European Journal of PROTISTOLOGY European Journal of Protistology 41 (2005) 139–150 Morphological investigation of three Tetramitus spp. which are phylogenetically very closely related: Tetramitus horticolus, Tetramitus russelli n. comb. and Tetramitus pararusselli n. sp. Johan F. De Jonckheere a,Ã , Susan Brown b , Julia Walochnik c , Horst Aspo¨ ck c , Rolf Michel d a Scientific Institute of Public Health, B-1050 Brussels, Belgium b CEH Dorset, Winfrith Technology Centre, Dorchester, Dorset DT2 8ZD, UK c Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, A-1095 Vienna, Austria d Zentrales Institut-BW –Koblenz, Lab Abt. I. (Mikrobiologie), 56070 Koblenz, Germany Received 3 September 2004; received in revised form 6 December 2004; accepted 8 December 2004 Abstract Two vahlkampfiid amoebae have been isolated which have the same 5.8S rDNA sequence as Tetramitus horticolus, but which differ in internal transcribed spacer (ITS) sequences from this species. The two new strains, 123R and Rhodos, are identical with each other in ITS sequences, except for a 1bp substitution in the ITS1 and an insertion–deletion sequence (indel) of 4 bp in the ITS2. Because of the close phylogenetic relationship between the two new isolates and T. horticolus we compared both isolates with the latter species by light and electron microscopy. While T. horticolus has a cyst pore structure not seen in any other vahlkampfiid, the two new strains have no pores in their cysts. Strains 123R and Rhodos are very similar in morphology, but significantly different in cyst diameter. Neither strain could be induced to form flagellates under laboratory conditions. Strain 123R seems to correspond to the description of Vahlkampfia russelli. Because of this, and its molecular identification as a strain of Tetramitus, we propose that this strain should become the neotype strain of V. russelli (Singh, 1952), now classified as T. russelli n. comb. Because of the small (but significant) differences of strain Rhodos from strain 123R we propose the former as the type strain of a new species, T. pararusselli n. sp. The addition of T. horticolus, T. russelli and T. pararusselli confirms that the phenotypic definition of the genus Tetramitus is obsolete. Instead the genus can be differentiated by the significant differences from other vahlkampfiid genera in rDNA sequence. r 2005 Elsevier GmbH. All rights reserved. Keywords: Vahlkampfiidae; New combination; New species; Cyst morphology; Tetramitus; 5.8S rDNA Introduction The identification of naked amoebae is notoriously difficult, and the amoebae of the family Vahlkampfiidae ARTICLE IN PRESS www.elsevier.de/ejop 0932-4739/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2004.12.001 Ã Corresponding author. Research Unit for Tropical Diseases, Christian de Duve Institute for Cellular Pathology, Avenue Hippo- crate, 74-75, B1200 Brussels, Belgium. E-mail address: jdjonckh@ben.vub.ac.be (J.F. De Jonckheere).