticociliates and Related Species 527

Figure 6. Phylogenetic relationships inferred from different gene sequences in thirteen species. A. Inferred from ITS2 primary sequences only. Numbers at the nodes represent bootstrap values (in %): 1st No. ¼ bootstrap values derived from ML method out of 100 replications, 2nd No. ¼ Bayesian credibility value using the MrBayes program, 3rd No. ¼ bootstrap values derived from MP method out of 1000 replications. B. Inferred from ITS2 primary sequences and putative secondary structure. Other symbols have the same meaning as in A. C. Inferred from ITS2 putative secondary structures. Tetrahymena americanis and Paramecium tetraurelia were selected as the outgroup taxa. The thick branches denote positions that are congruent in topologies A, B, C. D. Phylogenetic relationships inferred from SSU rRNA gene data among the scuticociliates and related species. Numbers at the nodes represent bootstrap values (in %): 1st No. ¼ bootstrap values derived from ML method out of 100 replications, 2nd No. ¼ Bayesian credibility value using the MrBayes program, 3rd No. ¼ bootstrap values derived from MP method out of 1000 replications. A karyorelictid , Loxodes striatus (U24248), was selected as the outgroup species. Species used in this study are shown in bold. *Support values o50%/0.50 and disagreement between a method and the reference ML tree at a given node.

longum, Paranophrys magna, Pseudocohnilembus even absent, in others. In addition, a complex hargisi, Pseudocohnilembus persalinus and Uro- network of interactions might take place to nema elegans) but may be short and divergent, or assemble the pre-rRNA structural features directly ARTICLE IN PRESS

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involved in processing steps into a relatively from several different lines of evidence in order to compact structure. For instance, an interaction establish robust phylogenies. between the 5.8S rRNA 30-end and LSU 50-end Billoud et al. (2000) focused on the rRNA which is an essential requirement, is also common molecules of Cirripedia (barnacles) comparing in yeast and vertebrates (Joseph et al. 1999). primary and secondary structure information. In Moreover, putative secondary structure models this study ‘‘molecular morphometrics’’, i.e. the are not necessarily the same as the ones that form measurable structural parameters of the mole- in vivo, and they are three-dimensional forms of cules (geometrical features, bond energies, base ITS2, not two-dimensional forms. Therefore, the composition, etc.), were used as specific char- model is constrained and may not accurately acters to construct a phylogenetic tree. Subbotin reflect the situation in life. Nevertheless, evidence et al. (2007) converted the original sequence data of co-variations in the ITS2 sequences was into 28 symbol codes using secondary structure recovered here, which supported our structural information. Although phylogenetic trees based on estimates. rRNA were successfully reconstructed, their meth- ods were not suitable for our study because of the difficulty of defining substructural homology in Phylogenetic Comparisons within ITS2 structure alignment. Wolf et al. (2005b) wrote Scuticociliatia and Related Species the software CBCAnalyzer for detecting species that are discriminated by their sexual incompat- We can use the available data and conserved