1. Eukuryof. Milmhrol.. Sl(5). 2004 pp. S75-581 0 2004 by the Society of Protozoologists Molecular Phylogenetic Analysis Places Percolomonas cosmopolitus within Heterolobosea: Evolutionary Implications SERGEY I. NIKOLAEV; ALEXANDRE P. MYLNIKOV,” CEDRIC BERNEY; JOSE FAHRNI; JAN PAWLOWSKI; VLADIMIR V. ALESHIN” and NIKOLAY B. PETROVaJ “Departmentof Evolutionary Biochemistry. A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia, and bInstitutefor Biology of Inland Waters, RAS, Yaroslavskaya obl,, Borok, Russia, and ‘Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland ABSTRACT. Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolornonus shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic recon- structions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group. Key Words. Helix 17, Heterolobosea, phylogeny, SSU rRNA, V3 region. HE free-living flagellate Percolornonas cosmopolitus is al. 1990). Later, the class Lyromonadea was especially estab- T common and widespread in surface coastal and oceanic lished to include P. lanterna (Cavalier-Smith 1993). Molecular waters. Initially, Ruinen (1938) described this organism as a phylogenetic studies revealed a high rate of divergence of the representative of the heterolobosean genus Tetramitus on the small-subunit ribosomal RNA (SSU rRNA) gene of P. lanterna basis of morphological and ecological characters, such as gen- compared to vahlkampfiids and have shown its basal position eral body form, flagellation, dimensions, and trophic behaviour. to the Vahlkampfiidae (Weekers, Kleyn, and Vogels 1997). More recently, on the basis of ultrastructural evidence, Fenchel Two anaerobic amoebae, Monopylocystis visvesvarai and Saw- and Patterson (1986) established for it a new genus Percolo- yeria marylandensis, were shown to be related to P. lanterna monas of uncertain phylogenetic position within the class Het- on the basis of SSU rRNA gene analyses (O’Kelly et al. 2003; erolobosea. Ultrastructural features of Percolomonas were con- Silberman et al. 2002). sidered primitive for eukaryotes by Cavalier-Smith (1 993), who Complementary to molecular evolution studies, there are proposed a new class Percolomonadea, constituting, together works on the organization of the flagellar apparatus of hetero- with the classes Heterolobosea and Lyromonadea, a new phy- loboseans (Broers et al. 1990; Brugerolle and Simpson 2004; lum Percolozoa. Later, the class Percolatea was erected to unite Fenchel and Patterson 1986; Simpson 2003). In some Perco- Percolornonus and Stephanopogon (Cavalier-Smith 2004). It lornonas species, the striated ciliary roots, which for a long time has been suggested that Percolomonas, lyromonads and (other) were thought to be absent in this genus, were described. As heteroloboseids belong to the taxon Excavata (Cavalier-Smith striated ciliary roots are known for all other flagellated Heter- 2002; Simpson and Patterson 1999), a group otherwise encom- olobosea, the authors disputed the validity of such groups as passing a diversity of aerobic and anaerobic flagellates. the Percolomonadea, Lyromonadea, and Percolozoa (Brugerolle The class Heterolobosea was established by Page and Blan- and Simpson 2004). ton (1985) to unify Acrasida (former slime molds) and Schi- There is a relatively large SSU rRNA database for represen- zopirenida (amoeboflagel1ates)-amoeboid organisms that tatives of the Heterolobosea (Brown and De Jonckheere 1999; share eruptive movement in the amoeboid stage, discoid mito- De Jonckheere and Brown 1999; Sogin et al. 1996; Weekers, chondrial cristae, and lack typical dictyosomes. Many hetero- Kleyn, and Vogels 1997), while the Discicristates (i.e. Eugle- loboseans are capable of altering from amoeboid to flagellate nozoa and Heterolobosea) are poorly represented in the actin stages; some are solely amoeboid. Now, it is clear that some gene database. In the current study, in order to ascertain the organisms known only as flagellates also belong to this group taxonomic position of Percolornonas, we obtained the actin and (Fenchel and Patterson 1986). Based on the capacity to form SSU rRNA gene sequences of P. cosmopolitus and performed so-called “fruiting bodies” or not, the class Heterolobosea was phylogenetic analyses including all heterolobosean sequences separated into two orders, Acrasida and Schizopyrenida, re- available to date. To overcome the problems