Multigene Phylogeny Resolves Deep Branching of Amoebozoa
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Accepted Manuscript Multigene phylogeny resolves deep branching of Amoebozoa Thomas Cavalier-Smith, Anna Maria Fiore-Donno, Ema Chao, Alexander Kudryavtsev, Cédric Berney, Elizabeth A. Snell, Rhodri Lewis PII: S1055-7903(14)00278-4 DOI: http://dx.doi.org/10.1016/j.ympev.2014.08.011 Reference: YMPEV 4995 To appear in: Molecular Phylogenetics and Evolution Received Date: 17 April 2014 Revised Date: 2 August 2014 Accepted Date: 11 August 2014 Please cite this article as: Cavalier-Smith, T., Fiore-Donno, A.M., Chao, E., Kudryavtsev, A., Berney, C., Snell, E.A., Lewis, R., Multigene phylogeny resolves deep branching of Amoebozoa, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/j.ympev.2014.08.011 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 Multigene phylogeny resolves deep branching of Amoebozoa Thomas Cavalier-Smitha, Anna Maria Fiore-Donnoa,b, Ema Chaoa, Alexander Kudryavtsevc, Cédric Berneya, d, Elizabeth A. Snella, Rhodri Lewisa a Department of Zoology, University of Oxford, Oxford, United Kingdom b Present address: Terrestrial Ecology Group, Biozentrum Köln, University of Cologne, Germany c Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Saint-Petersburg, Russia d Present address: Station Biologique de Roscoff, UMR7144/Groupe Evolution et PaléOcéans, CNRS/Université Paris VI, Roscoff, France Author for correspondence: Thomas Cavalier-Smith, [email protected] 2 ABSTRACT Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo- flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data. Keywords: amoebae phylogenomics; eukaryote-wide phylogeny; Nolandella; Himatismenida; Varipodida; Semiconosia 3 1 Introduction Amoebae are unicellular organisms that produce pseudopodia and change shape as they move: they were formerly all lumped as Sarcodina (Levine et al., 1980), a name signifying that their cells are a simple protoplasmic mass that uses pseudopodia for movement, not complex cilia as in flagellates and ciliates. From the 1970s to 1990s, increased application of electron microscopy and the first molecular phylogenies confirmed earlier suspicions that Sarcodina was too broadly defined and that the distinction between amoebae and flagellates was often artificial. It is now clear that amoebae are polyphyletic and belong in three evolutionarily distinct major groups not directly related to each other, each of which also includes diverse flagellate protists. One distinctive group named Heterolobosea (Page and Blanton, 1985) comprises protists whose amoebae moving by sudden, "eruptive" bulging and often having a separate non-amoeboid flagellate stage; these belong in the excavate phylum Percolozoa (Cavalier-Smith 2003), which also includes purely flagellate protozoa like Percolomonas and Stephanopogon (Nikolaev and Cavalier-Smith 2007). Very different are the tremendously diverse rhizarian ‘amoebae’ (Cavalier-Smith, 2002) with net-like or thread- like pseudopodia; Rhizaria belong in the "SAR" clade (Burki et al., 2007), now formally subkingdom Harosa of the kingdom Chromista (Cavalier-Smith, 2010a). The third major amoeboid group, phylum Amoebozoa (Cavalier-Smith, 1998), is the subject of this paper; they are of special evolutionary interest, being related to the opisthokont common ancestor of animals and fungi (Cavalier-Smith et al., 2004; Cavalier-Smith, 2013). Amoebozoa typically have non-eruptive, generally blunt and broad (i.e. lobose) pseudopodia; cells are naked or with a shell (testate) or bear a dorsal organic cell coat (Himatismenida), though some have other protoplasmic forms such as the massive macroscopic multinucleate plasmodia of many slime moulds (myxomycete Mycetozoa). Amoebozoa comprise two subphyla: the purely amoeboid Lobosa that never have cilia and 4 mostly have blunt