ibility. However, due to the low number of CBCs, structural elements to identify homology in ITS2 the block CBC Tree can be used only for evolution, but what is the phylogenetic utility of this reconstructing the phylogeny of a small set of molecule among scuticociliates and related spe- closely related taxa (Mu¨ ller et al. 2007). Some cies? Clearly, the variable ITS2 regions are more attempts have also been made to formalize the likely to accumulate mutations that could poten- secondary structure comparisons, although diffi- tially record the divergence of the major (e.g. family culties persist in determining a distance between level) lineages, meanwhile they are also more likely two related structures with variable topologies to accrue homoplastic changes in our studies. (Fontana et al. 1993; Nakaya et al. 1996). Recently, It is well known that alignment plays an several algorithms based on the ‘‘string’’ strategy important role in phylogenetic research in the have been proposed (Jiang et al. 2002; Zhang sense that alignment ambiguities may lead to et al. 1999). Wang et al. (2007) developed the string spuriously scored synapomorphies as well as strategy based on the highly divergent ITS1 to infer erroneously inferred indels. Since the secondary phylogeny among the Pectinidae (scallops). In our structure of ITS2 provides recognition and dock- study, we adopted the string strategy to reveal ing signals during the maturation of the precursor phylogenetic relationships among scuticociliates of ribosomal RNA, the structure is often more and related species (Fig. 6C). conservative than the primary sequence (Good The tree topologies based on the three datasets et al. 1997). As a result, one cannot just use suggest that a deep phylogenetic signal has been standard multiple sequence alignment techniques retained in the ITS2 sequences of those species such as Clustal W (Thompson et al. 1994), since used in our study. The availability of SSU rRNA these completely neglect structural information. In sequences allows us to compare our ITS2 results to our study, MARNA helped to generate multiple an independent phylogeny. The putative secondary alignment based on pairwise sequence—structure structure of ITS2 appears to be effective and helpful information of RNAs. Undoubtedly, the process in finding the clustering patterns, although the of alignment was greatly facilitated by the putative positions of ‘‘ambiguous’’ genera such as Dexio- secondary structure model predicted with com- trichides—Cardiostomatella complex, Pleuronema parative analyses (Fig. 6B). The most striking and Pseudocohnilembus as well as the family feature of the primary sequence tree is the Orchitophryidea require further consideration. position of Paramecium tetraurelia, which is As reported recently, the Dexiotrichides— nested within the scuticociliates, albeit with weak Cardiostomatella complex exhibits a mixture of bootstrap support (Fig. 6A). This is in contrast to hymenostome and scuticociliate features with the well-established position of Paramecium respect to: ciliary pattern; obliquely oriented Tetra- among the peniculines in trees based on other hymena-like membranelles; a deeply excavate molecular (SSU rRNA) and morphological data, buccal cavity; an evenly curved paroral mem- thus emphasizing the need for congruent results brane; a short fragment with densely packed ARTICLE IN PRESS

