Molecular phylogeny of choanoflagellates, the sister group to Metazoa M. Carr*†, B. S. C. Leadbeater*‡, R. Hassan‡§, M. Nelson†, and S. L. Baldauf†¶ʈ †Department of Biology, University of York, Heslington, York, YO10 5YW, United Kingdom; and ‡School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom Edited by Andrew H. Knoll, Harvard University, Cambridge, MA, and approved August 28, 2008 (received for review February 28, 2008) Choanoflagellates are single-celled aquatic flagellates with a unique family. Members of the Acanthoecidae family (Norris 1965) are morphology consisting of a cell with a single flagellum surrounded by characterized by the most distinct periplast morphology. This a ‘‘collar’’ of microvilli. They have long interested evolutionary biol- consists of a complex basket-like lorica constructed in a precise and ogists because of their striking resemblance to the collared cells highly reproducible manner from ribs (costae) composed of rod- (choanocytes) of sponges. Molecular phylogeny has confirmed a close shaped silica strips (Fig. 1 E and F) (13). The Acanthoecidae family relationship between choanoflagellates and Metazoa, and the first is further subdivided into nudiform (Fig. 1E) and tectiform (Fig. 1F) choanoflagellate genome sequence has recently been published. species, based on the morphology of the lorica, the stage in the cell However, molecular phylogenetic studies within choanoflagellates cycle when the silica strips are produced, the location at which the are still extremely limited. Thus, little is known about choanoflagel- strips are stored, and the mode of cell division [supporting infor- late evolution or the exact nature of the relationship between mation (SI) Text] (14). choanoflagellates and Metazoa. We have sequenced four genes from Speculation on a possible close evolutionary relationship be- a broad sampling of the morphological diversity of choanoflagellates tween choanoflagellates and Metazoa was strengthened early on by including most species currently available in culture. Phylogenetic the discovery of a colonial choanoflagellate, Proterospongia haeck- analyses of these sequences, alone and in combination, reject much eli, by Saville-Kent in 1880. This taxon superficially resembles a of the traditional taxonomy of the group. The molecular data also poriferan larva in that it appears to consist of flagellated cells strongly support choanoflagellate monophyly rejecting proposals protruding from a matrix bearing internal amoeboid-like cells (11). that Metazoa were derived from a true choanoflagellate ancestor. Although subsequent sightings of this species have not been au- Mapping of a complementary matrix of morphological and ecological thenticated, and its original description is somewhat enigmatic, traits onto the phylogeny allows a reinterpretation of choanoflagel- numerous other colonial forms are now known. Thus, cho- late character evolution and predicts the nature of their last common anoflagellates have long been treated in introductory biology texts ancestor. as a classic example of stepwise evolution of complexity leading to the true multicellularity of Metazoa. evolution ͉ morphology ͉ holozoa ͉ animals ͉ protists Most molecular phylogenetic trees that include multiple cho- anoflagellate species have been based on nuclear small subunit, EVOLUTION hoanoflagellates are a major group of heterotrophic ribosomal gene (SSU rDNA) sequences. These studies have mostly Cnanoflagellates, ubiquitously distributed in aquatic environ- recovered choanoflagellate monophyly but have been hampered by ments (1). These single-celled organisms were first described by a small sampling of species and some species misidentification (4–5, James-Clark in 1866, who was also the first to note the strong 7–9). Therefore, to examine major trends in choanoflagellate resemblance between the choanoflagellate cell morphology and evolution, we have constructed a taxonomically broad, multigene that of the collared cells (choanocytes) of sponges (Porifera) (2). phylogeny of the group and a complementary matrix of morpho- Based on these similarities, a close relationship between cho- logical and ecological traits. Phylogenetic analyses of a concate- anoflagellates and Metazoa was long postulated and has now been nated four-gene dataset show that the traditional taxonomy of confirmed (3–9). However, there is still very little molecular data choanoflagellates is flawed, and the evolution of some of their most from more than two or three choanoflagellate species. Thus, we notable morphological traits is more complex than initially thought. have a limited understanding of choanoflagellate phylogeny or how Results to interpret evolutionary trends within the group. Most