Figures
Abstract
Background
Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa—in particular determining the root—is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences.
Principal Findings
Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported.
Conclusions/Significance
We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids.
Citation: Noda S, Mantini C, Meloni D, Inoue J-I, Kitade O, Viscogliosi E, et al. (2012) Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α. PLoS ONE 7(1): e29938. https://doi.org/10.1371/journal.pone.0029938
Editor: John W. Stiller, East Carolina University, United States of America
Received: August 16, 2011; Accepted: December 8, 2011; Published: January 9, 2012
Copyright: © 2012 Noda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by a grant from the Biomass Engineering Program from RIKEN, Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Nos. 19780067 to SN, and 19380055 and 20658024 to MO, respectively), and a grant from the French Ministère de l'Education Nationale, de la Recherche et de la Technologie to CM, and by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Institut Pasteur of Lille, the University Lille–Nord de France. EV was the recipient of the JSPS Invitation Fellowship Program for Research in Japan FY2010 and the JSPS BRIDGE Fellowship Program FY2011. CM was the recipient of the JSPS Predoctoral-Postdoctoral Fellowship for Foreign Researchers FY2009. DM was supported by grants from the ERASMUS Program and Regione Automa della Sardegna. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The phylum Parabasalia, or commonly parabasalids, comprises a monophyletic but complex assemblage of diverse species of flagellated protists typified by the presence of a characteristic parabasal apparatus (Golgi complex associated with striated fibers), closed mitosis with an external spindle (cryptopleuromitosis), and anaerobic energy-generating organelles called hydrogenosomes [1], [2]. Based on morphological characters, more than 80 genera and 400 parabasalid species have been described thus far [1], [3]. Most parabasalids inhabit the digestive tract of animal hosts as commensals, parasites, or symbionts. In particular, symbiotic parabasalids found in the gut of termites and wood-eating cockroaches play a central role in the digestion of cellulose [4]. This symbiotic relationship is considered a key element in the evolution of social behavior in the hosts [5] and has ecological significance for the decomposition of plant litter in terrestrial ecosystems [6]. Several parabasalids are also of considerable medical and veterinary importance as pathogens—i.e., Trichomonas vaginalis and Tritrichomonas foetus [7], [8].
In addition to the functional host interactions of parabasalids, their biodiversity, conspicuous morphology, unique anaerobic biochemistry, and potentially crucial position in various schemes of eukaryotic evolution have long fascinated researchers. Indeed, Parabasalia is included in the supergroup Excavata and in Metamonada with Fornicata (e.g., Giardia) and Preaxostyla (e.g., Monocercomonoides and Trimastix) [9]. The monophyly of Excavata, although supported morphologically [9], [10], is debated still intensely, but multigene analyses interestingly suggest that Excavata stems from a very deep branching event within the history of eukaryotes [11]–[13].
Molecular phylogenetic studies based on small-subunit (SSU) rRNA gene sequences have focused mainly on cultured representatives of parabasalids [14]–[27]. In contrast, molecular studies of the parabasalids found in the gut of termites have been impeded because these organisms live in complex microbial communities and are very difficult to culture [28], [29]. SSU rRNA gene sequences from parabasalid symbionts, however, have been identified subsequently by culture-independent studies [30]–[50] (see also Table 1 for reference in each parabasalid species).
Because of a high level of sequence divergence of the SSU rRNA gene across parabasalid lineages, a lack of resolution and possible tree-construction artifacts have occurred, even though a large number of taxa have been investigated [26], [39], [43], [46]. To overcome this drawback, multiple protein-encoding genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, and tubulins have been examined [51]–[57]. In these phylogenetic reconstructions, however, taxon sampling was limited, and some conflicting results for relationships of major parabasalid groups were obtained depending on the genes. Worse, in the case of enolase, paralogous copies resulting from ancient duplications might confound phylogenetic interpretation [53]–[55], [57]. Among these indicators, GAPDH sequences that contain a greater phylogenetic signal have given well-resolved trees largely congruent with the SSU rRNA gene phylogeny. Consequently, the GAPDH sequences have been used gradually in phylogenetic reconstructions, and the data of many important taxa have accumulated to cover a wide range of parabasalid diversity [46], [47].
Taxonomic classifications of parabasalids have been proposed on the basis of marked morphological differences, particularly in the arrangement of the basal bodies of the flagella and associated cytoskeletal elements [1], [58], [59]. Parabasalids historically have been divided into two classes: Trichomonada and Hypermastigia [1]. Hypermastigia (or hypermastigids) comprises species of typically large-cell forms equipped with numerous flagella, whereas Trichomonada cells are usually smaller and simpler than those of hypermastigids, with up to six flagella. Molecular studies provide critical and sometimes unexpected insights into the evolution of parabasalids and globally conflict with established systematics. For instance, hypermastigids have been considered to have a polyphyletic origin; therefore, many authors have pointed out the need to revise parabasalid systematics on the basis of these molecular data [16], [17], [20], [37], and indeed some systematic revisions have been started [60], [61]. Consequently, Cepicka et al. [26] have proposed dividing the parabasalids into six classes: Trichonymphea, Spirotrichonymphea, Cristamonadea, Tritrichomonadea, Hypotrichomonadea, and Trichomonadea. The traditional hypermastigids are assigned to the former three classes, which almost exclusively comprise species occurring in the gut of termites and wood-eating cockroaches. Nevertheless, these revisions do not solve all the problems with the systematics of parabasalids, and great uncertainty remains with respect to the phylogenetic relationships among and within these classes [20], [24], [26], [38], [39], [46], [57].
In phylogenetic analyses based on SSU rRNA gene or concatenated data of protein sequences, Trichonymphea, the most morphologically complex group of parabasalids, frequently occurs as the most basal lineage of Parabasalia [31]–[33], [35], [54], [55]. Support for this rooting often has been poor, however, and the differences among several alternative positions in parabasalid lineages have been insignificant [20], [33], [35], [55]. This uncertainty has been explained by the absence of any close outgroup species to Parabasalia [20]. The parabasalid lineage usually branches out deeply in eukaryote phylogenies, and the branch leading to Parabasalia is very long. Such a long branch may attract the fast-evolving Trichonymphea, causing artificial rooting. Furthermore, the gene encoding GAPDH, like many other glycolytic enzymes, of Parabasalia has been derived from a bacterium via lateral gene transfer (LGT) [51], [62]–[64], which disturbs root inference in comparisons to other eukaryotes.