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dikinetids at the anterior end of SK1 (Song et al. (adjacent to the middle of M2 versus adjacent to 2005). Our analysis including putative secondary the anterior end of M2). The ITS2 trees revealed structure prediction supports the scheme pre- similar relationships (Fig. 6A—C), whereas in the sented by Li et al. (2006) in which this assemblage tree based on SSU rRNA gene sequence data may represent an evolutionary divergent offshoot (Fig. 6D) Metanophrys as well as Pseudocohni- and a new order Loxocephalida, comprising lembus branched basally within the order Philas- Dexiotrichides—Cardiostomatella complex, within terida except for the two genera Miamiensis and the subclass Scuticociliatia is suggested. This . In addition, Song and Wilbert (2000) hypothesis awaits further reinvestigation to test noted that the genus Anophryoides should be the topology of existing phylogenetic trees. synonymized with Paranophrys, and that it differs Pleuronema branches at a deep level in the SSU from Mesanophrys in the structure of the buccal rRNA tree and groups with Schizocalyptra in a apparatus. The ITS2 region of Anophryoides is clade that is basal to the other scuticociliates with more similar to the consensus sequence of strong support (Fig. 6D). By contrast, in the ITS2 Mesanophrys spp. and Orchitophrya than to that secondary structure trees, Pleuronema is grouped of Paranophrys, indicating that Anophryoides with Cardiostomatella and Dexiotrichides in a might be phylogenetically closer to Mesanophrys. clade that is sister to the ‘‘true’’ scuticociliates Since a few taxa are nested together, it is difficult s.s. (Fig. 6C). During the divisional processes of to determine which tree has the most potential to Pleuronema, considerable parental membranellar accurately demonstrate scuticociliate phylogeny. dedifferentiation occurs with changes in shape as Clearly more data are needed in order to resolve well as orientation (Ma et al. 2003). This is an the early branches and the evolutionary relation- alternative kind of ‘‘scuticobuccokinetal’’ type, ships among the Orchitophryidea. which is quite different from that of Dexiotrichides To summarize, ITS2 combines information most or Cardiostomatella (Song et al. 2005). These data germane to genus- and species-level systematics, suggest that Pleuronema should be placed within although it may also be useful at family or even the ‘‘true’’ scuticociliates s.s., although a reliable higher levels and thus be useful for resolving alignment could not be obtained among the scuticociliate phylogeny. In order to produce a lineages within this complex clade because more stable phylogeny of the scuticociliates, Pleuronema was the only representative of the however, sequence data for additional key taxa order Pleuronematida (Fig. 6B). are needed. No single gene is thought to contain a Members of the genus Pseudocohnilembus are sufficient number of informative sites to produce an characterized by their two highly specialized paroral unequivocal phylogeny. The results of the present membranes parallel to each other and about equal study suggest that ITS2 gene sequence data might in length (Song et al. 2002). Based on molecular provide important phylogenetic evidence to sup- data, Pseudocohnilembus, Paranophrys and Uro- plement that from other genes, and at a relatively nema were apparently closely related (Shang et al. small cost considering its short length. 2006). The data presented here demonstrate that Pseudocohnilembus is a sister group to Meta- nophrys andbothformanisolatedpositioninthe Methods SSU rRNA tree (Fig. 6D); whereas Pseudocohnilem- bus clusters with the clade containing Mesanophrys DNA extraction, amplification and sequencing: Cardiosto- and Anophryoides in the ITS2 trees (Fig. 6C). matella vermiformis (Kahl 1928) and Pseudocohnilembus The monophyly of the family Orchitophryidea is persalinus Evans & Thompson, 1964 were obtained from the cell bank of the senior author’s laboratory and are available in doubt and phylogeny within this group is not upon request. The morphologies of these two isolates, plus well resolved. Lynn and Small (2002) placed those of eight other scuticociliates used in the present study Paranophrys, Mesanophrys, Metanophrys, Ano- are shown in Figure 7. phryoides and Orchitophrya in the family Orchito- DNA was extracted following Chen and Song (2002) and used as a template for polymerase chain reactions (PCR) phryidea. The molecular data reported here, utilizing the primers ITS-F (50-GTA GGT GAA CCT GCG GAA however, suggest that the family Orchitophryidea GGA TCA TTA-30) and ITS-R (50-TAC TGA TAT GCT TAA GTT appears to be paraphyletic. Paranophrys, Uro- CAG CGG-30) that are complementary to conserved regions nema and Parauronema always form a clade, the and which encompass the 30 end of SSU rRNA, the whole of 0 sister group of which is still ambiguous yet the ITS1, 5.8S rRNA and ITS2 regions, and the 5 end of LSU rRNA (Goggin and Murphy 2000). PCR conditions were as Metanophrys differs from the closely related follows: predenaturation at 94 1C for 5 min followed by thirty genus Paranophrys only in the terminal position cycles of denaturation at 94 1C for 1 min; annealing at 63 1C for of the anterior end of the paroral membrane 1 min; extension at 72 1C for 1 min; a final extension at 72 1C ARTICLE IN PRESS