caused by the high spectively. The order Schizopyrenida is traditionally divided level of substitution rate heterogeneity across taxa in the SSU into two families, Vahlkampfiidae and Gruberellidae, on the rRNA (Petrov and Aleshin 2002; Philippe and Germot 2000; basis of the presence of a flagellate stage, the trophozoite mor- Rokas and Holland 2000), conventional methods of phyloge- phology, and mode of mitosis. The family Vahlkampfiidae in- netic inference were coupled with a comparative analysis of cludes several genera, among which are Vahlkarnpjia, Tetram- molecular synapomorphies at the level of the SSU rRNA sec- itus, Naegleria, and Heteramoeba. The genus Vahlkarnpja was ondary structure. primarily established for vahlkampfiid amoebas without a fla- gellate stage in their life cycle (Page and Blanton 1985). Later, MATERIALS AND METHODS two species (i.e. Paravahlkampjia ustiana and Neovahlkarnpjia Cell cultures and DNA extraction, amplification, cloning, darnariscotta) were excluded from the genus due to their high and sequencing. The cells of Percolomonas cosrnopolitus rates of morphological and genetic divergence (Brown and De (Ruinen 1938) were obtained from White Sea coastal waters. Jonckheere 1999). Some authors included the anaerobic and They have been cultivated on the mineral Schmaltz-Pratt me- amitochondriate Psalteriomonas lanterna in the class Hetero- dium (artificial sea water) at 20 “C, 20%0 salinity, with the bac- lobosea, as a member of the order Schizopyrenida (Broers et terium Aerobacter aerogenes as a food source. DNA was iso- lated by phenol extraction and precipitated with ethanol. A par- I Corresponding Author: N. PETROV-Telephone number: 7-095- tial fragment of the actin gene was amplified using the forward 939 1440. FAX number 7-095-9393 181 ; E-mail: [email protected] primers Act-F1 (5’-CNG ARG CDC CAT TRA AYC-3’) and 575 576 J. EUKARYOT. MICROBIOL., VOL. 51, NO. 5, SEPTEMBER-OCTOBER 2004 Act-N2 (5’-AAC TGG GA(CT) GA(CT) ATG GA-3’) and the approximately unbiased (AU) test (Shimodaira 2002) using reverse primer Act-l354r (5’-GGA CCA GAT TCA TCA TAY CONSEL (Shimodaira and Hasegawa 2001). Elements of the TC-3‘). The entire SSU rRNA gene was amplified using uni- SSU rRNA molecule secondary structure were modeled with versal eukaryotic primers for nuclear SSU rRNA coding regions mfold (Zuker, Mathews, and Turner 1999) and visualized with (Medlin et al. 1988). PCR products were purified by agarose RnaViz (De Rijk and De Wachter 1997). Actin and SSU rRNA gel electrophoresis, cloned in the pGEM-T Vector System (Pro- alignments are available upon request from the authors. Species mega), and manually sequenced on both strands using thefmol names and corresponding GenBank accession numbers of all DNA sequencing system (Promega). The length of the ampli- taxa included in our analyses are given in Fig. 1 and 2. fied sequences of actin and SSU rRNA genes of P. cosmopol- itus were 784 and 1,8 10 nucleotides, respectively. The sequenc- RESULTS es have been submitted to the GenBank database under acces- Morphology. The cultured strain of P. cosmopolitus used in sion numbers AY283751 for the actin gene and AF519443 for this study fully corresponds morphologically to the one exam- the SSU rRNA gene. ined in a previous ultrastructural work (Fenchel and Patterson Phylogenetic and secondary structure analyses. The par- 1986), which provided the detailed description of the species. tial actin sequence of P. cosmopolitus was manually fitted to Phylogenetic analyses. Our BA of actin genes (Fig. 1) re- an alignment of 15 eukaryotic actin sequences. In the analyses, veals a close relationship between P. cosmopolitus and two spe- 254 amino acid positions were included. A Bayesian analysis cies of the vahlkampfiid, heterolobosean genus Naegleria, with (BA) of the data was conducted using MrBayes, version 2.01 a posterior probability (PP) of 0.94. Moreover, the Percolo- (Huelsenbeck and Ronquist 2001) with four simultaneous runs monas + Naegleria clade forms a sister-group to the Eugle- of the Markov chain Monte Car10 algorithm. The chains were nozoa (PP of 1.00), supporting the monophyly of discicristates. run for 600,000 generations with sampling every 10 genera- However, because no other actin genes of Heterolobosea are tions. The states of the chains before they reached stationarity available to date, this analysis does not