articles - Genetics
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articles - Genetics
Vol 443 | 19 October 2006 | doi:10.1038/nature05110 ARTICLES Reconstructing the early evolution of Fungi using a six-gene phylogeny Timothy Y. James1, Frank Kauff1, Conrad L. Schoch2*, P. Brandon Matheny3*, Valérie Hofstetter1*, Cymon J. Cox1{, Gail Celio4, Cécile Gueidan1, Emily Fraker1, Jolanta Miadlikowska1, H. Thorsten Lumbsch5, Alexandra Rauhut6, Valérie Reeb1, A. Elizabeth Arnold1{, Anja Amtoft7, Jason E. Stajich8, Kentaro Hosaka2{, Gi-Ho Sung2, Desiree Johnson2, Ben O’Rourke2, Michael Crockett2, Manfred Binder3, Judd M. Curtis3, Jason C. Slot3, Zheng Wang3{, Andrew W. Wilson3, Arthur Schüßler9, Joyce E. Longcore10, Kerry O’Donnell11, Sharon Mozley-Standridge12, David Porter12, Peter M. Letcher13, Martha J. Powell13, John W. Taylor14, Merlin M. White15, Gareth W. Griffith16, David R. Davies17, Richard A. Humber18, Joseph B. Morton19, Junta Sugiyama20, Amy Y. Rossman21, Jack D. Rogers22, Don H. Pfister23, David Hewitt23, Karen Hansen23, Sarah Hambleton24, Robert A. Shoemaker24, Jan Kohlmeyer25, Brigitte Volkmann-Kohlmeyer25, Robert A. Spotts26, Maryna Serdani26, Pedro W. Crous27, Karen W. Hughes28, Kenji Matsuura29, Ewald Langer30, Gitta Langer30, Wendy A. Untereiner31, Robert Lücking5, Burkhard Büdel6, David M. Geiser32, André Aptroot33, Paul Diederich34, Imke Schmitt5{, Matthias Schultz35, Rebecca Yahr1{, David S. Hibbett3, François Lutzoni1, David J. McLaughlin4, Joseph W. Spatafora2 & Rytas Vilgalys1 The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree. Fungi, Viridiplantae and Animalia are all large clades descended from unicellular, flagellated, aquatic forms that radiated extensively on land. For both plants and animals, biologists have developed unified hypotheses regarding the evolution of morphology and ecology from ancestral to highly derived traits. For example, among green plants, morphologically simple photosynthetic forms, such as unicellular green algae, gave rise to multicellular forms such as bryophytes, and were followed by a radiation of complex flowering forms with highly derived sexual mechanisms at the tips of the plant phylogeny1,2. Similarly, animals seem to have evolved increasingly complex tissue systems and development from a simple, flagellated, protistlike ancestor similar to extant Choanoflagellida3. 1 Department of Biology, Duke University, Durham, North Carolina 27708-0338, USA. 2Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 973312902, USA. 3Department of Biology, Clark University, Worcester, Massachusetts 01610, USA. 4Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108, USA. 5Field Museum of Natural History, Chicago, Illinois 60605-2496, USA. 6Fachbereich Biologie, Abteilung Pflanzenökologie und Systematik, 67653 Kaiserslautern, Germany. 7 Institute of Systematic Botany, New York Botanical Garden, Bronx, New York 10458-6126, USA. 8University Program in Genetics and Genomics, Duke University, Durham, North Carolina 27708-0338, USA. 9Institute of Botany, Darmstadt University of Technology, D-64287 Darmstadt, Germany. 10Department of Biological Sciences, University of Maine, Orono, Maine 04469, USA. 11National Center for Agricultural Utilization Research, USDA Agricultural Research Service, Peoria, Illinois 61604, USA. 12Department of Plant Biology, University of Georgia, Athens, Georgia 30605, USA. 13Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487, USA. 14Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA. 15Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045-7534, USA. 16Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DA, UK. 17Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK. 18United States Plant, Soil and Nutrition Laboratory, USDA-ARS Plant Protection Research Unit, Ithaca, New York 14853-2901, USA. 19Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia 26506-6057, USA. 20TechnoSuruga, Chiyoda-ku, Tokyo 101-0052, Japan. 21Systematic Botany and Mycology Laboratory, USDA Agricultural Research Service, Beltsville, Maryland 20705, USA. 22Department of Plant Pathology, Washington State University, Pullman, Washington 99164, USA. 23Harvard University Herbaria, Cambridge, Massachusetts 02138, USA. 24Biodiversity (Mycology and Botany), Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada. 25Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, North Carolina 28557, USA. 26Mid-Columbia Agricultural Research and Extension Center, Oregon State University, Hood River, Oregon 97031, USA. 27Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, 3508 AD Utrecht, The Netherlands. 