Approaches using the sequence data of multiple genes increase the number of informative characters for phylogenetic inference. In parallel, increasing the number of sampling taxa (or species) is also important. Assembling datasets rich in both genes and taxa is likely to produce more accurate and robust results, although controversy exists about which strategy (increasing the number of genes or taxa) contributes most to the accurate inference [65]–[67]. In general, recent genome sequencing efforts have increased the number of genes for some species, but the genome analyses of either cultured microbial species or yet-uncultivated species are still poor. In terms of taxon sampling, data availability is also restricted, particularly for yet-uncultivated species. The goal of this study is to improve the phylogenetic framework of the relationships among parabasalid groups as well as of the evolution of parabasalid biodiversity by examining two additional genes encoding actin and elongation factor (EF)-1α in diverse parabasalid taxa of both cultivated and yet-uncultivated species. These two proteins are functionally independent from each other and from GAPDH. Their genes are expressed usually highly, and this feature is particularly important for obtaining gene sequences from a small number of manually isolated cells of yet-uncultivated species by reverse transcription (RT)-PCR and designing PCR primers based on EST (expressed sequence tag) data of termite-gut symbionts [68], [69]. Protein sequences of these two molecular markers were examined in representative species that have been investigated already with both SSU rRNA gene and GAPDH. In particular, we examined many members of Cristamonadea and Trichonymphea because both classes contain a large number of genera that were relatively easy to distinguish owing to their conspicuous morphologies. We inferred the phylogenetic relationships among parabasalids and examined the root position of this protistan group based on the concatenation of SSU rRNA gene, actin, EF-1α, and GAPDH sequences.
Results
Actin, EF-1α, and GAPDH sequences
In this study, sequences of genes encoding actin and EF-1α were determined from 29 species of parabasalids (Table 1). Among these species, 22 are symbionts in the gut of termites, whereas seven were cultured representatives from other animals. For each gene, several sequences showing more than 98% amino acid identity were obtained from most of these species. These differences can be explained by sequence variation among duplicated gene copies in the genome, at least in the cases of single-cell analyses, and/or by intra-species variation of the gene in the population when multiple cells were used for the analysis. The GAPDH sequences were also obtained from two previously unstudied species, Hoplonympha sp. and Staurojoenina assimilis because they have been argued to be basal in Trichonymphea [70]–[72]. Two distinct sequences with 89% amino acid identity were obtained from the latter species, which is typical for the GAPDH gene in parabasalids [46], [55]. Maximum protein sequence differences between parabasalian species were 23%, 33%, and 45% for actin, EF-1α, and GAPDH, respectively.
Figure 1 shows the phylogenetic trees inferred from the sequences of the three proteins. The sequences of the multiple gene copies identified in this study as well as those found in the genome of T. vaginalis [73] showed close phylogenetic relatedness, and thus did not confound the phylogenetic reconstruction—at least the reconstruction needed to infer the relationships among major parabasalian groups, although some inter- or intra-species relationships seemed to be difficult to clarify. GAPDH yielded a relatively good resolution, with many of the branches receiving high statistical support, whereas the actin and EF-1α trees were resolved rather poorly, particularly within Cristamonadea in both cases and within Trichonymphea in the case of actin. Spirotrichonymphea, Trichonymphea, Cristamonadea, and Hypotrichomonadea all were monophyletic, with significant supports for the former two in the three trees. Tritrichomonadea and Trichomonadea were paraphyletic in both the actin and EF-1α trees. A remarkable difference among the three protein-based trees was the position of Spirotrichonymphea. Indeed, it was sister to Trichonymphea in the GAPDH tree and to Cristamonadea in the EF-1α tree (both were supported but only fairly), whereas it was sister to neither Trichonymphea nor Cristamonadea in the actin tree. Depending on the placement of Spirotrichonymphea, relationships among the six parabasalian classes were considerably different for the three proteins, except that Cristamonadea and Tritrichomonadea were grouped together in the GAPDH and actin trees and the group of Trichomonadea and Trichonymphea was separated from the other classes in the actin and EF-1α trees. The results indicated that the analysis of single proteins gave poor resolution and would cause incorrect inferences of relationships because it is unclear which of the individual sequences resolves the species phylogeny most accurately.
Unambiguously aligned protein sequences of 278 (A), 280 (B) and 274 (C) sites were used for phylogenetic inference. The species names of the parabasalids except for the genus Trichonympha are shown in Table 1. The GAPDH sequences of Trichonympha acuta and Eucomonympha imla and the EF-1α sequence of Trichomonas tenax (not shown in Table 1) were also included in the analyses. Although the EF-1α sequences published for Pentatrichomonas and Tritrichomonas [57] were not included in C because of their shorter sequence length, the analysis with a reducing number of sites (219 sites) demonstrated that they were related very closely to the sequences from the same taxa shown in C. The sequence accession number was indicated for each taxon. The sequences used for the concatenation are in bold. The trees were estimated in RAxML and the numbers near the nodes indicate the bootstrap values. Values below 50% are not shown. Vertical bars to the right of the trees represent the parabasalian classes according to [26]: C, Cristamonadea; S, Spirotrichonymphea; Tn, Trichonymphea; Tt, Tritrichomonadea; H, Hypotrichomonadea; and Tm, Trichomonadea. Scale bars correspond to 0.10 substitutions per site.
Sequence concatenation and relationships among parabasalian classes
To overcome the poor resolution with single markers, one representative of the multiple gene copies in each of the 28 common parabasalian species was used for sequence concatenation of the three proteins and SSU rRNA gene and subsequent analysis (Figure 2). A significant resolution—particularly in the relationships among the six parabasalian classes (>80% bootstrap values)—was obtained from this concatenated dataset. The monophyly of each parabasalian class was completely supported except in the Tritrichomonadea. In the apical part of the tree, Cristamonadea and Tritrichomonadea formed a well-supported clade, which formed a sister group with Spirotrichonymphea. These three classes were grouped further with Hypotrichomonadea, and the four were separated completely from Trichonymphea and Trichomonadea.
