The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

Sabeeha S. Merchant^1*, Simon E. Prochnik^2*, Olivier Vallon³, Elizabeth H. Harris⁴, Steven J. Karpowicz¹, George B. Witman⁵, Astrid Terry², Asaf Salamov², Lillian K. Fritz-Laylin⁶, Laurence Maréchal-Drouard⁷, Wallace F. Marshall⁸, Liang-Hu Qu⁹, David R. Nelson¹⁰, Anton A. Sanderfoot¹¹, Martin H. Spalding¹², Vladimir V. Kapitonov¹³, Qinghu Ren¹⁴, Patrick Ferris¹⁵, Erika Lindquist², Harris Shapiro², Susan M. Lucas², Jane Grimwood¹⁶, Jeremy Schmutz¹⁶, Pierre Cardol^3,17, Heriberto Cerutti¹⁸, Guillaume Chanfreau¹, Chun-Long Chen⁹, Valérie Cognat⁷, Martin T. Croft¹⁹, Rachel Dent²⁰, Susan Dutcher²¹, Emilio Fernández²², Hideya Fukuzawa²³, David González-Ballester²⁴, Diego González-Halphen²⁵, Armin Hallmann²⁶, Marc Hanikenne¹⁷, Michael Hippler²⁷, William Inwood²⁰, Kamel Jabbari²⁸, Ming Kalanon²⁹, Richard Kuras³, Paul A. Lefebvre¹¹, Stéphane D. Lemaire³⁰, Alexey V. Lobanov³¹, Martin Lohr³², Andrea Manuell³³, Iris Meier³⁴, Laurens Mets³⁵, Maria Mittag³⁶, Telsa Mittelmeier³⁷, James V. Moroney³⁸, Jeffrey Moseley²⁴, Carolyn Napoli³⁹, Aurora M. Nedelcu⁴⁰, Krishna Niyogi²⁰, Sergey V. Novoselov³¹, Ian T. Paulsen¹⁴, Greg Pazour⁴¹, Saul Purton⁴², Jean-Philippe Ral⁴³, Diego Mauricio Riaño-Pachón⁴⁴, Wayne Riekhof⁴⁵, Linda Rymarquis⁴⁶, Michael Schroda⁴⁷, David Stern⁴⁸, James Umen¹⁵, Robert Willows⁴⁹, Nedra Wilson⁵⁰, Sara Lana Zimmer⁴⁸, Jens Allmer⁵¹, Janneke Balk¹⁹, Katerina Bisova⁵², Chong-Jian Chen⁹, Marek Elias⁵³, Karla Gendler³⁹, Charles Hauser⁵⁴, Mary Rose Lamb⁵⁵, Heidi Ledford²⁰, Joanne C. Long¹, Jun Minagawa⁵⁶, M. Dudley Page¹, Junmin Pan⁵⁷, Wirulda Pootakham²⁴, Sanja Roje⁵⁸, Annkatrin Rose⁵⁹, Eric Stahlberg³⁴, Aimee M. Terauchi¹, Pinfen Yang⁶⁰, Steven Ball⁶¹, Chris Bowler^28,62, Carol L. Dieckmann³⁷, Vadim N. Gladyshev³¹, Pamela Green⁴⁶, Richard Jorgensen³⁹, Stephen Mayfield³³, Bernd Mueller-Roeber⁴⁴, Sathish Rajamani⁶³, Richard T. Sayre³⁴, Peter Brokstein², Inna Dubchak², David Goodstein², Leila Hornick², Y. Wayne Huang², Jinal Jhaveri², Yigong Luo², Diego Martínez², Wing Chi Abby Ngau², Bobby Otillar², Alexander Poliakov², Aaron Porter², Lukasz Szajkowski², Gregory Werner², Kemin Zhou², Igor V. Grigoriev², Daniel S. Rokhsar^2,6 and Arthur R. Grossman²⁴

