Oxymonads Are Closely Related to the Excavate Taxon Trimastix Joel B. Dacks,* Jeffrey D. Silberman,²1 Alastair G. B. Simpson,³2, Shigeharu Moriya,§ Toshiaki Kudo,§ Moriya Ohkuma,§ and Rosemary J. Red®eld\ *Program in Evolutionary Biology, Canadian Institute for Advanced Research, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada; ²Josephine Bay Paul Center in Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts; ³School of Biological Sciences, University of Sydney, New South Wales, Australia; §Institute of Physical and Chemical Research and Japan Science and Technology Corporation, Wako, Saitama, Japan; and \Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada Despite intensive study in recent years, large-scale eukaryote phylogeny remains poorly resolved. This is particularly problematic among the groups considered to be potential early branches. In many recent systematic schemes for early eukaryotic evolution, the amitochondriate protists oxymonads and Trimastix have ®gured prominently, having been suggested as members of many of the putative deep-branching higher taxa. However, they have never before been proposed as close relatives of each other. We ampli®ed, cloned, and sequenced small-subunit ribosomal RNA genes from the oxymonad Pyrsonympha and from several Trimastix isolates. Rigorous phylogenetic analyses indicate that these two protist groups are sister taxa and are not clearly related to any currently established eukaryotic lineages. This surprising result has important implications for our understanding of cellular evolution and high-level eukaryotic phylogeny. Given that Trimastix contains small, electron-dense bodies strongly suspected to be derived mitochondria, this study constitutes the best evidence to date that oxymonads are not primitively amitochondriate. Instead, Trimastix and oxymonads may be useful organisms for investigations into the evolution of the secondary amitochondriate condition. All higher taxa involving either oxymonads or Trimastix may require modi®cation or abandonment. Affected groups include four contemporary taxa given the rank of phylum (Metamonada, Loukozoa, Trichozoa, Percolozoa), and the informal excavate taxa. A new ``phylum-level'' taxon may be warranted for oxy- monads and Trimastix. Introduction Recent years have seen increasing uncertainty Cleveland 1956). Some oxymonads, such as Saccino- about the broadest-scale structure of the eukaryotic evo- baculus and Oxymonas, undergo self-fusion of gametes lutionary tree, particularly the identity of the deepest (autogamy). These taxa are thought to have a one-step extant branches. These dif®culties have been revealed meiosis, in which a single reductive division produces by the implementation of novel analysis methods (Stiller two daughter cells, instead of the two divisions and four and Hall 1999), the use of different models of evolution daughter cells typical of meiosis in other organisms (but (Silberman et al. 1999), and the use of genes giving see Haig 1993). Other oxymonads, such as Pyrsonym- con¯icting results (Embley and Hirt 1998). Another ma- pha, do not undergo true sexual reproduction, but, rather, jor problem is the absence of many key protist groups have a ploidy cycle in which their initially high ploidy from most or all molecular phylogenies. Oxymonads is reduced by a series of apparently meiotic divisions and Trimastix are two such key groups. and then restored by multiple rounds of DNA replication Oxymonads are a group of structurally distinct, ob- (Hollande and Carruette-Valentin 1970). Unlike most ligately symbiotic ¯agellates (usually with four ¯agella eukaryotes, oxymonads also lack mitochondria and Gol- per cell), most of which are cellulose digesters found in gi dictyosomes (Brugerolle 1991). This cytological sim- the hindgut of termites and wood-eating cockroaches. plicity, especially the lack of mitochondria, led to oxy- First described by Leidy in 1877, oxymonads are best monads being advanced as one of the most primitive known for their atypical sexual cycles, described in a groups of eukaryotes (Cavalier-Smith 1981). long series of papers by Cleveland (summarized by The relationships of oxymonads with other eukary- otes are uncertain and contentious. In the modern era, 1 Present address: UCLA Institute of Geophysics and Planetary they have generally been allied with the other cytolog- Physics and Department of Microbiology and Immunology, University ically simple, amitochondriate, tetra¯agellate protists, of California±Los Angeles. i.e., the retortamonads and diplomonads. These groups 2 Present address: Department of Biochemistry and Molecular Bi- formed the widely accepted phylum Metamonada, unit- ology, Dalhousie