Proc. Nail. Acad. Sci. USA Vol. 89, pp. 9764-9768, October 1992 Evolution A broad molecular phylogeny of ciliates: Identification of major evolutionary trends and radiations within the phylum (large subunit rRNA/sequence/evolution) ANNE BAROIN-TOURANCHEAU, PILAR DELGADO*, ROLAND PERASSO, AND ANDRE ADOUTTE Laboratoire de Biologie Cellulaire 4, Centre National de la Recherche Scientifique, Unite Associte 1134, Bftiment 444, Universit6 Paris-Sud, 91405 Orsay Cedex, France Communicated by Andre' Lwoff, June 4, 1992 (receivedfor review April 3, 1992) ABSTRACT The cellular architecture of ciliates is one of with a typical set of cytoskeletal fibers (see refs. 1 and 2). the most complex known within eukaryotes. Detailed system- Within the phylum, diversification is first manifested by the atic schemes have thus been constructed through extensive overall pattern of implantation of the cilia over the cell comparative morphological and ultrastructural analysis of the surface and in a region specialized for food ingestion, the oral ciliature and of its internal cytoskeletal derivatives (the infra- apparatus. This has formed the basis of all the early system- ciliature), as well as of the architecture of the oral apparatus. atics of the groups (3) and of the "classical" phylogenetic In recent years, a consensus was reached in which the phylum hypotheses, which viewed ciliate evolution as progressing was divided in eight classes as defined by Lynn and Corliss from cells with simple, apical, and symmetrical oral appara- [Lynn, D. H. & Corliss, J. 0. (1991) in Microscopic Anatomy tuses with homogeneously distributed cilia, to cells with ofInvertebrates: Protozoa (Wiley-Liss, New York), Vol. 1, pp. complex, dissymetrical oral apparatuses and uneven distri- 333-467]. By comparing partial sequences of the large subunit bution of cilia. The organization of the oral apparatus led to rRNA molecule, and by using both distance-matrix and max- the widely adopted three-subphylum system (4)-Kinetof- imum-parsimony-tree construction methods (checked by boot- ragminophora, Oligohymenophora, and Polyhymenophora, strapping), we examine the phylogenetic relationships of 22 cited in the order thought to reflect an increase in complexity species belonging to seven of these eight classes. At low and evolutionary trend. Karyorelictids, ciliates with a very taxonomic levels, the traditional grouping of the species is peculiar, nearly diploid nondividing macronucleus, were generally confirmed. At higher taxonomic levels, the branching later erected as a fourth subphylum, thought to correspond to pattern of these seven classes is resolved in several deeply the earliest emerging line among ciliates (5). The detailed separated major branches. Surprisingly, the first emerging one comparative analysis of ultrastructural organization of basal contains the heterotrichs and is strongly associated with a bodies and their derivatives led to a substantially different karyorelictid but deeply separated from hypotrichs. The lito- three-subphylum system-Rhabdophora, Postciliodesmato- stomes, the oligohymenophorans, and the hypotrichs separate phora, and Cyrtophora (6). Although quite different at very later in a bush-like topology hindering the resolution of their high taxonomic levels, the two systems presently recognize order of diversification. These results show a much more which amount to different ones ancient origin of heterotrichs than was classically assumed, almost the same classes, eight indicating that asymmetric, abundantly ciliated oral appara- in Lynn and Corliss' most recent treatment (1). tuses do not correspond to "highly evolved" traits as previously The updating of these phylogenies with rRNA molecular thought. They also suggest the occurrence of a major radiative analyses has started (7-12). Small- and large-subunit (SSU explosion in the evolutionary history ofthe ciliates, yielding five and LSU) rRNA phylogenies appeared to be promising ofthe eight classes of the phylum. These classes appear to differ because the phylum was sufficiently old and molecularly essentially according to the cytoskeletal architecture used to diversified. Some major groups have hence been identified shape and sustain the cellular cortex (a process of essential which, to a large extent, are in good agreement with the adaptative and morphogenetic importance in ciliates). traditional systematics at "low" and "intermediate" taxo- nomic levels. The depth of their divergence also confirmed the large intra- and intergroup genetic distances observed The phylum Ciliophora constitutes a large group of unicel- through the study of enzyme polymorphism (13), structural lular eukaryotes containing over 7000 species which have protein comparisons (14), and DNADNA hybridization (15). colonized a remarkable diversity of ecological niches. The Major questions concerning high-level (i.e., interclass) rela- typical ciliate cell displays one of the most highly differen- tionships remain open, however. It is such questions that are tiated and elaborate organizations among eukaryotes both in addressed in the present work. Partial sequences of the 28S terms of the variety of physiological functions carried out by LSU rRNA from 22 species belonging to seven classes (all the the single cell and in terms ofthe diversity and complexity of classes except the Phyllopharyngea) and including one kary- the organelles making up the cell. In addition, a bewildering orelictid have been obtainedt and treated with distance- variety ofcell shapes has been elaborated within the phylum. matrix and parsimony tree-building algorithms to yield phy- Members of the group, however, are united by several clear logenetic schemes, the robustness ofwhich was evaluated by synapomorphies which have long been considered as a bootstrap analysis. There are three major interests in such an testimony of its monophyly. Foremost among these are the approach. (i) It allows an extensive test of the congruence nuclear dimorphism, with germinative micronuclei and veg- between morphologically and molecularly based phyloge- etative macronuclei; a sexual process ofreproduction involv- netic schemes in a group of unicellular eukaryotes especially ing conjugation; and a complex pellicular and subpellicular rich in traits. It should provide insights on structure comprising cilia, often organized along longitudinal morphological (ii) anteroposterior rows (kineties), with basal bodies associated Abbreviations: LSU, large subunit; SSU, small subunit. *Present address: Departamento de Microbiologia, Facultad de The publication costs of this article were defrayed in part by page charge Biologia, Universidad de Sevilla, Apdo 1095, 41080 Seville, Spain. payment. This article must therefore be hereby marked "advertisement" tThe sequences have been deposited in the GenBank data base in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession nos. M98361-M98388). 9764 Downloaded by guest on September 26, 2021 Evolution: Baroin-Tourancheau et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9765 a remarkable experience of diversification at the unicellular comparison in this area can also be carried out in two level, instructing us on how the major morphological and subdomains in which the variations in rate of substitution and morphogenetic strategies of ciliates evolved and, in particu- in length are moderate. An additional 100-nucleotide con- lar, how their hyperdeveloped cytoskeleton was elaborated. served region, 2000 nucleotides downstream from the 5' end, (iii) It may shed light on the evolution of the peculiar genetic has also been sequenced for a subset of species. Therefore, code observed within the phylum (reviewed in ref. 16). depending on the species sample under analysis, increasing lengths of sequence can be analyzed. (Sequences have been MATERIALS AND METHODS submitted to the GenBank/EMBL data bank and an aligned Sources of Ciliate Cultures. Fresh cultures of Blepharisma set is available upon request to the authors.) The sequences japonicum, Chaenea vorax, Loxodes striatus, and Uronychia taken from the EMBL data bank are those of Tetrahymena sp. were generously supplied by G. Fryd-Versavel (Labora- thermophila (X54512) and Tetrahymena pyriformis (X54004). toire de Biologie Evolutive et Dynamique des Populations, Alignment of sequences, computation of the observed and Universitd Paris-Sud, Orsay, France). Fresh cultures of the corrected numbers of nucleotide differences obtained by Colpidium campylum, Didinium nasutum, Enchelys pelu- using Kimura's Knuc correction (20), derivation of the re- cida, Pseudomicrothorax sp., Stylonychia lemnae, Urostyla sulting matrices, and formatting for the various tree-building sp., Euplotes aediculatus, Stentor coeruleus, Coleps sp., programs were carried out with the MUST package developed Paraurostyla sp., and Pleuronema marinum were grown in in our laboratory by H. Philippe (unpublished work). Den- the laboratory. Identification by protargol impregnation was drograms were constructed by two distance-matrix methods carried out by F. Iftode (this laboratory) and reference slides [neighbor-joining according to Saitou and Nei (21) and pro- have been conserved. Frozen cells of Isotricha prostoma grammed by H. Philippe; FITCH program of Felsenstein's were a gift from B. Vigues (Laboratoire de Zoologie et PHYLIP 3.2 package (Department of Genetics, University of Protistologie, Universitd Blaise Pascal, Clermont-Ferrand, Washington, Seattle, WA)] and by the maximum-parsimony France). Total RNA from