Accepted Manuscript Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opis- thokonts (animals, fungi, choanozoans) and Amoebozoa Thomas Cavalier-Smith, Ema E. Chao, Elizabeth A. Snell, Cédric Berney, Anna Maria Fiore-Donno, Rhodri Lewis PII: S1055-7903(14)00279-6 DOI: http://dx.doi.org/10.1016/j.ympev.2014.08.012 Reference: YMPEV 4996 To appear in: Molecular Phylogenetics and Evolution Received Date: 24 January 2014 Revised Date: 2 August 2014 Accepted Date: 11 August 2014 Please cite this article as: Cavalier-Smith, T., Chao, E.E., Snell, E.A., Berney, C., Fiore-Donno, A.M., Lewis, R., Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/ j.ympev.2014.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 1 Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts 2 (animals, fungi, choanozoans) and Amoebozoa 3 4 Thomas Cavalier-Smith1, Ema E. Chao1, Elizabeth A. Snell1, Cédric Berney1,2, Anna Maria 5 Fiore-Donno1,3, and Rhodri Lewis1 6 7 1Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK. 8 2Present address: Department of Life Sciences, The Natural History Museum, Cromwell Road, 9 London SW7 5BD, UK. 3Present address: Zoology Institute, Terrestrial Ecology Group, Faculty 10 of Mathematics and Natural Sciences, University of Cologne, Biozentrum Köln, Zülpicher Str. 47 11 b, D-50674 KÖLN, Germany. 12 13 Author for correspondence: Thomas Cavalier-Smith, email: [email protected]. 14 15 ABSTRACT 16 Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three 17 groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. 18 Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were 19 previously argued to have evolved independently from the little-studied, largely flagellate, 20 protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and 21 the more primitive excavate flagellates that have ventral feeding grooves and the most primitive 22 known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of 23 Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three 24 planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote- 25 wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa 26 independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal 27 pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to 28 opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca 29 (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts 30 plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, 31 Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon 32 instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in 33 Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between 34 homogeneous-model maximum-likelihood trees and evolutionarily more realistic site- 35 heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 2 36 ‘podiate’ clade, one of only three eukaryotic ’supergroups’. Our trees indicate that evolution of 37 sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously) 38 generated podiate eukaryotes from Malawimonas-like excavate flagellates. 39 40 Key words: cell evolution, eukaryote phylogeny, podiates, Protozoa, transcriptome sequencing, 41 Sulcozoa 42 43 1. Introduction 44 Phylogenetically, all eukaryotes have been assigned to just three supergroups: podiates, 45 corticates (kingdoms Plantae and Chromista), and Eozoa (excavates and Euglenozoa) (Cavalier- 46 Smith, 2013a). The entirely heterotrophic podiates, the focus of this paper, include Animalia, 47 Fungi, and three protozoan phyla (Sulcozoa, Amoebozoa, Choanozoa); they are so called 48 because of the general presence of pseudopodia except in the derived Fungi that lost them 49 (Cavalier-Smith, 2013a). Originally ‘excavates’ excluded Euglenozoa (Simpson and Patterson 50 1999), but later these distinctive flagellates were included despite not sharing excavate 51 morphology (Cavalier-Smith 2002, 2003, Simpson 2003) under the influence of a probably 52 erroneous assumption about the location of the eukaryotic root; here we follow Cavalier-Smith 53 (2010a, 2013a) in excluding Euglenozoa from excavates. Multigene trees usually show corticates 54 as a clade, but are contradictory concerning the boundary between the excavate Eozoa and the 55 putatively basal