Reference: Biol. Bull. 196: 381-384. (June 1999) Free-Living Flagellates From Anoxic Habitats and the Assembly of the Eukaryotic Cell DAVID J. PATTERSON, ALASTAIR G. B. SIMPSON, AND NIMALIKA WEERAKOON School of Biological Sciences, University of Sydney, NSW 2006, Australia Comparative studies of protists conducted from the 1970s to 1990s led to models of their evolutionary relationships and, in turn, to models of the sequence in which eukaryotic cells were assembled (1, 2). In part, this was a result of ultrastructural studies of cell organization which had re- vealed that many familiar clusters of protists were polyphy- letic. Such studies provided evolutionary biologists with a fairly robust catalog of about 70 types of eukaryotes (2, 3). Spurred on by ideas that early eukaryotes were created by symbiotic associations of previously independent organ- isms, investigators explored the relationships among many of these groups by a combination of ultrastructural and molecular comparisons; the latter were mostly based on the small subunit ribosomal RNA (4). An important feature of the emerging tree was that a number of lineages had arisen from early, pre-mitochondrial stages in eukaryote evolution and have survived to the present. These purportedly primi- tively amitochondriate protists included the microsporidia, pelobionts, diplomonads, retortamonads, oxymonads, ent- amoebae, trichomonads, and other parabasalids. Some or all of these taxa, in various combinations, have been referred to as the “Archezoa.” This group has proven to be composi- tionally unstable, and it is paraphyletic. Nonetheless, the identification of this group assisted in the emergence of models of how eukaryotic cells might have developed. The models involved the autogenous emergence of the endoplas- mic reticulum, nuclei, and dictyosomes and the autogenous or xenogenous evolution of flagella and microtubular and non-microtubular cytoskeletal structures, before the symbi- otic acquisition of mitochondria and plastids. One strength This paper was originally presented at a workshop titled Evolution: A Molecular Point of View. The workshop, which was held at the Marine Biological Laboratory, Woods Hole, Massachusetts, from 24-26 October 1997, was sponsored by the Center for Advanced Studies in the Space Life Sciences at MBL and funded by the National Aeronautics and Space Administration under Cooperative Agreement NCC 2-896. of this model, with intermediate stages of eukaryote evolu- tion being represented by extant “relict” organisms, is its testability through further phylogenetic analysis. Over the last few years, further molecular studies seem to have refuted elements of this hypothesis. There is evidence that many archezoan groups are secondarily amitochondri- ate (4-S) although the diplomonads and parabasalids have clung more resolutely to the base of the eukaryotic tree than other taxa. The deep branching status of the pelobionts also remains in dispute (9-11). Largely because of the lack of appropriate investigations, there is no evidence that retorta- monads or oxymonads have had a mitochondriate ancestry. The case for primitively amitochondriate status remains unresolved for a number of taxa. If most or all of the groups assigned to the Archezoa are secondarily amitochondriate, then it significantly reduces our ability to present detailed and testable models of the sequence of innovations that led to the eukaryotic cell. We have addressed the possibility that there may be more primitively amitochondriate taxa that have not yet been described. We have used anoxic culture conditions to isolate amitochondriate (and mitochondriate) flagellates from low-oxygen freshwater, brackish, and ma- rine habitats (12). We have been able to record previously unknown types of free-living protists from these habitats. Several of the organisms in question are described below and illustrated (Figs. 1, 2). Mustigamoeba schizophrenia (Figs. 1.1, 2.1) is a pelo- biont. In addition to lacking mitochondria, dictyosomes, and non-microtubular cytoskeletal structures, this species has some ultrastructural peculiarities including paired nuclei and basal bodies that lack the triplets of microtubules which characterize basal bodies in almost all other cells (10). These idiosyncrasies have been interpreted as evidence of a primitive status. However, other closely related species have now been examined and have more conventional ar- rangements of both organelles. This leaves open the alter- 381