The concatenated dataset comprising 278 amino acid sites of GAPDH, 280 amino acid sites of actin, 274 amino acid sites of EF-1α, and 1338 nucleotide sites of SSU rRNA gene sequences was analyzed in 28 parabasalian species. The tree was estimated in RAxML using separate models with the parameters and branch length optimized for each gene partitions individually. The supporting values (bootstrap in RAxML/Bayesian posterior probability) are indicated at the nodes. Values below 50% or 0.5 are indicated with hyphens. When the site-heterogeneous CAT model was used in each partition, the identical tree topology with similar bootstrap values was obtained (data not shown). Vertical bars to the right of the tree represent the parabasalian classes. The scale bar corresponds to 0.10 substitutions per site.
Alternative phylogenetic relationships of parabasalian classes were examined with the concatenated dataset using the Shimodaira-Hasegawa (SH) and approximately unbiased (AU) tests, in which all possible pairings of the classes that did not appear in the tree based on the concatenated dataset were compared (Table 2). The SH test did not reject any pairing comprised of two from four classes, Cristamonadea, Tritrichomonadea, Spirotrichonymphea, and Hypotrichomonadea, although the AU test did not rejected only two pairing ((C+S) and (S+H), see Table 2). The results suggested that some ambiguities remained in the relationships of these four classes.
Congruency of the inferred relationships of the six parabasalian classes was examined using SH tests. The branching order of the six classes obtained by the sequence concatenation was not significantly worse using all the datasets of single proteins or the SSU rRNA gene (P>0.05), whereas the relationships inferred from GAPDH and SSU rRNA gene sequences were rejected using the concatenated dataset (P = 0.006 and 0.004, respectively), suggesting some phylogenetic noise in these two datasets. Nevertheless, the removal of either the GAPDH or the SSU rRNA gene sequences from the concatenated dataset did not seriously change the overall relationship of the six classes except that Tritrichomonadea became paraphyletic in both cases and Spirotrichonymphea and Hypotrichomonadea formed a sister group with only a weak support in the case of SSU rRNA gene sequence removal (see additional file 1: Figures S1 and S2).
Root of Parabasalia
The root of Parabasalia was investigated through analyses with outgroup taxa. Because the GAPDH gene of Parabasalia likely has been acquired from a bacterium via LGT [51], [62]–[64], a sequence concatenation of EF-1α, actin, and SSU rRNA gene of 30 common parabasalian species was analyzed with representatives of diverse eukaryotic lineages as outgroup taxa (Figure 3). The root of Parabasalia was located at the position dividing the Trichonymphea plus Trichomonadea group from the others (position k in Figure 3). The monophyly of Parabasalia was supported fully. Except for the paraphyly of Tritrichomonadea, the relationships among the parabasalian classes were quite similar to those revealed in the analyses without outgroup taxa, although the supporting values of the branching orders of Cristamonadea, Tritrichomonadea, Spirotrichonymphea, and Hypotrichomonadea were decreased. Notably, each of the groupings of these four classes and the sister-group relationship between Trichomonadea and Trichonymphea had considerable support (94/1 and 74/1, respectively), indicating the significance of the parabasalian root position.
Unambiguously aligned amino acid sites of actin (268) and EF-1α (274), and nucleotide sites of SSU rRNA (1265) gene sequences were concatenated and analyzed in 30 parabasalian species and 23 diverse eukaryotes as outgroups. The tree was estimated with RAxML using the CAT model (CATMIX). The parameters and branch length were optimized for each gene partition individually. The supporting values (bootstrap in RAxML/Bayesian posterior probability) are indicated at the nodes. Values below 50% or 0.5 are indicated with hyphens. Vertical bars to the right of the tree represent the parabasalian classes. The 11 possible root positions are indicated in red letters. The scale bar corresponds to 0.10 substitutions per site.
Because some outgroup taxa had very long branches, which might confound the inference of the parabasalian root, these long-branch outgroup taxa were removed from the analyses in a stepwise manner (see Table S1). A series of analyses removing long-branch outgroup taxa from 23 to 13 did not substantially affect the root position, the relationships among the parabasalian classes, or their support values, indicating that these long-branch outgroup taxa did not disturb the inference.
We compared 11 possible root positions (a–k in Figure 3) using the SH and AU tests (Table 3). The root positions at the nodes leading to Trichonymphea (position g), Trichomonadea (f), and Hypotrichomonadea (e), the node dividing the group of these three classes from the others (j), and the clade of Histomonas plus Dientamoeba (b) were not rejected in the SH test (P>0.05), while only the positions g and e were not rejected in the AU test. The results indicated that although the inferred root position k was the most likely, some ambiguity remains.
When GAPDH was included in the analysis of the sequence concatenation with a number of eukaryotic taxa as outgroups (data not shown), the identical root position was obtained with considerable support values. Because Preaxostyla such as Trimastix is the only known eukaryotic group that shares a common origin of the GAPDH gene of Parabasalia (LGT in their common ancestor or LGTs from closely related bacteria) [62], [63], Trimastix was used as an outgroup taxon for parabasalian root analysis of the sequence concatenation including GAPDH (Figure 4). Again, the identical root position was inferred, and significant support values were obtained both at the node uniting Trichonymphea and Trichomonadea and at the node grouping the other four classes. The relationships among the six classes were identical to those shown in Figures 2 and 3.
Unambiguously aligned amino acid sites of GAPDH (257), actin (268), and EF-1α (274), and nucleotide sites of SSU rRNA (1338) gene sequences were concatenated and analyzed in 28 parabasalian species with Trimastix as an outgroup. The tree was estimated in RAxML using the CAT model (CATMIX). The parameters and branch length were optimized for each gene partition individually. The supporting values (bootstrap in RAxML/Bayesian posterior probability) are indicated at the nodes. Values below 50% or 0.5 are indicated by hyphens. Vertical bars to the right of the tree represent the parabasalian classes. The scale bar corresponds to 0.10 substitutions per site.
