View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology, Vol. 12, 1773–1778, October 15, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01187-9 The Closest Unicellular Relatives of Animals B.F. Lang,1,2 C. O’Kelly,1,3 T. Nerad,4 M.W. Gray,1,5 Results and Discussion and G. Burger1,2,6 1The Canadian Institute for Advanced Research The evolution of the Metazoa from single-celled protists Program in Evolutionary Biology is an issue that has intrigued biologists for more than a 2 De´ partement de Biochimie century. Early morphological and more recent ultra- Universite´ de Montre´ al structural and molecular studies have converged in sup- Succursale Centre-Ville porting the now widely accepted view that animals are Montre´ al, Que´ bec H3C 3J7 related to Fungi, choanoflagellates, and ichthyosporean Canada protists. However, controversy persists as to the spe- 3 Bigelow Laboratory for Ocean Sciences cific evolutionary relationships among these major P.O. Box 475 groups. This uncertainty is reflected in the plethora of 180 McKown Point Road published molecular phylogenies that propose virtually West Boothbay Harbor, Maine 04575 all of the possible alternative tree topologies involving 4 American Type Culture Collection Choanoflagellata, Fungi, Ichthyosporea, and Metazoa. 10801 University Boulevard For example, a monophyletic MetazoaϩChoanoflagel- Manassas, Virginia 20110 lata group has been suggested on the basis of small 5 Department of Biochemistry subunit (SSU) rDNA sequences [1, 6, 7]. Other studies and Molecular Biology using the same sequences have allied Choanoflagellata Dalhousie University with the Fungi [8], placed Choanoflagellata prior to the Halifax, Nova Scotia B3H 4H7 divergence of animals and Fungi [9], or even placed Canada them prior to the divergence of green algae and land plants [10]. Moreover, Ichthyosporea [11], a newly cre- ated taxon that was provisionally designated DRIPs (re- ferring to the four initial members, Dermocystidium, ro- Summary sette agent, Ichthyophonus, and Psorospermium [6]) and later Mesomycetozoa [12], has tentatively been Molecular phylogenies support a common ancestry placed somewhere near [12] (in one case, immediately between animals (Metazoa) and Fungi [1–3], but the before [6]) the animal-fungal divergence. Testifying to evolutionary descent of the Metazoa from single-celled the taxonomic uncertainty surrounding the Ichthyo- eukaryotes (protists) and the nature and taxonomic affili- sporea, one class of this phylum, i.e., the Amoebidiales, ation of these ancestral protists remain elusive. We ad- had traditionally been classified as trichomycete fungi. dressed this question by sequencing complete mito- Conflicting scenarios as to the relationship among chondrial genomes from taxonomically diverse protists Choanoflagellata, Ichthyosporea, and animals were to generate a large body of molecular data for phyloge- critically addressed in the course of a recent analysis netic analyses. Trees inferred from multiple concate- using complete SSU and large subunit (LSU) rRNA data, nated mitochondrial protein sequences demonstrate examined either individually or in combination [13]. This that animals are specifically affiliated with two mor- particular study demonstrates the weakness of tree as- phologically dissimilar unicellular protist taxa: Mono- sessments that are uniquely based on nonparametric siga brevicollis (Choanoflagellata), a flagellate, and bootstrap values and lends credence to the view that Amoebidium parasiticum (Ichthyosporea), a fungus- the precise interpretation of such values is not only diffi- like organism. Statistical evaluation of competing evo- cult [14] but also often leads to overconfidence in the lutionary hypotheses [4] confirms beyond a doubt that wrong tree [4]. For example, the monophyly of Choano- Choanoflagellata and multicellular animals share a flagellataϩIchthyosporea is supported by a high boot- close sister group relationship, originally proposed strap value (94%) based on the LSU data and by more than a century ago on morphological grounds a marginal bootstrap value (61%) in the combined [5]. For the first time, our trees convincingly resolve LSUϩSSU data set, whereas the Kishino-Hasegawa the currently controversial phylogenetic position of (KH) and Shimodaira-Hasegawa (SH) tests did not re- the Ichthyosporea, which the trees place basal to Cho- cover support for this topology with any combination of anoflagellata and Metazoa but after the divergence of data used [13]. Based upon the highly conflicting results, Fungi. Considering these results, we propose the new the authors come to the conclusion that the available taxonomic group Holozoa, comprising Ichthyosporea, data may be