¹ Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA 90095, USA.
² U.S. Department of Energy (DOE) Joint Genome Institute (JGI), Walnut Creek, CA 94598, USA.
³ CNRS, Université Paris 6, Institut de Biologie Physico-Chimique, 75005 Paris, France.
⁴ Department of Biology, Duke University, Durham, NC 27708, USA.
⁵ Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
⁶ Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
⁷ Institut de Biologie Moléculaire des Plantes, CNRS, 67084 Strasbourg Cedex, France.
⁸ Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143, USA.
⁹ Biotechnology Research Center, Zhongshan University, Guangzhou 510275, China.
¹⁰ Department of Molecular Sciences and Center of Excellence in Genomics and Bioinformatics, University of Tennessee, Memphis, TN 38163, USA.
¹¹ Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA.
¹² Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011, USA.
¹³ Genetic Information Research Institute, Mountain View, CA 94043, USA.
¹⁴ The Institute for Genomic Research, Rockville, MD 20850, USA.
¹⁵ Plant Biology Laboratory, Salk Institute, La Jolla, CA 92037, USA.
¹⁶ Stanford Human Genome Center, Stanford University School of Medicine, Palo Alto, CA 94304, USA.
¹⁷ Plant Biology Institute, Department of Life Sciences, University of Liège, B-4000 Liège, Belgium.
¹⁸ University of Nebraska-Lincoln, School of Biological Sciences–Plant Science Initiative, Lincoln, NE 68588, USA.
¹⁹ Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK.
²⁰ Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
²¹ Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.
²² Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Campus de Rabanales, 14071 Córdoba, Spain.
²³ Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
²⁴ Department of Plant Biology, Carnegie Institution, Stanford, CA 94306, USA.
²⁵ Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México 04510 DF, Mexico.
²⁶ Department of Cellular and Developmental Biology of Plants, University of Bielefeld, D-33615 Bielefeld, Germany.
²⁷ Department of Biology, Institute of Plant Biochemistry and Biotechnology, University of Münster, 48143 Münster, Germany.
²⁸ CNRS UMR 8186, Département de Biologie, Ecole Normale Supérieure, 75230 Paris, France.
²⁹ Plant Cell Biology Research Centre, The School of Botany, The University of Melbourne, Parkville, Melbourne, VIC 3010, Australia.
³⁰ Institut de Biotechnologie des Plantes, UMR 8618, CNRS/Université Paris-Sud, Orsay, France.
³¹ Department of Biochemistry, N151 Beadle Center, University of Nebraska, Lincoln, NE 68588–0664, USA.
³² Institut für Allgemeine Botanik, Johannes Gutenberg-Universität, 55099 Mainz, Germany.
³³ Department of Cell Biology and Skaggs Institute for Chemical Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
³⁴ PCMB and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA.
³⁵ Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
³⁶ Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena, 07743 Jena, Germany.
³⁷ Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.
³⁸ Department of Biological Science, Louisiana State University, Baton Rouge, LA 70803, USA.
³⁹ Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA.
⁴⁰ Department of Biology, University of New Brunswick, Fredericton, NB, Canada E3B 6E1.
⁴¹ Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴² Department of Biology, University College London, London WC1E 6BT, UK.
⁴³ Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS/USTL, IFR 118, Université des Sciences et Technologies de Lille, Cedex, France.
⁴⁴ Universität Potsdam, Institut für Biochemie und Biologie, D-14476 Golm, Germany.
⁴⁵ Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, USA.
⁴⁶ Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
⁴⁷ Institute of Biology II/Plant Biochemistry, 79104 Freiburg, Germany.
⁴⁸ Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, NY 14853, USA.
⁴⁹ Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney 2109, Australia.
⁵⁰ Department of Anatomy and Cell Biology, Oklahoma State University, Center for Health Sciences, Tulsa, OK 74107, USA.
⁵¹ Izmir Ekonomi Universitesi, 35330 Balcova-Izmir Turkey.
⁵² Institute of Microbiology, Czech Academy of Sciences, Czech Republic.
⁵³ Department of Plant Physiology, Faculty of Sciences, Charles University, 128 44 Prague 2, Czech Republic.
⁵⁴ Bioinformatics Program, St. Edward's University, Austin, TX 78704, USA.
⁵⁵ Department of Biology, University of Puget Sound, Tacoma, WA 98407, USA.
⁵⁶ Institute of Low-Temperature Science, Hokkaido University, 060-0819, Japan.
⁵⁷ Department of Biology, Tsinghua University, Beijing, China 100084.
⁵⁸ Institute of Biological Chemistry, Washington State University, Pullman, WA99164, USA.
⁵⁹ Appalachian State University, Boone, NC 28608, USA.
⁶⁰ Department of Biology, Marquette University, Milwaukee, WI 53233, USA.
⁶¹ UMR8576 CNRS, Laboratory of Biological Chemistry, 59655 Villeneuve d'Ascq, France.
⁶² Cell Signaling Laboratory, Stazione Zoologica, I 80121 Naples, Italy.
⁶³ Graduate Program in Biophysics, Ohio State University, Columbus, OH 43210, USA.

Fig. 1. A schematic of a Chlamydomonas cell (from transmission electron micrographs) showing the anterior flagella rooted in basal bodies, with intraflagellar transport (IFT) particle arrays between the axoneme and flagellar membrane, the basal cup-shaped chloroplast, central nucleus and other organelles. An expanded cross section of the flagellar axoneme, as redrawn from (48), shows the nine outer doublets and the central pair (9+2) microtubules; axoneme sub-structures are color-coded and labeled (see inset). [View Larger Version of this Image (63K GIF file)]