Addition of α-tubulin and β-tubulin sequences to the concatenated dataset, which reduced the number of the parabasalian taxa available for the analysis to 12, demonstrated that Trichonymphea was the most basal class (see Figure S3), which is consistent with the conclusions of previous studies [26], [55], [56]. To investigate the effects of the number of parabasalian taxa on the inference of their root position, a fixed number of randomly chosen parabasalian taxa were excluded from the concatenated dataset of EF-1α, actin, and SSU rRNA gene sequences and analyzed repeatedly (Table 4). As the number of examined taxa became small, the root position gradually shifted to Trichonymphea, suggesting that the limited numbers of taxa sampling caused the wrong rooting at Trichonymphea.
Discussion
Importance of taxon sampling and new phylogenetic markers
The analyses of many important genera with multi-gene sequences in this study greatly expand our understanding of the evolution of parabasalian biodiversity. Actin and EF-1α sequences, although reported previously in some taxa [57], [74]–[77], are obtained newly in this study in all the examined taxa except for Trichomonas and Pentatrichomonas. For the first time, the concatenated dataset for 28 or 30 common taxa outlines the robust relationship of most of the major parabasalian groups and a more plausible new root position, thereby largely overcoming the problems encountered in previous molecular phylogenetic studies.
The increased number of parabasalian taxa sampled for the analyses is likely a key parameter in the improvement of Parabasalia rooting. A long-standing debate exists in phylogenetics about whether improved accuracy results from increasing the number of examined taxa (species) or the number of genes (informative characters) [65]–[67], [78]. Studies of empirical data often emphasize the importance of the number of taxa sampled. Particularly, if a small number of taxa that tend to cause long-branch attraction are evaluated with a large number of characters, some slight systematic biases can become magnified and misinterpreted as phylogenetic signals and may cause unfortunately well-resolved, but wrong inferences [78]. In this situation, the addition of taxa in the analysis is an efficient approach. Indeed, multiple changes in an alignment site are detected more easily, and the model parameters for the inference are optimized more precisely when many taxa are analyzed [66], [78].
Previous molecular phylogenetic studies typically have favored rooting at the branch leading to Trichonymphea. Hampl et al. [20], however, considered this rooting as an artificially generated wrong inference, even though they did not suggest any robust alternative position. The taxa exclusion analyses described in this study (see Table 4) strongly support the importance of the number of sampled taxa for the inference of the parabasalian root. As the number of examined taxa was reduced, the root gradually shifted to Trichonymphea. The addition of the protein sequences of α- and β-tubulins to the concatenated dataset for the reduced number of taxa (12) unfortunately resulted in rooting at the branch leading Trichonymphea (additional file 1: Figure S3), probably because some systematic biases caused the wrong inference in the limited taxon sampling. Altogether, we conclude that the increased number of sampling taxa improves the accuracy of the rooting of parabasalids, although closely related species to parabasalids as suitable outgroups are still unavailable.
This study provides a significant resolution of the relationship of the major groups of parabasalids. Except for GAPDH, previously examined protein sequences (tubulins and enolase) have been demonstrated to generate only low levels of phylogenetic signals [54], [55], [57], and indeed, concatenated analyses of these protein sequences have yielded conflicting results with poor resolution [26], [55], [56]. Individually, each of these proteins also demonstrated poorly resolved and conflicting relationships of major parabasalian groups (see Figure 1). Therefore, the addition of the actin and EF-1α protein sequences to the sequence concatenation is important for resolution. We conclude that multiple sequences from a single species do not have a strong impact on the phylogenetic analyses based on concatenated sequences. In the enolase case, anciently diverged sequences from a single parabasalid species emerged as two completely distinct positions in the parabasalian phylogeny [53]–[55], [57]. In this study, the sequences from a single species always occurred as close neighbors. The sequences from taxa in a single class formed, in most cases, a monophyletic or at least paraphyletic lineage. The relationships of closely related genera inferred with a certain protein, though poorly resolved, are also observed in many cases in the trees of the other two proteins (see Figure 1). Furthermore, the primers used to amplify actin and EF-1α genes are designed based on EST data of gut symbionts [68], [69] and these primers are matched completely to the EST sequences, but two distinct sequences showing distinct phylogenetic positions have not been obtained at all from a single species. Therefore, this minimized the likelihood that the sequences determined in this study contain anciently diverged paralogs.
Nevertheless, the phylogenetic relationship within some parabasalian classes such as Cristamonadea was resolved still poorly, probably owing to the limited phylogenetic information contained in actin and EF-1α sequences (maximum sequence differences of 17% and 15% between Cristamonadea members, respectively). Moreover, the possibility of several alternative phylogenetic relationships could not be rejected (see Table 2). Further study of other protein markers with sufficient taxon sampling is still needed. Recently, a conserved single-copy gene-encoding largest subunit of RNA polymerase II has been suggested as a useful marker [57]; however, only cultured parabasalids have been investigated so far and studies with yet-uncultivated parabasalids are needed. In the present study, Spirotrichonymphea and Hypotrichomonadea are represented by only two taxa and a further taxon sampling may improve the resolution; however, Hypotrichomonadea comprises only two genera [26] both of which are included in the analyses and Spirotrichonymphea cells, usually small in size, are very hard to distinguish by light microscopy if multiple species occur simultaneously in the gut of termites (and they very often do).
Phylogenetic relationships among parabasalids and their morphology
These improvements in molecular phylogeny of parabasalids provide us new insights about their evolutionary relationships. A salient point in the present study is the placement of Spirotrichonymphea, which branches out distantly from Trichonymphea and Cristamonadea in the tree based on the sequence concatenation, although the possibility of the sister-group relationship with the latter class cannot be excluded completely. In previous studies, Spirotrichonymphea has been placed ambiguously somewhere among parabasalids. The branch leading to Spirotrichonymphea is very long in phylogenetic trees based on the SSU rRNA gene sequence, suggesting its artificial placement [20], [35], [38]. Some studies have shown its affinity to Cristamonadea [20], [26], [37], and others have located it in a more basal position, near Trichonymphea [35], [55]. In the present study, such conflicting results were obtained in the single-gene phylogenies (see Figure 1).