and broad cytoplasmic projections, and Conosa with both amoeboid and flagellate lineages and often more pointed or even mildly branched subspeudopodia. In species number, perhaps over 2400 (Pawlowski et al., 2012), Amoebozoa are a major protist phylum, yet are underrepresented in molecular databases (Pawlowski, 2008; Pawlowski and Burki, 2009). Currently, their phylogeny is based either on few species in well-resolved but extremely sparse multigene trees, or on a plethora of species in basally ill-resolved and often partially contradictory small subunit ribosomal RNA gene (18S rDNA) trees, leaving several phylogenetic questions debatable. These include: (1) their monophyly or otherwise (here, monophyly = holophyly of Ashlock (1971) and Cavalier-Smith (2010b)); (2) the phylogenetic validity of the present classification into classes and orders based primarily on locomotory modes and ultrastructure as interpreted in the light of single-gene trees (Smirnov et al., 2011: Cavalier-Smith 2013); (3) the unstable position of Archamoebea (e.g. Entamoeba, Mastigamoeba, Phreatamoeba); and (4) that of the even harder to place Himatismenida, with a unique dorsal cell coat (e.g. Cochliopodium, Ovalopodium and Parvamoeba). Existing data for multigene analyses are taxonomically biased (Fig. 1) and too sparse (only a dozen species) to answer these questions, so we have partially sequenced seven new transcriptomes and thereby answer all four. Most completed or in progress genome sequences belong to subphylum Conosa and only two to subphylum Lobosa – only one to each class, a deficiency especially serious for the diverse and contested class Discosea, and none to the conosan classes Variosea or Protostelea. Comprehensive reviews are available for Amoebozoa (Pawlowski, 2008; Schilde and Schaap, 2013; Smirnov et al., 2011), so we outline as essential background only recent studies defining these key questions that we now resolve. 5 1.1 Are Amoebozoa monophyletic? Amoebozoa encompass such a variety of forms and life-styles that the group has been criticized for lack of unifying ultrastructural characters (Parfrey et al., 2006; Yoon et al., 2008). Numerous 18S rDNA phylogenies including a wide array of Amoebozoa failed to resolve their basal branching or consistently show monophyly (e.g. Altan et al., 2012; Kudryavtsev et al., 2011; Pombert et al., 2013; Ptáčková et al., 2013). Several recent eukaryote-wide multigene phylogenies (>10 genes) based on 5-10 Amoebozoa clearly recovered their monophyly (Baurain et al., 2010; Brown et al., 2013; Derelle and Lang, 2012; Grant et al., 2012; Lasek-Nesselquist and Gogarten, 2013), but others failed to do so, at least in part of the analyses (Burki et al., 2013; Grant et al., 2012; Parfrey et al., 2010; Yoon et al., 2008). An anaerobic amoeboflagellate of unstable position, Breviata anathema (first misidentified as the amoebozoan Mastigamoeba invertens), was thought to be either weakly related to the flagellate apusomonads (Walker et al., 2006) or the most divergent branch of Amoebozoa (Cavalier-Smith et al. 2004; Paps et al., 2013; Shalchian-Tabrizi et al., 2008) or even to belong within Amoebozoa as sister to Archamoebea (Shalchian-Tabrizi et al., 2008). Recently, it was convincingly shown that Breviatea (Breviata, Subulatomonas, Pygsuia) are not amoebozoans, but a distinct sulcozoan clade between Amoebozoa and opisthokonts that is probably sister to opisthokonts plus apusomonads (Brown et al., 2013; Cavalier-Smith et al., submitted; Katz et al., 2011). Exclusion of Breviatea was a major step in defining the boundaries of Amoebozoa, but multigene analyses (>100 genes) with broader sampling are necessary to establish more convincingly whether remaining Amoebozoa are monophyletic. 1.2 The deepest division in Amoebozoa: Conosa/Lobosa or Tubulinea/other Amoebozoa? Recently a new classification