Fig. 2. Evolutionary relationships of 20 species with sequenced genomes (54, 55) used for the comparative analyses in this study include cyanobacteria and nonphotosynthetic eubacteria, Archaea and eukaryotes from the oomycetes, diatoms, rhodophytes, plants, amoebae and opisthokonts. Endosymbiosis of a cyanobacterium by a eukaryotic protist gave rise to the green (green branches) and red (red branches) plant lineages, respectively. The presence of motile or nonmotile flagella is indicated at the right of the cladogram. [View Larger Version of this Image (33K GIF file)]

Fig. 3. Linkage group I depicted as a long horizontal rod, with genetically mapped scaffolds shown as open rectangles below (the scaffold number is under each scaffold, and arrows indicate the orientation of the scaffold where it is known; other scaffolds were placed in their most likely orientation on the basis of genetic map distances. The scale of each map is determined by molecular lengths of the mapped scaffolds. Short and long red ticks are drawn on scaffolds every 0.2 Mb and 1.0 Mb, respectively. We assumed small 50 kb gaps between scaffolds. Genetic distances between markers (centimorgans), where they are known, are shown by two-headed arrows above the scaffold, with the gene symbol and any synonyms in parentheses shown at the top. Genomic regions are labeled below the scaffolds: 5S, rDNA, mito (insertion of mitochondrial DNA). Chlamydomonas genes with homologs in other organisms/lineages ("Cuts" as defined in the text and Fig. 5) are shown as tracks of vertical bars: light red, genes shared between Chlamydomonas and humans, but not occurring in nonciliated organisms; dark red, genes in CiliaCut; light green, genes shared between Chlamydomonas and Arabidopsis, but not in nonphotosynthetic organisms; dark green, genes in GreenCut; magenta, predicted tRNAs, including those that represent SINE sequences; dark blue, small nucleolar RNAs (snoRNAs). [View Larger Version of this Image (17K GIF file)]

Fig. 4. (A) Scatter plot of best BLASTP hit score of Chlamydomonas proteins to Arabidopsis proteins versus best BLASTP hit score of Chlamydomonas proteins to human proteins. Functional or genomic groupings are colored [see inset key in (A)]: Chlamydomonas flagellar proteome (42) high confidence set (chlamyFPhc); CiliaCut; Arabidopsis stroma plastid proteome (stromaPP); Arabidopsis thylakoid plastid proteome (thylakoidPP); eyespot proteome; GreenCut; remaining proteins are gray. (B) Chlamydomonas protein paralogs were grouped into families together with their homologs from human and Arabidopsis. The outer circle represents the proteins in Chlamydomonas, 7476 (out of 15,143 total), that fall into 6968 families. Another 7937 proteins cannot be placed in families. Counts of families (and the numbers of proteins from each species in them) with proteins from Chlamydomonas and human only, Chlamydomonas and Arabidopsis only, and Chlamydomonas and human and Arabidopsis, are shown in the inner circles and the overlap between the two inner circles, respectively. Cre, Chlamydomonas; Hsa, human; Ath, Arabidopsis. [View Larger Version of this Image (37K GIF file)]

Fig. 5. Summary of genomic comparisons to photosynthetic and ciliated organisms. (A) GreenCut: The GreenCut comprises 349 Chlamydomonas proteins with homologs in representatives of the green lineage of the Plantae (Chlamydomonas, Physcomitrella, and Ostreococcus tauri and O. lucimarinus), but not in nonphotosynthetic organisms. Genes encoding proteins of unknown function that were not previously annotated were given names on the basis of their occurrence in various cuts. CGL refers to conserved only in the green lineage. The GreenCut protein families, which also include members from the red alga Cyanidioschyzon within the Plantae, were assigned to the PlantCut (blue plus green rectangles). CPL refers to conserved in the Plantae. GreenCut proteins also present in at least one diatom (Thalassiosira and Phaeodactylum) were assigned to the DiatomCut (yellow plus green rectangle). CGLD refers to conserved in the green lineage and diatoms. Proteins present in all of the eukaryotic plastid-containing organisms in this analysis were assigned to the PlastidCut (green rectangle). CPLD refers to conserved in the Plantae and diatoms. The criteria used for the groupings associated with the GreenCut are given in the lower table. (B) CiliaCut: The CiliaCut contains 195 Chlamydomonas proteins with homologs in human and species of Phytophthora, but not in nonciliated organisms. This group was subdivided on the basis of whether or not a homolog was present in Caenorhabditis, which has only nonmotile sensory cilia. The 133 CiliaCut proteins without homologs in Caenorhabditis were designated the MotileCut (orange rectangle). Unnamed proteins in this group were named MOT (motility). Proteins with homologs in Caenorhabditis are associated with nonmotile cilia (white and yellow areas). Proteins in this group that were not already named were named SSA. The CentricCut (yellow plus light orange box) is made up of 69 CiliaCut homologs present in the centric diatom Thalassiosira. These proteins can be divided into those also in the MotileCut (38 proteins; light orange box) or those not present in the MotileCut (31 proteins; yellow box). [View Larger Version of this Image (32K GIF file)]