Historically, Spirotrichonymphea and Trichonymphea have been considered evolutionary closely related to each other because of the similarity of their morphogenesis. Indeed, in both groups, two symmetrical sets of basal bodies, basal fibers, and flagellar bands are separated equally at cell division and then completed in the sister cells [1]. In Spirotrichonymphea, however, flagella are arranged uniquely in left-handed spiral bands originating at the cell apex. Brugerolle [79] has emphasized some common ultrastructural features between Spirotrichonymphea and trichomonads (members in the former family Trichomonadidae), such as the organization of a privileged basal body (#2 in his description) bearing preaxostylar fibers connected to the pelta-axostyle junction. According to our molecular phylogeny based on the concatenated dataset, Spirotrichonymphea and Trichonymphea are neither sister nor sequentially branching lineages. Therefore, we suggest that their common characteristics in morphogenesis have evolved convergently.
In addition to Spirotrichonymphea and Trichonymphea, lophomonads (Joenia, Joenoides, Joenina, and Deltotrichonympha; members in the former order Lophomonadida) included within Cristamonadea are parabasalids exhibiting a hypermastigid nature. In our trees, they are related usually distantly to Spirotrichonymphea and Trichonymphea. During cell division, lophomonads retain only four privileged basal bodies, and the flagella are reconstructed in the daughter cells, whereas Spirotrichonymphea and Trichonymphea permanently maintain a multiflagellar state [1]. This feature specific to lophomonads supports their independent emergence from the other two groups. As shown in the present study and previously [46], these lophomonad genera branch out basally in Cristamonadea, although Deltotrichonympha forms a distinct lineage from the other lophomonads in SSU rRNA gene sequence analyses [46], [80] and seem to have emerged more recently. In addition, simpler devescovinids and multinucleated calonymphids likely emerged later, and Tritrichomonadea, which is sister to Cristamonadea, contains the most rudimentary Histomonas and Dientamoeba. Therefore, the apical group of parabasalids comprising Cristamonadea and Tritrichomonadea have undergone dynamic morphological transitions of multiplication and reduction of flagellar and cytoskeletal systems as well as transitions to multinucleated status. Recently, a lophomonad species (Lophomonas striata) in a cockroach has been shown to be sister to Trichonymphea, not to nest within Cristamonadea, and thus “lophomnads” are completely polyphyletic [80].
In Trichonymphea, several morphological peculiarities have distinguished the families Hoplonymphidae (Hoplonympha) and Staurojoeninidae (Staurojoenina) from the other members of this class. Their flagellar areas are restricted to the anterior rostrum and form two and four symmetrical longitudinal rows in Hoplonymphidae and Staurojoeninidae, respectively [1], [70], [81], [82]. In the other Trichonymphea members, flagella in the rostrum form a so-called rostral tube, which is composed of two half-round plates of parabasal fibers. Furthermore, the similarity of Hoplonymphidae and Staurojoeninidae with trichomonads or Spirotrichonymphea members sometimes has been argued [70]–[72]. In our study, relationships within Trichonymphea were resolved fully and Trichonymphea was divided into two robust groups (see Figures 2 and 3). Each of the two groups contains either Hoplonymphidae or Staurojoeninidae as the basal lineage. If flagellar organization in the limited number of longitudinal rows is primitive, the rostral tube and flagella in the post-rostral area likely developed convergently in these two groups.
Our phylogenetic inference mostly corroborates the new classification proposed by Cepicka et al. [26]. Former classifications that elevate a group corresponding to Cristamonadea to the ordinal level [60], [61] have resulted in the paraphyly or polyphyly of members outside Cristamonadea, Spirotrichonymphea, and Trichonymphea, as previous studies have stated repeatedly [20], [26], [39], [46]. These members now are divided into the three classes—Tritrichomonadea, Hypotrichomonadea, and Trichomonadea—which comprise trichomonads and monocercomonads (members in the former families Trichomonadidae and Monocercomonadidae, respectively). These three classes clearly form distinct lineages in the trees reported in this study. Therefore, this reclassification indeed marks significant progress for parabasalian systematics. Nevertheless, some uncertainties remain. In our analyses, Tritrichomonadea was either monophyletic or paraphyletic at the base of Cristamonadea, but neither relationship was supported significantly. Because formal taxonomic units should be monophyletic, the revision of the taxonomic status of Tritrichomonadea (and also Cristamonadea) may be necessary as discussed previously [26]; for instance, these two classes can be united when data for more robust phylogenies become available. Moreover, several newly proposed classes have not received monophyletic confidence definitively owing to the lack of robust molecular phylogeny, and molecular phylogenetics often denies morphology-based classifications from family to genus or species levels [26], [44], [46]. A large number of species examined with the SSU rRNA gene sequences, which provide only a low level of phylogenetic resolution, have not yet been analyzed using protein sequences. These species are, for example, Honigbergiella, Ditrichomonas, Lacusteria, and Hexamastix (Trichomonadea) or Simplicimonas (Tritrichomonadea).
Evolutionary implications
The new root position that we have uncovered suggests that the ancient, most primitive parabasalid has a trichomonad-like character (Figure 5), although other possibilities cannot be excluded. Supporting this conclusion, all the possible alternative basal lineages of parabasalids (root positions b, e, f, and j in Figure 3; see also Table 3) are close to or representative of the classes including trichomonads except for the Trichonymphea lineage, which likely results from an incorrect inference as discussed above. In particular, trichomonads in Tritrichomonadea, Hypotrichomonadea, and Trichomonadea are specified by the presence of a costa and undulating membrane. Their recurrent flagellum is associated with the cell body, forming an undulating membrane underlain by a striated fiber, the costa. The structures of the costa and undulating membrane exhibit variations among these three classes. The costa has a similar banding pattern in members of Tritrichomonadea and Hypotrichomonadea (A-type striation according to Honigberg et al. [72]), whereas the costa of Trichomonadea shows a different banding pattern (B-type striation). The undulating membrane is rail-like (in Tritrichomonas) or lamelliform (in Simplicimonas) in Tritrichomonadea and lamelliform in both Hypotrichomonadea and Trichomonadea. The primitive parabasalid likely possessed the lamelliform undulating membrane, and the rail-like undulating membrane evolved later. The homologous protein components of both types of costa suggest their common origin [83]. The differentiation of the two types of costa probably occurred very early in parabasalid evolution; however, the A-type striation pattern may be primitive because it also occurs in the parabasal fibers of most parabasalids. The common ancestor of parabasalids very likely possessed a parabasal apparatus and hydrogenosomes because they are common characters of parabasalids (Figure 5).
