Ancyromonadida: A New Phylogenetic Lineage Among the Protozoa Closely Related to the Common Ancestor of Metazoans, Fungi, and Choanoflagellates (Opisthokonta) Michael S. Atkins, 1 Andrew G. McArthur, 2 Andreas P. Teske 1 1 Biology Department MS 33, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA 2 The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543-1015, USA Received: 27 March 2000 / Accepted: 12 June 2000 Abstract. Molecular and morphological evidence points to the ancyromonad Ancyromonas as a plausible candidate for the closest relative to the common ancestor of metazoans, fungi, and choanoflagellates (the Opistho- konta). Using 18S rDNA sequences from most of the major eukaryotic lineages, maximum-likelihood, mini- mum-evolution, and maximum-parsimony analyses yielded congruent phylogenies supporting this hypoth- esis. Combined with ultrastructural similarities between Ancyromonas and opisthokonts, the evidence presented here suggests that Ancyromonas may form an indepen- dent lineage, the Ancyromonadida Cavalier-Smith 1997, closer in its relationship to the opisthokonts than is its nearest protist relatives, the Apusomonadida. However, the very low bootstrap support for deep nodes and hy- pothesis testing indicate that the resolving power of 18S rDNA sequences is limited for examining this aspect of eukaryotic phylogeny. Alternate branching positions for the Ancyromonas lineage cannot be robustly rejected, revealing the importance of ultrastructure when examin- ing the origins of multicellularity. The future use of a multigene approach may additionally be needed to re- solve this aspect of eukaryotic phylogeny. Key words: Protozoa — Flagellate — Ancyromo- nas — Apusomonas — Opisthokont — 18S small sub- unit ribosomal DNA — Phylogeny Introduction Animals and true fungi, along with their protist relatives, the choanoflagellates and chytrids, comprise the largest major eukaryotic lineage, with over 1 million species (Patterson 1999). Together, and exclusive of green plants, this group was hypothesized to form a single clade, the Opisthokonta, based on shared ultrastructural characteristics (Cavalier-Smith and Chao 1995). These characteristics include the presence of a unicellular mo- tile stage (sperm in the case of animals) bearing a single posterior flagellum, similarities in flagellar root structure between choanoflagellates and chytrids (Cava- lier-Smith 1987), and flattened mitochondrial cristae (Cavalier-Smith and Chao 1995). This hypothesis has found strong support from molecular research with the discovery of an insertion in the amino acid sequence of protein synthesis elongation factor 1 (EF-1), shared exclusively by animals and fungi (Baldauf and Palmer 1993), and in the molecular phylogenies of ribosomal RNAs (Van de Peer and De Wachter 1997; Wainright et al. 1993), actin, -tubulin, -tubulin, and EF-1 proteins (Baldauf 1999), and other protein phylogenies (Nikoh et al. 1994). Recent molecular evidence also suggests that the Opisthokonta should include the Myxospora (previ- ously considered a group of protozoa) as a subgroup within the Cnidaria (Smothers et al. 1994) and the Mi- crospora as close relatives to the fungi (Hirt et al. 1999; Roger 1999). While all opisthokonts have origins among the proto- zoa, it is uncertain whether these origins are mono- or Correspondence to: Michael S. Atkins; e-mail: matkins@whoi.edu J Mol Evol (2000) 51:278–285 DOI: 10.1007/s002390010089 © Springer-Verlag New York Inc. 2000