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530 M. Miao et al.

Figure 7. Morphology of scuticociliates included in the present studies. A. Cardiostomatella vermiformis; B. Dexiotrichides pangi; C. Pleuronema coronatum; D. Metanophrys similis; E. Pseudocohnilembus persalinus; F. Parauronema longum; G. Uronema elegans; H. Mesanophrys carcini; I. Paranophrys magna; J. Pseudocohnilembus hargisi. Scale bars: 100 mminA,40mm in the other micrographs.

for 10 min. Taq polymerase (TaKaRa, Otsu, Japan) was added structures were established by submissions of the primary only after reaction mixtures had reached the initial 94 1C sequence of all the species to the RNA folding website heating step. Purified PCR product of appropriate size was supporting MFOLD version 2.3 (available from http://bioinfo. inserted into the pUCm-T vector (Sangon, Toronto, Ontario, math.rpi.edu/mfold/rna/form1.cgi, Zuker et al. 1999) using the Canada) and sequenced by the Takara sequencing facility in default parameters for folding (except T ¼ 25 1C). All sequences Dalian, China. The product was cloned and two individual were constrained according to the rules suggested by Vaughn colonies were picked and sequenced. The sequence of one et al. 1984), e.g., forcing specified pairing relations between the clone of each species used in the phylogeny has been 50-end of the LSU and 30-end of the 5.8S regions. Only simple deposited in GenBank (see Table 1 for accession numbers). canonical base parings, including GU, were considered. Sequence analyses: ITS2 sequences of eleven scutico- Comparisons among the results for the various species were ciliates obtained from GenBank, together with new sequences made in order to reveal the folding pattern common to them all, of Pseudocohnilembus persalinus and Cardiostomatella ver- which in turn established the conserved structural model of the miformis, were used in the present investigation (Table 1). The Scuticociliatia and hence homology for phylogeny. ITS2 sequence of one other scuticociliate is available in Parts of ITS2 secondary structures were also inspected for GenBank, namely Schizocalyptra aeschtae, however this was compensating base changes (CBCs). A CBC is a pairing not included in the present study because it is exceptionally position in a helix where the sequences of related species divergent and does not show a motif in common with differ at both positions yet retain the pairing potential sequences from those of the scuticociliates in general (data (Coleman et al. 1998). For example, at a site on a stem with not shown). The secondary structure of the 5.8S rRNA had a C that is paired with a G on the opposing portion of the two internal paired regions and at least one 5.8S rRNA—LSU stem, a CBC for a mutation of the C to a T would be an A interaction (50-URUYUGYWUCAGUGU versus ACCUGA- transition at the paired site. Hemi-CBCs mean compensatory WRUCARDYA-30)(Coleman 2005; Gottschling and Plo¨ tner changes on only one side of a helix pairing. Obviously, the 2004). The 50 and 30 ends of the ITS2 sequences were occurrence of either AU/GU or GC/GU change is of weaker determined via Rfam (available on the web http://www. comparative value, on a statistical basis, than a truly sanger.ac.uk/Software/Rfam/)(Griffiths-Jones et al. 2003) compensatory change. Such mutations are considered to and the European large subunit ribosomal RNA database support secondary structure assumptions. (http://rrna.uia.ac.be/lsu/)(Wuyts et al. 2001). In Helices II and III, the frequencies of bases at each Putative secondary structure modeling and sequence position and mutual information of base-paired regions alignment based on secondary structure: Secondary were calculated using the program RNA Structure Logo ARTICLE IN PRESS

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ITS2 Structure of Scuticociliates and Related Species 531