The tree shows the relationships of the six parabasalian classes. Flagellar multiplication in a single mastigont system has occurred independently in the boxed classes. The multiplications have occurred ancestrally in two classes (marked with filled circles) and probably twice within the other class (open circle). Triangles indicate the occurrence of cytoskeletal simplification in undulating membrane (UM) and costa. See the text for details.
Based solely on comparisons of morphological characters, the most ancient lineage of parabasalids historically has been believed to be monocercomonads owing to their cytoskeletal simplicity, and to the assumption that complex structure and morphology developed later during parabasalian evolution [58], [59]. Earlier molecular phylogenetic studies challenged this simple-to-complex evolutionary scheme, and have suggested almost the reverse scheme owing to the basal placement of the most elaborate Trichonymphea. The new root position described in this study indicates that the evolution of parabasalids is principally simple-to-complex, but the complexity has emerged independently in multiple lineages in different modes of flagellar system multiplication.
Possible occasional reversions to simpler forms make the matter more complicated. Monocercomonads, the most rudimentary parabasalids, are polyphyletic, as clearly shown in the present study as well as by previous work [16], [18], [24]–[26]. Indeed, some monocercomonads such as Monocercomonas and Histomonas are related closely to Tritrichomonas. The monocercomonad Hypotrichomonas forms a clade with Trichomitus. Other monocercomonads such as Ditrichomonas and Honigbergiella are likely related to Trichomonadea according to analyses based on SSU rRNA gene sequences [16], [17], [24]. Therefore, secondary reduction of cellular complexity seems to have occurred in each of the three classes—Tritrichomonadea, Hypotrichomonadea, and Trichomonadea (see Figure 5).
A growing number of free-living parabasalids have been investigated recently based on the SSU rRNA gene sequence in Honigbergiella, Lacusteria, and Pseudotrichomonas in addition to Ditrichomonas and Monotrichomonas [26], [27], although phylogenetic positions of these free-living species was not examined in this study. The free-living species seemingly are dispersed in SSU rRNA gene-based phylogenetic trees but many form a paraphyletic assemblage near the origin of Trichomonadea [27]. Considering the new root position of Parabasalia inferred by this study, it is possible that free-living species represent the most basal lineages of Parabasalia (Figure 5). This possibility needs to be investigated further, because it is of ecological and evolutionary significance for the origin of parabasalids as well as parasitic trichomonads as discussed previously [27].
The hypermastigid nature of flagellar multiplication in a single mastigont has evolved independently at least three times, and these multiplications led to the Trichonymphea, Spirotrichonymphea, and lophomonads in Cristamonadea (see Figure 5). Because these three classes are found only in the gut of termites and cockroaches, each ancestor of these three classes (presumably a trichomonad-like species) established a symbiotic relationship with a host ancestor and evolved and diversified in the gut of their hosts as previously described for Trichonymphea members [47]. Adaptation to the gut environment significantly affects their morphological evolution. Indeed, the development of their flagella and associated cytoskeletal system is advantageous for the improvement of fitness in this niche, because it allows vigorous movement that prevents them from flowing out of the gut and facilitates their access to food in the gut. Their habitats also likely affect cell size, which is associated with cytoskeletal development. The cell of the gut symbionts is large enough to incorporate masticated wood particles by phagocytosis, whereas parasitic and free-living species absorb smaller molecules or tiny bacterial cells as food. Meanwhile, members of the genera Pseudotrypanosoma and Trichomitopsis in Trichomonadea are also found among the gut symbionts of termites, and their large cell sizes seem to represent a consequence of their adaptation to the gut environment, although they apparently do not develop a complex flagellar system [33], [36].
Conclusions
This study provides a revised, taxonomically broad phylogenetic framework for Parabasalia. We consider both the increasing number of taxa sampled and the use of new protein markers as particularly important factors in the accuracy and robustness of our inferences. Culture-independent analyses of the termite-gut symbionts are critical for collecting data for the large number of examined taxa, and such techniques are powerful for the investigation of additional data and taxa. The evolution of Parabasalia is complex in terms of morphology owing to a number of independent multiplications and simplifications of flagella and associated cytoskeletal structures. Morphology-based systematics sometimes has hampered the understanding of the true nature of parabasalian evolution. Likely, their ecology greatly affects evolution through adaptation to the niches and co-diversification with their hosts.
Materials and Methods
Cultivation and DNA extraction of trichomonads
Culturable strains used were as follows: Tetratrichomonas gallinarum strain A6 (cf. [17], [84]); T. foetus strain KV1 (ATCC 30924); Monocercomonas sp. strain NS-1PRR (ATCC 50210); Trichomitus batrachorum strain G11 (ATCC 30066); Hypotrichomonas acosta strain L3 (ATCC 30069). The origins of their isolation are shown in Table 1. All strains were grown axenically at 37°C or 27°C in trypticase-yeast extract-maltose (TYM) medium [85] without agar supplemented with 10% (v/v) heat-inactivated horse serum (Gibco-BRL), 100 U/mL of penicillin G, and 50 µg/mL of streptomycin sulfate. Genomic DNA was isolated as described [86]. DNAs of Dientamoeba fragilis strain Bi/PA (ATCC 30948) and Histomonas meleagridis strain HmZL [18] were provided by C. G. Clark (London School of Hygiene and Tropical Medicine, London, UK) and F. Delbac (LMGE, CNRS UMR 6023, Aubière, France), respectively.
