0012-4966/03/1112- © 2003 MAIK “Nauka / Interperiodica” 0553 Doklady Biological Sciences, Vol. 393, 2003, pp. 553–556. Translated from Doklady Akademii Nauk, Vol. 393, No. 4, 2003, pp. 570–573. Original Russian Text Copyright © 2003 by Nikolaev, Berney, Fahrni, Mylnikov, Petrov, Pawlowski. Several years ago, the taxonomy of protists (unicel- lular eukaryotes) was still based exclusively on mor- phological and ultrastructural characters. For example, the taxonomic group Heliozoa was distinguished on the basis of the body shape and the presence of radial axopodia emerging from the protist body and rein- forced from inside by microtubules. For a long time, this group was considered to be monophyletic. Studies on ultrastructural organization demonstrated that the mitochondrial cristae in centrohelid and desmothoracid Heliozoa were lamellar and tubular, respectively. Because it is generally believed that the shape of the mitochondrial cristae is one of the most conserved characters related to the basal radiation of eukaryotes [1], this finding cast doubt on the suggestion that cen- trohelid and desmothoracid Heliozoa are closely related [2]. On the other hand, it was suggested on the basis of these data that centrohelid Heliozoa were closely related to gymnophryid amoebas (with lamellar mitochondrial cristae), whereas desmothoracid Helio- zoa (with tubular mitochondrial cristae) were classified as a taxon related to Cercomonadea [3]. Although the majority of protistan taxa identified on the basis of ultrastructural characters and the shape of mitochondrial cristae have retained their status, the use of molecular data for phylogenetic reconstruction pro- vides many examples of uniting protists with different shapes of mitochondrial cristae into monophyletic groups. For example, the use of molecular data based on the marker 18S rRNA suggested that there was a morphologically heterogeneous group combining the following taxa: Cercomonadea, Chlorarachnea, shell and naked filose amoebas, and Phytomyxea [4, 5]. Later, a representative of gymnophryid amoebas (Gym- nophrys cometa) which has lamellar mitochondrial cristae was added to this group of protists with tubular mitochondrial cristae. The life cycle of most represen- tatives of this new group of Cercozoa is complicated; it includes the double-flagellar amoeboid and the cyst stages. The results of molecular analysis suggest that this life cycle is original. Therefore, the life cycles of both amoeboid organisms and Flagellata have under- gone secondary reduction, with of one of stages being lost [6]. The complicated life cycle and the presence of extrusomes of the kinetocyst type that give grounds to suggest that desmothoracid Heliozoa and Cercozoa are closely related. Because recent molecular phylogenetic studies failed to demonstrate that centrohelid Heliozoa was a closely related to any other group of protists [7], the goal of this work was to test the hypothesis that des- mothoracid Heliozoa were closely related to the main group of Cercozoa. To test this hypothesis, we sequenced the 18S rRNA gene in two representatives of desmothoracid Heliozoa, Clathrulina elegans (AY305009) and Hedriocystis sp. (AY305010). The resulting sequences were aligned with the sequences of the 18S rRNA gene in representatives of different groups of protists and subjected to phylogenetic analy- sis. The results of the study provide unambiguous evi- dence that desmothoracid Heliozoa and gymnophryid amoebas are closely related subgroups of Cercozoa. The 18S rRNA gene of Clathrulina elegans was obtained by synthesis of cDNA on an RNA template using a universal eukaryotic primer sB [8]. The result- ing cDNA was used for amplification of the 18S rRNA gene using universal primers sA and sB [8], and the obtained product was reamplified using two specific primers, s14nBr (5'-AGCCGC A(AG)(AG)CTC CAC TCC TGG-3') and s13Cl (5'-CCG ACT AGA(AT)AT GGG TAT T(AC)G-3') along with universal primers sA and sB. The complete sequence of the 18S rRNA gene of Hedriocystis sp. was obtained by amplification of three overlapping gene segments limited by the follow- GENERAL BIOLOGY Genetic Relationships between Desmothoracid Heliozoa and Gymnophryid Amoebas as Evidenced by Comparison of the Nucleotide Sequences of 18S rRNA Genes S. I. Nikolaev*, S. Berney**, J. Fahrni**, A. P. Mylnikov***, N. B. Petrov*, and J. Pawlowski** Presented by Academician A.A. Bogdanov June 30, 2003 Received July 8, 2003 *Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia **Department of Zoology and Animal Biology, University of Geneva, Switzerland ***Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Yaroslavl oblast, 152742 Russia