(http://www.cbc.dtu.dk/gorodkin/appl/slogo.html)(Gorodkin liella pacifica (AY541685), Pleuronema coronatum (AY103188), et al. 1997). Structural motifs among ‘‘typical’’ scuticociliates Pseudocohnilembus marinus (Z22880), P. persalinus were identified based on the structure logo. For each struc- (AY551906), Pseudokeronopsis carnea (AY881633), P. flava tural domain, the position, number of base-pairs, unpaired (DQ227798), Pseudovorticella sinensis (DQ845295), Schizo- bases in bulge and/or interior loops, and the GC content of calyptra aeschtae (DQ777744), Tetrahymena australis base-paired regions were investigated and compared. (M98015), Thyrophylax vorax (AY541686), Uronema elegans Construction of phylogenetic trees: Three different (AY103190), U. marinum 1 (DQ867073), U. marinum 2 phylogenetic analyses were carried out from different data- (DQ867072), Uronemella filificum (EF486866), and Zootham- sets: Dataset I. Only primary sequences of ITS2 were aligned nopsis sinica (DQ190469). A karyorelictid ciliate, Loxodes using a computer-assisted procedure, Clustal W, ver. 1.80 striatus (U24248), was selected as the outgroup species. (Thompson et al. 1994). Dataset II. Primary sequences and secondary structures were combined, and the alignment could be obtained at the MARNA web server (Siebert and Backofen 2005)(http://biwww2.informatik.uni-freiburg.de/ Acknowledgements Software/MARNA/index.html). As the default setting, the base deletion was scored 2.0, base mismatch 1.0, arc removing The work is supported by the ‘‘National Natural 2.0, arc breaking 1.5, and mismatch 1.8 with ensemble of shaped structures. The two alignments were used to develop Foundation of Science of China’’ (Project no. phylogeny following multiple algorithms. The alignment was 30670280), and the Darwin Initiative Program submitted to the best-fit model from MRMODELTEST (Nylan- (Project no. 14-015) which is funded by the UK der 2004) obtained by the Akaike Information Criterion (AIC) in Department for Environment, Food and Rural order to perform a search with the branch and bound Affairs. We would like to express gratitude to algorithm in PAUP*(V.4.0b10) (Swofford 2002). Heuristic searches and a 100-fold bootstrap analysis were applied for Dr. Marc Gottschling (Freie Universita¨ t Berlin, maximum likelihood analysis using the PhyML V3.0 program Germany), Prof. Annette W. Coleman (Brown (via http://www.phylogeny.fr/phylo_cgi/phyml.cgi). A Bayesian University, USA), Prof. Marco Oliverio (La Inference (BI) tree was run in the computer program, MrBayes Sapienza University, Italy), Dr. Peter Foster (Nat- v3.0b4 (Huelsenbeck and Ronquist 2001), using the Markov ural History Museum, London) and Prof. Guanpin Chain Monte Carlo (MCMC) algorithm. The chain length was 5,000,000 generations with trees sampled every 100 genera- Yang (Ocean University of China, China) for their tions and an initial burn-in of 10,000. Maximum-parsimony valuable comments on the manuscript. We are (MP) calculation was analyzed in PAUP (V.4.0b10), and also grateful to Mr. Yangang Wang and Hongan bootstrap re-sampled 1000 times. Dataset III. Only putative Long postgraduates of the Laboratory of Proto- secondary structures of ITS2, which were presented as string in bracket notation, were used for structural alignment. In the zoology, OUC, for sampling. bracket notion, base pairs are denoted by matching brackets, dots denote unpaired bases. The distance matrix was produced by the Vienna RNA distance server (http://bioweb.- pasteur.fr/seqanal/interfaces/rnadistance.html) and the full References string algorithm was selected. The two distance matrices were subjected to phylogenetic analysis by neighbor joining Alverez I, Wendel JF (2003) Ribosomal ITS sequences and using MEGA 3.1. Tetrahymena americanis (GenBank plant phylogenetic inference. Mol Phylogenet Evol 29: 417—434 accession number AY833381) and Paramecium tetraurelia Billoud B, Guerrucci M, Masselot M, Deutsch JS (2000) (GenBank accession number AY833390) were chosen as Cirripede phylogeny using a novel approach: molecular out-groups to the 13 species in above analyses. Different morphometrics. Mol Biol Evol 17: 1435—1445 trees were compared to assess the stability of internal branches in the resulting topology. Chen Z, Song W (2002) Phylogenetic positions of Aspidisca Phylogenetic trees based on SSU rRNA gene sequences steini and Euplotes vannus within the order Euplotida were also constructed for comparative purposes. 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Nucleic Acids Res 35: (DQ885987), Glaucoma chattoni (X56533), Ichthyophthirius 3322—3329 multifiliis (U17354), Lembadion bullinum (AF255358), Licno- Coleman AW, Preperata RM, Mehrotra B, Mai JC (1998) phora macfarlandi (AF527758), Loxophyllum rostratum Derivation of the secondary structure of the ITS-1 transcript in (DQ411864), L. jini (EF123708), Mesanophrys carcini Volvocales and its taxonomic correlations. Protist 149: 135—146 AY103189, Metanophrys similis (AY314803), (AY550080), Paramecium woodruffi (AF255362), Corliss JO (1979) The Ciliated Protozoa: Characterization, Paranophrys magna (AY103191), Parauronema longum Classification, and Guide to the Literature, 2nd Ed. Pergamon (AY212807), Philasterides dicentrarchi (AY642280), Plagiopy- Press, London ARTICLE IN PRESS

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