Manipulation of termite symbionts
Table 1 lists the gut symbionts investigated in this study and their host termites. All taxa were found stably in the hindgut flora of the respective termites and were easily recognizable on the basis of their morphological characters [1], [3]. According to a comprehensive list of flagellate species in the gut of termites [3], cristamonads inhabiting each termite species are as follows: P. adamsoni and I. minor harbors only J. pulchella and M. cuspidata, respectively; H. mossambicus harbors only J. intermedia and G. herculea; K. flavicollis harbors two Foaina spp. in addition to J. annectens; C. cavifrons harbors species of Foaina in addition to the three cristamonads examined in this study; and C. domesticus harbors species of Devescovina and Stephanonympha in addition to F. nana. As cristamonad symbionts, N. koshunensis harbors species of Foaina as well as Devescovina and Stephanonympha [37], and G. satsumensis harbors species of Foaina and Devescovina in addition to Macrotrichomonas sp. [46]. The SSU rRNA and GAPDH genes of the cristamonad species mentioned above (except Stephanonympha sp.) were analyzed simultaneously using the same cell preparations [46]. M. darwiniensis harbors species of Koruga, Mixotricha, and Metadevescovina in addition to Deltotrichonympha [3], and the SSU rRNA gene sequence of Deltotrichonympha sp. obtained from our preparation was almost identical to those of Deltotrichonympha spp. (AJ583378, AJ583378, and AB326380). C. formosanus harbors only three parabasalian species [3], all of which were examined in this study. As species in Trichonymphea that show conspicuous morphology and thus are easily recognizable, I. minor harbors only S. assimilis [3], R. speratus harbors only T. agilis and T. mirabilis [3], H. sjoestedti harbors only species of Trichonympha, Eucomonympha, and Hoplonympha [87]. All of these genera were examined in this study.
M. darwiniensis, P. adamsoni, K. flavicollis, and H. mossambicus were collected in Australia, Australia, France, and Kenya, respectively, and generously provided by C. Bordereau (Université de Bourgogne, France). C. cavifrons collected in the United States was provided by M. F. Dolan (University of Massachusetts, USA). I. minor collected in Japan was provided by W. Ohmura (Forestry and Forest Products Research Institute, Japan). G. satsumensis, C. domesticus, C. formosanus, R. speratus, and H. sjoestedti were collected in Japan.
The cells of parabasalian symbionts in the hindgut suspension of each termite were isolated manually and washed extensively under a microscope equipped with a micromanipulator (Cell Tram, Eppendorf) as described elsewhere [88], [89]. A single cell or a pool of 10–30 cells showing typical morphology were isolated and used as templates for RT-PCR (see Table 1). In some cases, the isolated cells were subjected to isothermal whole-genome amplification (WGA) as previously described [90], [91], and the amplified genome DNA was used as a template for PCR (see Table 1).
Cloning and sequencing of actin, EF-1α, and GAPDH genes
The protein-coding genes were amplified using RT-PCR from the isolated cells of termite-gut symbionts using protein-specific primers for the amino-terminal conserved region and the oligo-dT primer as previously described [55]. The design of the actin and EF-1α primers was based on the comparisons of the EST data of the gut symbionts of termites [68], [69] and sequences of various eukaryotes because we found sequence mismatches between previously used primers and the EST data. The following protein-specific primers were used: actin-F1, 5′-TGGGANGANATRGARAARATYTGG3′ and EF1F1, 5′-AARGCDGARCGNGARCGDGG-3′. The protein-specific primers for the carboxy-terminal conserved region used for PCR were actin-R1, 5′-GAAGCAYTTNCKRTGNACDAT-3′ and EF1R1, 5′-GRAAYTTRCANGCDATRTG-3′. If sufficient amplification product was not obtained during the first PCR, a second amplification was performed using the primer actin-F2, 5′-ATRGARAARATYTGGCAYCA-3′ and the oligo-dT primer for the actin gene, and the primer EF1F2, 5′-CGDGGDATYACNATYGAYAT-3′ and the EF1R1 or oligo-dT primer for the EF-1α gene. The GAPDH gene was amplified with RT-PCR using previously designed primers [55]. The amplification products were separated using agarose gel electrophoresis, purified, and cloned into pCR2.1-TOPO (Invitrogen). Clones containing inserts of the expected size were picked and partially sequenced, and the complete DNA sequence of each representative clone was obtained via primer walking. The sequences obtained in this study have been deposited in the DNA Databank of Japan and the accession numbers are shown in Figure 1.
Phylogenetic analyses
The protein sequences identified in this study and publicly available were used for the analyses. The genome sequence of T. vaginalis G3 [73] was searched for homologous sequences with the three proteins. The EST sequences of Pentatrichomonas hominis in the public database (FL516063–FL518016) were also searched, and the identified ESTs were assembled to produce in silico-translated amino acid sequences. All the non-identical amino acid sequences showing significant identities to sequences of other parabasalids (such as >50% identity) and covering most of the protein region were included in the analyses. In the case of P. hominis, only a single unique sequence was found in each protein. The recently reported GAPDH sequences of H. meleagridis [56] were also used.
These protein sequences were aligned using ClustalW2 [92] and refined manually. Only unambiguously aligned positions were used for phylogenetic inference. Because any outgroup sequences may cause long-branch attraction owing to their distant relationships to parabasalids, the phylogenetic tree of a single protein was inferred without outgroups. The appropriate model of sequence evolution was selected using the program ProtTest 2.4 [93]. For the tree of each single protein dataset, maximum likelihood (ML) analysis was carried out with RAxML 7.2.6 [94] using the PROTGAMMAWAG model. Four gamma-distibuted substition rate categories were used in this and the following analyses.
The alignments of the protein sequences and previously aligned sequences of the SSU rRNA gene [46] were concatenated manually. When the analyses included GAPDH, the taxa Deltotrichonympha sp. and D. fragilis were excluded because of the lack of their GAPDH sequences. The outgroup taxa and their sequences used for the analyses are shown in Table S2. The sequence alignments used for the analyses shown in Figures 2 and 3 are available as supplementary data (Datasets S1 and S2).