University, Halifax, Nova Scotia, Canada. ed by their shared possession of four anterior basal bod- Abbreviations: ML, maximum likelihood; sp., species; ssu rDNA, ies and lack of organelles (Cavalier-Smith 1981, 1998). small-subunit ribosomal RNA gene. However, the distinctive presence of a motile axostyle, Key words: ssu rDNA, eukaryote evolution, protist, phylogenet- a cytoskeletal backbone running the length of oxymonad ics, metamonad, mitochondria, sex. cells, sets the oxymonads apart from the other meta- Address for correspondence and reprints: Joel B. Dacks, Program monads. In his 1991 review, Brugerolle suggested that in Evolutionary Biology, Canadian Institute for Advanced Research, there was ``a probable long evolutionary distance be- Department of Biochemistry and Molecular Biology, Dalhousie Uni- tween this group and the other two.'' Recent elongation versity, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: [email protected]. factor (EF-1 alpha) phylogenies that include the ®rst Mol. Biol. Evol. 18(6):1034±1044. 2001 gene sequence data from oxymonads (Moriya, Ohkuma, q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 and Kudo 1998; Dacks and Roger 1999) indicate that a 1034 An Oxymonad-Trimastix Clade 1035 close relationship with diplomonads is unlikely. Newer four rDNA sequences to each other and to other eu- accounts of eukaryotic diversity instead place oxymon- karyotic taxa was determined by phylogenetic analysis. ads with Heterolobosea and Stephanopogon in the con- tentious phylum Percolozoa (Cavalier-Smith 1999, Materials and Methods 2000) or simply describe them as ``eukaryotic taxa with- Protist Isolation and Gene Ampli®cation out known sister groups'' (Patterson 1999). The genus Trimastix was ®rst described by Kent in Pyrsonympha cells were obtained from specimens 1880 but has only recently become the subject of de- of the Western subterranean termite (Reticulitermes hes- tailed study by evolutionary protistologists. Trimastix perus), a species known to harbor the oxymonads Pyr- are free-living anaerobes/microaerophiles with four ¯a- sonympha and Dinenympha (Grosovsky and Margulis gella and a broad ventral feeding groove. Ultrastructural 1982), collected from a natural colony near Kelowna, examinations have revealed that Trimastix lack classical Canada. Termite gut contents were diluted into modi®ed mitochondria, having instead small, membrane-bounded Trager's media (Buhse, Stamler, and Smith 1975). The organelles resembling hydrogenosomes (O'Kelly 1993; largest cells with typical Pyrsonympha morphology Brugerolle and Patterson 1997; Simpson, Bernard, and were selected away from nonoxymonad ¯agellates by Patterson 2000). The discovery of these organelles micromanipulation, washed, and reselected. Due to the prompted Cavalier-Smith (1997) to group Trimastix with dif®culty of manipulation and identi®cation, the cells the hydrogenosome-bearing parabasalids in a new phy- were identi®able only as Pyrsonympha sp. lum, Trichozoa. However, detailed ultrastructural ex- About 50±75 cells were pelleted by centrifugation aminations also demonstrated that Trimastix shares a at 3,000 rpm for 1 min, and DNA was extracted using large number of cytoskeletal similarities with a seem- standard techniques (Maniatis, Fritsch, and Sambrook ingly diverse collection of mitochondriate and amito- 1982). The 39 region of the Pyrsonympha sp. ssu rDNA chondriate protists that also have feeding grooves: the gene (639 nt) was ampli®ed by PCR, using eukaryotic retortamonads, core jakobids (Reclinomonas, Jakoba, speci®c primer 59N (TGAAACTTAAAGGAATTGA- and Histiona), Malawimonas, Carpediemonas, some di- CGGA) and primer B from Medlin et al. (1988). Cycling plomonads, and some Heterolobosea (O'Kelly, Farmer, parameters began with an initial denaturation of 958C and Nerad 1999; O'Kelly and Nerad 1999; Patterson for 1 min, followed by 1 min at 458C and 3 min at 728C. 1999; Simpson and Patterson 1999). Trimastix has been This cycle was repeated an additional 29 times with the included with these groups in the informal assemblage initial heating step at 948C for 10 s, and was followed ``excavate taxa,'' envisaged as a monophyletic or para- by a ®nal cycle with extension time increased to 4 min phyletic group (Simpson and Patterson 1999). Cavalier- to promote the complete extension of products. The re- Smith (1999) recently rejected Trichozoa and instead sulting PCR products were cloned into a pGem-T vector erected a new phylum, Loukozoa, based on the shared (Promega BioTech, Madison, Wis.) and sequenced on presence of a ventral feeding groove (and referring to an ABI sequencer. the presence of either mitochondria or mitochondrion Once the identity of