Molecular phylogeny of choanoflagellates, the sister group to Metazoa

Abstract

Choanoflagellates are single-celled aquatic flagellates with a unique morphology consisting of a cell with a single flagellum surrounded by a "collar" of microvilli. They have long interested evolutionary biologists because of their striking resemblance to the collared cells (choanocytes) of sponges. Molecular phylogeny has confirmed a close relationship between choanoflagellates and Metazoa, and the first choanoflagellate genome sequence has recently been published. However, molecular phylogenetic studies within choanoflagellates are still extremely limited. Thus, little is known about choanoflagellate evolution or the exact nature of the relationship between choanoflagellates and Metazoa. We have sequenced four genes from a broad sampling of the morphological diversity of choanoflagellates including most species currently available in culture. Phylogenetic analyses of these sequences, alone and in combination, reject much of the traditional taxonomy of the group. The molecular data also strongly support choanoflagellate monophyly rejecting proposals that Metazoa were derived from a true choanoflagellate ancestor. Mapping of a complementary matrix of morphological and ecological traits onto the phylogeny allows a reinterpretation of choanoflagellate character evolution and predicts the nature of their last common ancestor.

Fig. 1.

Morphological variation within choanoflagellates. Shadowcast whole mounts of cells or thecae viewed with transmission electron microscopy. (A) Monosiga ovata. (c) collar; f, flagellum. Bar = 2 μm. (B) Salpingoeca urceolata. Empty flask-shaped theca is shown. Arrow denotes inner flange that connects cell (absent) to theca. (Scale bar, 1 μm.) (C) Salpingoeca infusionum. Empty cup-shaped organic theca is shown. (p) peduncle (stalk). (Scale bar, 1 μm.) (D) Salpingoeca amphoridium. Colonial “proterospongia” stage is shown. Note six regularly placed cells held together by fine posterior threads. (Scale bar, 5 μm.) (E) Acanthoeca spectabilis. Immediately after division (nudiform) showing two cells, each with a forwardly directed flagellum (arrows in F) is shown. The juvenile (j) is above the cell remaining in the parent lorica. (Scale bar, 2 μm.) (F) Stephanoeca diplocostata. Tectiform division showing inverted juvenile cell (j) being pushed into an accumulation of costal stripsis shown. Arrows denote transverse (ring) costae. (Scale bar, 2 μm.)

Fig. 2.

Molecular phylogeny of choanoflagellates based on a concatenated four-gene dataset. The tree shown was derived by Bayesian inference based on a combined tubA, hsp90, SSU, and LSU rDNA nucleotide sequence alignment. Branches are drawn proportional to the number of nucleotide substitutions per site as indicated by the scale bar at the lower left. Branches receiving 1.00 biPP and 100% mlBP support are denoted by an *. biPP and mlBP values are otherwise given above and below branches respectively. Additional values are shown for four important deep branches in the choanoflagellate and Metazoa grouping, indicating support values obtained when the unstable, long-branched taxa Corallochytrium limacisporum and Nuclearia simplex were excluded.

Fig. 3.

Matrix of morphological and ecological characters for choanoflagellates. Characters (27) were scored for all taxa for which information was available either from the literature or our own observations. Species are arranged in order corresponding to the molecular phylogeny (Fig. 2) as indicated by the simplified schematic to the left of the matrix. Accumulation of costal strips: (1) around surface of juvenile; (2) inside the top of the parent collar. Order of costal strip production: (1) longitudinal first, helical second; (2) transverse first, longitudinal second. Lorica size: (1) <8 μm; (2) 9–12 μm; (3) 12–15 μm; and (4) >15 μm.