European Journal of PROTISTOLOGY European Journal of Protistology 44 (2008) 45–53 A molecular phylogenetic investigation of Pseudoamphisiella and Parabirojimia (Protozoa, Ciliophora, Spirotrichea), two genera with ambiguous systematic positions Zhenzhen Yi a , Weibo Song a,Ã , Alan Warren b , David McL. Roberts b , Khaled A.S. Al-Rasheid c , Zigui Chen a , Saleh A. Al-Farraj c , Xiaozhong Hu a a Laboratory of Protozoology, KLM, Ocean University of China, Qingdao 266003, China b Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK c Zoology Department, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Received 6 April 2007; received in revised form 27 June 2007; accepted 6 August 2007 Abstract The gene coding for the small subunit ribosomal RNA molecule was sequenced in three marine spirotrichs: Pseudoamphisiella lacazei, P. alveolata, and Parabirojimia similis, the systematic positions of which remain unclear in spite of recent progress. Phylogenetic trees were constructed by means of neighbor-joining (NJ), least-squares (LS), maximum parsimony (MP), and Bayesian inference (BI) methods in order to test previously inferred phylogenetic assignments of Pseudoamphisiella and Parabirojimia based on morphological and morphogenetic data. The main results are that: (1) all trees constructed have similar topologies in which the genus Parabirojimia derives at the base of the class Stichotrichia, which suggests that it might represent a unique taxon at about suborder or even-order level near urostylids; (2) the present study supports the conclusion reported by previous researchers that urostylids could be a paraphyletic assemblage and that Uroleptus and Paruroleptus should be removed from the order Urostylida; (3) Pseudoamphisiellidae/Pseudoamphisiella always clusters with Prodiscocephalus borrori rather than with urostylids, which indicates that they might represent an ancestral form for both discocephaline and urostylid species; (4) the monophyly of the traditional suborder Discocephalina and typical euplotids is consistently rejected. Based on both morphological/morphogenetical and molecular data obtained, a new suborder Parabirojimina n. subord. is suggested, which currently includes one family, Parabirojimidae n. fam., and the single genus Parabirojimia. r 2007 Elsevier GmbH. All rights reserved. Keywords: Urostylida; Discocephalina; Parabirojimina n. suborder; Parabirojimidae n. fam.; Small subunit rRNA; Phylogeny Introduction In a recent widely accepted ciliate classification system (Lynn and Small 2002), the class Spirotrichea is subdivided into 6 subclasses: Protocruziidia, Phacodi- niidia, Hypotrichia, Stichotrichia, Oligotrichia and Choreotrichia. However, phylogenetic relationships among families and genera within the Spirotrichea remain confused, especially among the hypotrichs and stichotrichs, despite the fact that several revisions and numerous morphological and morphogenetic studies of organisms within these taxa have been performed in recent years (for example, Berger 1999, 2006; Berger and ARTICLE IN PRESS www.elsevier.de/ejop 0932-4739/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2007.08.002 Ã Corresponding author. Tel.: +86 532 8203 2283. E-mail address: wsong@ouc.edu.cn (W. Song).