For the analyses of only parabasalids, the ML tree was estimated in RAxML using mixed models (GTRGAMMA for the SSU rRNA gene and PROTGAMMAWAG for each protein sequence). Parameters and branch length were optimized for each of the partitions individually and bootstrap values were obtained from 1000 replicates. The ML estimation and the bootstrap were also conducted with the site-heterogeneous CAT model in RAxML. Bayesian analysis was performed in MrBayes 3.1.2 [95] using separate models (GTR+I+Γ for the SSU rRNA gene and WAG+I+Γ for each protein sequence), and parameters and branch lengths were optimized for each partition individually. The starting tree was random, and four simultaneous Markov chains in duplicate were run for 10,000,000 generations. Log likelihoods stabilized well before 2,000,000 generations, and the remaining generations were used to measure Bayesian posterior probabilities. The homogeneity of sequence composition in each protein or gene was evaluated in the χ2 test implemented in TREE-PUZZLE 5.2 [96].
For the analyses with outgroup taxa, the ML tree was estimated as described above but using the CAT model (GTRMIX and PROTMIXWAG in RAxML) instead of the site-homogeneous model, because the site-heterogeneous CAT model appears to be more robust than site-homogeneous models against artifacts by long-branch attraction [97]. Bootstrap analyses of 1000 replicates were conducted with the CAT model in RAxML. Bayesian analysis was performed in MrBayes as described above.
Differences in alternative tree topologies were compared with the SH test implemented in CONSEL [98] using the site-wise log-likelihood outputs obtained with RAxML. Using only the data from parabasalids, an alternative tree topology was obtained under the constraint of a given phylogenetic hypothesis by the RAxML analysis with the same substitution model described above. For the root of parabasalids, the outgroup was grafted onto 11 possible root positions of parabasalids and tree topology was obtained under each constraint, and these root positions were evaluated using the SH test. To measure the effect of the number of analyzed taxa on the parabasalian root position, randomly chosen parabasalian taxa were excluded and analyzed repeatedly with different sets of fixed numbers of excluded taxa.
Supporting Information
Figure S1.
Exclusion of GAPDH from the sequence concatenation of parabasalids. The GAPDH sequence was excluded from the dataset used for the inference shown in Figure 2, and the sequences of Deltotrichonympha sp. and Dientamoeba were added to the analysis. The tree was inferred using RAxML with the parameters and branch length optimized for each partition. The bootstrap values above 50% are indicated at the nodes. Note that the relationship of the six parabasalian classes and the root position did not change after the exclusion except for the paraphyly of Tritrichomonadea.
https://doi.org/10.1371/journal.pone.0029938.s001
(PDF)
Figure S2.
Exclusion of SSU rRNA gene from the sequence concatenation of parabasalids. The SSU rRNA gene sequence was excluded from the dataset used for the inference shown in Figure 2, and the tree was inferred using RAxML with the parameters and branch length optimized for each partition. The bootstrap values above 50% are indicated at the nodes. Note that the relationship of the six parabasalian classes and the root position did not change after the exclusion except for the paraphyly of Tritrichomonadea and the sister-group relationship of Spirotrichonymphea and Hypotrichomonadea. The removal of the SSU rRNA gene sequence was particularly important because the base composition of this gene was heterogeneous in the two Spirotrichonymphea members and Histomonas. All taxa were homogeneous in amino acid composition in the three protein sequences.
https://doi.org/10.1371/journal.pone.0029938.s002
(PDF)
Figure S3.
Maximum likelihood tree of the sequence concatenation of actin, EF-1α, α-tubulin, β-tubulin, and SSU rRNA gene in 13 parabasalian taxa and outgroup eukaryotes. Unambiguously aligned sites of α-tubulin (351) and β-tubulin (145) were concatenated with the dataset used for the analysis shown in Figure 3. The sequence data for both tubulins used for the concatenation are the same as described previously [55]. The tree was inferred using RAxML with the parameters and branch length optimized for each partition. The bootstrap values above 50% are indicated at the nodes. Because the sequences of α- and β-tubulin available for Pentatrichomonas and Entamoeba are short, we excluded them from the analysis. Trichonymphea was the most basal parabasalian lineage in this analysis; however, this rooting was likely a wrong inference caused by the limited taxon sampling (see the main text). Spirotrichonymphea instead of Tritrichomonadea was sister to Cristamonadea, but this change was not supported at all. The taxa exclusion analyses (see Table 4) indicated that when the number of parabasalian taxa was reduced, the frequency of the pairing of Cristamonadea and Spirotrichonymphea increased up to four times in 10 repeated analyses, suggesting an artificial pairing owing to the limited number of examined taxa.
https://doi.org/10.1371/journal.pone.0029938.s003
(PDF)
Table S1.
Removal of long-branch outgroup taxa and the effect on bootstrap values for major nodes within Parabasalia.
https://doi.org/10.1371/journal.pone.0029938.s004
(DOC)
Table S2.
Species used for the outgroup and their sequence accession numbers used for the concatenation.
https://doi.org/10.1371/journal.pone.0029938.s005
(DOC)
Dataset S1.
NEXUS-format alignment of sequence concatenation of GAPDH, actin, EF-1α, and SSU rRNA gene used for the analyses shown in Figure 2 .
https://doi.org/10.1371/journal.pone.0029938.s006
(TXT)
Dataset S2.
NEXUS-format alignment of sequence concatenation of actin, EF-1α, and SSU rRNA gene used for the analyses shown in Figure 3 .
https://doi.org/10.1371/journal.pone.0029938.s007
(TXT)
Acknowledgments
We especially thank T. Kurisaki for assistance in DNA sequencing. We also thank W. Ohmura, C. Bordereau, and M. F. Dolan for providing termite samples, and C.G. Clark and F. Delbac for providing trichomonad DNAs. We thank the RIKEN Integrated Cluster of Clusters for computing resources used for some of the analyses. We thank anonymous reviewers for their helpful comments on previous versions of the manuscript.
Author Contributions
Conceived and designed the experiments: SN EV MO. Performed the experiments: SN CM DM JI OK. Analyzed the data: SN JI MO. Wrote the paper: SN EV MO.
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