Europ. J. Protistol. 36, 293- 302 (2000) September 25, 2000 http://www.urban fisc her.de/jour nal s/ejp European Journal of PROTISTOLOGY Phylogenetic Position of the Ciliates Phacodinium (Order Phacodiniida) and Protocruzia (Subclass Protocruziidia) and Systematics of the Spirotrich Ciliates Examined by Small Subunit Ribosomal RNA Gene Sequences Mann Kyoon Shin', Ui Wook Hwanq '. Won Kim ', Andre-Denis G. Wright 3 .4, Connie Krawczyk 4 ,5 and Denis H. , Department of Biological Sci ence, Coll ege of Natural Sci ence, Un iver sity of Ul san, Ul san 680-749, Korea 2 Department of Mo lec ular Biology, C oll ege of Natural Sc ienc e, Seo ul Na tional University, Seoul 151-742, Korea 3 Present address: An ima l Pro duction, CS IRO, Pr iv ate Bag, PO Wembley, WA 6014, Austra lia 4 Department of Zoo logy, Un iversity of Guelph, Gu elph, ONN1G 2W1, Canada; Fax: 51 9-767-1656; e-mail: ddr@ uoguel ph.ca 5 Present address: Department of Medi cal Bio phys ics, Onta ri o Cancer In stitu te, 61 0 University Ave., Toront o M5G 2C1, Canad a Summary The small subunit rRNA (SSrRNA) genes were sequenced for Protocruzia sp2, Phacodinium metchnicoffi, Holosticha multistylata, and Halteria grandin ella. All four genera are placed within the Class Spirotrichea with strong bootstrap support in both distance matrix and parsimony tree con- struction methods and by maximum likelihood analysis using quartet puzzling. Protocruzia sp2 groups with Pro tocruzia spl with 100% bootstrap support, and the 5.6% genetic difference between them strongly argues that they are different species although they arc morphologically quite similar. The Protocruzia species branch first in the spirotrich clade at a deep level, supporting their recogni- tion as a Subclass Protocruziidia. Phacodinium branches after Euplot es at a deep level, confirming the conclusion that others have reached that this genu s is related to the hypotrich and stichotrich spirotrichs. Phacodin ium is as- signed to its own family and order, and we conclude th at the deep branching within the spirotrichs argues for its own subclass, the Subclass Phacodiniidia. Consistent with the partial SSrRNA sequences and with the sequence of a polymerase gene, Halteria groups within the stichotrich clade, supporting the argument that the oligotrichs are not monophyletic as cu rr entl y conceived. Finally, Holo sticha, which has been assigned to the Order Urost ylida, groups outside the stichotrichs with parsimony analysis, which is consistent with this ordinal assignment. However, it is associated with Halteria and Oxytricha granulifera in ':'corresponding author © 2000 by Urban & FischerVerlag the other analyses. Additional stichotrich sequences obvi- ously are required before we can confidently begin revision of the Subclass Stichotrichia. Key word s: PCR; Ciliophora; Stichotrich; Hypotrich; Puzzling; Halteria. Introduction The hypotrich (e.g., euplotid) and stichotrich (e.g., oxytrichid) ciliates are among the most easily recogniz- able ciliates. They are characterized by a dorsoventral flattening of the body with the presence of numerous compound somatic ciliary structures or cirri on the ventral surface and a wreath-like set of oral poly- kinetids along the left side of the oral region [6]. Of the 8,000 species of described ciliates, nearly 500 species belon g to these two groups, which also typically show a replication band that passes through the macronucleus during the S-phase of DNA synthesis [41]. This repli- cation band is also exhibited during macronuclear S- phase in choreo trich and oligotrich ciliates, although they do not possess typical cirri and dorsoventrally- flattened bodies. This suggests that these two groups should be included in the same clade as the hypotrich and stichotrich ciliates [33]. Lynn and Small [34] have thu s included the hypotrichs (e.g., Euplotes), sti- 0932-4739/00/36/03-293 $ 15.00/0