podiate phylum Sulcozoa. The problem lies in the uncertain phylogenetic 56 position of the excavate flagellate Malawimonas (Brown et al., 2013; Derelle and Lang, 2012; 57 Hampl et al., 2009; Zhao et al., 2012; Zhao et al., 2013). Some multigene trees show podiates as 58 a clade with Malawimonas one node deeper (e.g. Brown et al., 2013); others place Malawimonas 59 within podiates, typically as sister to the sulcozoan flagellate Collodictyon (e.g. Zhao et al., 60 2012, 2013). Either position is consistent with the postulate that the evolutionary transitions 61 between the three supergroups (Fig. 1) all involved biciliate ventrally grooved cells 62 morphologically similar to Malawimonas (Cavalier-Smith, 2013a). Podiates are proposed to 63 have arisen from a Malawimonas-like ancestor by evolving ventral pseudopodia and novel dorsal 64 semi-rigid pellicle and abandoning swimming in the plankton in favour of a benthic habitat, 65 gliding on the posterior cilium over surfaces in search of prey (Cavalier-Smith, 2013a). 66 To clarify the deep branching of podiates one must determine the relationships of the 67 various groups of Sulcozoa, a recently established protozoan phylum of mainly gliding 68 flagellates. Sulcozoa are morphologically unified by a unique cell structure, combining ventral 69 feeding groove, pseudopodia to catch prey (not for locomotion), and rigid dorsal pellicle 70 (Cavalier-Smith, 2013a). Sulcozoa comprise subphyla Apusozoa (apusomonads and breviates) 3 71 and Varisulca, proposed as the most basal podiate group (Cavalier-Smith, 2013a). Sulcozoa are 72 phylogenetically important as likely ancestors of two major groups, whose cell structure is 73 proposed to have been radically simplified from the complex cytoskeleton of the ventral feeding 74 groove of Sulcozoa and excavates: opisthokonts, whose ancestor lost the anterior cilium and 75 became uniciliate like human sperm, and Amoebozoa, which developed pseudopodia for 76 efficient locomotion (Cavalier-Smith, 2013a). A 16-gene tree weakly grouped three sulcozoan 77 orders as a clade, but was based on only three or four genes for apusomonads and planomonads 78 (Katz et al., 2011), the best studied gliding sulcozoan flagellates; a sulcozoan clade was not seen 79 in a 30-gene tree with four Sulcozoa, because Malawimonas was weakly within Sulcozoa (Grant 80 et al., 2012). A study of 159 genes weakly excluded Malawimonas from Sulcozoa and podiates, 81 but showed two or three distinct sulcozoan clades at the base of podiates, though sampling only 82 five Sulcozoa from three orders, and only Collodictyon representing subphylum Varisulca 83 (Brown et al., 2013). Brown et al. (2013) concluded that Apusozoa were paraphyletic; their trees 84 also confirmed earlier evidence that Collodictyon branched deeper still, as the most divergent 85 podiate lineage to date, making Sulcozoa the ancestral (paraphyletic) podiate group. 86 Most Sulcozoa are gliding not swimming flagellates: apusomonads (Cavalier-Smith and 87 Chao, 2010) and planomonads (Cavalier-Smith et al., 2008) and Mantamonas (Glücksman et al., 88 2011) (the latter two grouped as Glissodiscea) glide over surfaces on their posterior cilium held 89 rigidly behind their cell like a ski. Sulcozoa also include Diphylleida (Brugerolle et al., 2002; 90 Cavalier-Smith, 2003), alga-eating grooved swimming flagellates with two or four cilia, which 91 emit pseudopodia from their groove and whose cytoskeleton is substantially modified compared 92 with gliding Sulcozoa (Cavalier-Smith and Chao 2010; Cavalier-Smith 2013a). Diphylleids, 93 sometimes included in Apusozoa or Excavata (Cavalier-Smith, 2003), were recently grouped 94 with Glissodiscea and the non-flagellate Rigifilida (Yabuki et al., 2013) as Varisulca (Cavalier- 95 Smith, 2013a). Previously Glissodiscea included the gliding flagellate Discocelia (Vørs, 1988; 96 Cavalier-Smith, 2013a), but rRNA trees put it in Cercozoa (Cavalier-Smith et al. in prep.). 97 Breviate amoebae lost the posterior cilium and glide instead by the remaining anterior cilium 98 held rigidly ahead (Heiss et al., 2013), and have become secondarily anaerobic. Ciliary gliding, 99 pseudopodia,