insufficient to resolve the question of Choanoflagellata, and Metazoa. Our findings provide whether Choanoflagellata, Ichthyosporea, or the two insight into the nature of the animal ancestor and have combined are the closest living relatives of Metazoa. broad implications for our understanding of the evolu- Basal animal phylogeny has also been investigated via tionary transition from unicellular protists to multicel- single nucleus-encoded proteins, but again, the support lular animals. for this deep divergence relies solely on the interpreta- tion of often weak and variable bootstrap or quartet- puzzle support values. For instance, trees based on 6 Correspondence: [email protected] Hsp70 proteins [15] indicate a closer relationship of Current Biology 1774 Figure 1. Maximum Likelihood Tree of Con- catenated Proteins Encoded by mtDNA The sequences of 11 well-conserved proteins (Cox1,2,3, Cob, Atp6,9, and Nad1,3,4,4L,5) were concatenated. A ⌫ distribution model of site variation was used (see Experimental Procedures). Percent bootstrap support for PROML (300 samples) is shown above each branch, and that for PUZZLEBOOT/BIONJ (1000 samples) is shown below each branch. The scale bar denotes genetic distance. Taxon designations are as follows (GenBank Accession numbers within parentheses): Magnetospirillum magnetotacticum (NC_002725); Rickettsia prowazekii (NC_000963); Chrysodidymus synuroideus (NC_002174); Phytophthora infestans (NC_002387); Hyaloraphidium curvatum (NC_003048); Spizellomyces punctatus (NC_003052); Schizophyllum commune (NC_003049); Podospora anserina (NC_001329); Rhizopus stolonifer (unpub- lished; see Supplementary Material); Allo- myces macrogynus (NC_001715); Sarcophy- ton glaucum (AF064823, AF063191); Metridium senile (NC_000933); Homo sapiens (J01415); Monosiga brevicollis (this publica- tion); Prototheca wickerhamii (NC_001613); Marchantia polymorpha (NC_001660); Amoe- bidium parasiticum (this publication); Por- phyra purpurea (NC_002007); and Chondrus crispus (NC_001677). Choanoflagellata (represented by the taxon Monosiga For phylogenetic analyses, we used our own gener- ovata) to animals than to Fungi. However, the bootstrap ated mitochondrial protein sequences, including data value for this relationship is low (58%) and, as the au- reported here from M. brevicollis and A. parasiticum,as thors state [15], “rigorous statistical tests such as the well as sequences determined by others and retrieved Kishino-Hasegawa test were also carried out but were from public data repositories. The analyses included unable to provide statistical support for any of the alter- about 3000 aligned amino acid positions from 11 well- native trees.” Another recent phylogenetic study used conserved proteins whose sequences were concate- elongation factor 2 (EF-2), ␣- and ␤-tubulin, and actin nated (see Experimental Procedures section for details). proteins [16]. In all four trees, bootstrap and quartet- Figure 1 depicts the tree obtained with the maximum puzzle indices are only shown at selected branches and likelihood (ML) method implemented in PROML [18], are weak in support of either the fungal-animal diver- with site heterogeneity modeled by the discrete ⌫ distri- gence or the branching order within the animals, thus bution. Because the ML method is computationally calling into question the overall topology of these trees. highly demanding, the number of taxa was limited to 20. Finally, in this study among-site rate heterogeneity was Taxon selection was based on two criteria; we excluded not taken into consideration, and no statistical tree se- taxa that either completely lack mitochondrial nad genes lection tests were performed. or that display highly accelerated rates of evolution of We posit that one important reason for the contro- mitochondrial proteins (see Experimental Procedures versy about early animal evolution is that the available for details). Notably, within the Metazoa, essentially all sequence data have been insufficient to yield unambigu- taxa evolve quickly, with the exception of the sea anem- ous resolution of the taxa in question. To provide a one Metridium [19] and the leather coral Sarcophyton suitable data set, we sequenced complete mitochon- [20]. Therefore, only one metazoan (i.e., human) se- drial genomes from diverse protist phyla [17], including quence having a relatively long branch length has been the choanoflagellate Monosiga brevicollis and the ich- included in the analyses. The same data set was also thyosporean Amoebidium parasiticum, and thereby analyzed with an ML distance method (TREE-PUZZLE generated the first mitochondrial gene sequences for and BIONJ [21, 22]), PUZZLEBOOT [23] for bootstrap- Choanoflagellata and Ichthyosporea.