importantly, without knowing whether choanoflagellates constitute a monophy- Molecular Phylogeny of Choanoflagellates. Large fragments of the letic group, it is difficult to know the relevance of such trends to the nuclear SSU and large subunit (LSU) ribosomal RNA, alpha- early evolution of Metazoa. tubulin (tubA), and the 90-kDa heat shock protein (hsp90) coding Choanoflagellates are characterized by a distinctive and re- markably uniform cell body (protoplast) morphology. This com- Author contributions: M.C., B.S.C.L., and S.L.B. designed research; M.C., R.H., and M.N. prises a spherical to ovoid cell with a single anterior flagellum performed research; M.C., B.S.C.L., and S.L.B. analyzed data; M.C., B.S.C.L., and S.L.B. wrote surrounded by a collar of narrow actin-based microvilli (Fig. 1A) the paper. (10). In contrast to the uniformity of the choanoflagellate cell, The authors declare no conflict of interest. the morphology of the external covering (periplast) is varied and This article is a PNAS Direct Submission. sometimes striking, ranging from simple organic sheaths to Data Deposition: Sequences reported in this paper have been deposited in the GenBank complex silica ‘‘cages’’ up to a 100 ␮m or more in length. database (accession numbers EU011922–EU011972). Periplast morphology has formed the basis of the conventional ʈTo whom correspondence should be addressed. E-mail: [email protected]. classification of choanoflagellates into three families (11). Members §Present address: Department of Aquatic Sciences, Faculty of Resource Science and Tech- of the Codonosigidae family (Kent 1880) (Fig. 1A) have a thin nology, Universiti Malaysia Sarawak, Malaysia. fibrillar coat, the glycocalyx. This surrounds the cell and may extend ¶Present address: Department of Evolution, Genomics and Systematics, Evolutionary Biol- posteriorly to join a substantial stalk composed of carbohydrate ogy Centre, Uppsala University, Norbyva¨gen 18D, 752–36 Uppsala, Sweden. microfibrils (12). Species of the Salpingoecidae family (Kent 1880) *M.C. and B.S.C.L. contributed equally to this work. possess a substantial microfibril-based theca. This may be flask- This article contains supporting information online at www.pnas.org/cgi/content/full/ (Fig. 1B), cup- (Fig. 1C) or tube-shaped and is attached to the 0801667105/DCSupplemental. substratum by a stalk similar to that found in the Codonosigidae © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801667105 PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16641–16646 Downloaded by guest on September 25, 2021 f c c Fig. 1. Morphological variation within cho- anoflagellates. Shadowcast whole mounts of cells or thecae viewed with transmission elec- tron microscopy. (A) Monosiga ovata. (c) col- lar; f, flagellum. Bar ϭ 2 ␮m. (B) Salpingoeca p urceolata. Empty flask-shaped theca is shown. Arrow denotes inner flange that con- AB Cnects cell (absent) to theca. (Scale bar, 1 ␮m.) (C) Salpingoeca infusionum. Empty cup- f shaped organic theca is shown. (p) peduncle ␮ f (stalk). (Scale bar, 1 m.) (D) Salpingoeca am- phoridium. Colonial ‘‘proterospongia’’ stage is shown. Note six regularly placed cells held together by fine posterior threads. (Scale bar, 5 ␮m.) (E) Acanthoeca spectabilis. Immedi- ately after division (nudiform) showing two cells, each with a forwardly directed flagel- lum (arrows in F) is shown. The juvenile (j) is above the cell remaining in the parent lorica. (Scale bar, 2 ␮m.) (F) Stephanoeca diplo- costata. Tectiform division showing inverted juvenile cell (j) being pushed into an accumu- lation of costal stripsis shown. Arrows denote DE Ftransverse (ring) costae. (Scale bar, 2 ␮m.) genes were amplified by PCR from total genomic DNA for 16 (assigned to Clades 1 and 2, respectively). Within Clade 3, the two choanoflagellate species (Table S1). SSU rDNA was sequenced major morphological types of acanthoeicids, nudiform, and tecti- directly from PCR products for all species, from which no evidence form, are both recovered as strongly supported monophyletic of polymorphism was detected. LSU rRNA and both protein genes subgroups (1.00 biPP, 89–100% mlBP) (Fig. 2). This is particularly were amplified in multiple overlapping fragments, all of which significant with respect to the morphologically disparate nudiform matched completely in overlapping regions. Thus, there is no species for it demonstrates that they are an evolutionarily coherent evidence for the existence of paralogs for any of these genes in any assemblage. of the examined species. Two colony-forming Proterospongia species are found in Clade 1, Phylogenetic analyses of each of the four genes independently although they do not group together, whereas a third colony- show similar results for each gene