28 Botany Department, University of Tennessee, Knoxville, Tennessee 37996, USA. 29Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan. 30Institut für Biologie, Universität Kassel, D-34132 Kassel, Germany. 31Department of Botany, Brandon University, Brandon, Manitoba R7A 6A9, Canada. 32Department of Plant Pathology, Penn State University, University Park, Pennsylvania 16802, USA. 33Adviesbureau voor Bryologie en Lichenologie, NL-3762 XK Soest, The Netherlands. 34Musée national d’histoire naturelle, L-2160 Luxembourg. 35Biozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Systematik der Pflanzen, D-22609 Hamburg, Germany. {Present addresses: Biometry and Molecular Research, Department of Zoology, Natural History Museum, London SW7 5BD, UK (C.J.C.); Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA (A.E.A.); Department of Botany, The Field Museum, Chicago, Illinois 60605-2496, USA (K.H.); Department of Biological Sciences, Roy J. Carver Center for Comparative Genomics, University of Iowa, Iowa City, Iowa 52242, USA (Z.W.); Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, D-07745 Jena, Germany (I.S.); Royal Botanic Garden Edinburgh, Edinburgh EH3 5LA, UK (R.Y.). *These authors contributed equally to this work. 818 ©2006 Nature Publishing Group ARTICLES NATURE | Vol 443 | 19 October 2006 Currently, no accepted phylogenetic hypothesis exists for the evolution of form and nutritional mode for the earliest fungi. Traditional views of fungal phylogeny indicate that fungi with flagellated cells (Chytridiomycota) are the sister group of the remaining phyla of non-flagellated fungi (Zygomycota, Glomeromycota, Ascomycota and Basidiomycota), implying a single loss of the flagellum coincident with a shift to land. Key adaptations to the terrestrial habit in the fungi include the evolution of a filamentous growth form and the development of aerially dispersed spores. However, recent phylogenetic studies question the monophyly of the basal phyla Chytridiomycota and Zygomycota4,5. Resolving the phylogeny of the basal groups of the Fungi and their relationships to Ascomycota and Basidiomycota is necessary to understand the sequence of events leading to the colonization of land and the evolution of terrestrial ecosystems. Here we present a multilocus phylogeny of the kingdom Fungi, including representatives of all currently recognized phyla. This analysis provides a robust kingdom-level phylogeny and suggests that there were at least four independent losses of flagella during the early evolution of the Fungi. We estimated the phylogeny of the Fungi using data from six gene regions: 18S rRNA, 28S rRNA, 5.8S rRNA, elongation factor 1-a (EF1a), and two RNA polymerase II subunits (RPB1 and RPB2). Incongruence among gene regions was tested by maximum likelihood bootstrap (MLBS) analyses of each data partition. This strategy allowed us to identify potential contaminant sequences in addition to conflicting phylogenetic signal. Very little conflicting signal among genes was detected, allowing construction of one super-matrix combining the data for all six gene regions for 199 fungal taxa, 29 of which used data from genome sequencing projects (Supplementary Notes 1). Only 6% of the cells in the super-matrix were missing data, and the number of aligned nucleotides was 6,436. The data were analysed by bayesian methods using a heterogeneous amino-acid and nucleotide model (see Supplementary Notes 2 for a nucleotide-only analysis). Support was estimated at nodes by bayesian posterior probabilities (BPP), MLBS and analysis of individual gene partitions (Supplementary Notes 3). Chytridiomycota is not monophyletic The combined gene phylogeny of the Fungi supported monophyly of the Ascomycota, Basidiomycota and Glomeromycota (Fig. 1). The Ascomycota and Basidiomycota formed a clade of ‘dikarya’ (that is, fungi characterized by having a portion of their life cycle with paired nuclei). Phylogenetic analyses also supported, by BPP, a clade uniting the dikarya and Glomeromycota, in agreement with previously published 18S rRNA phylogenies6,7. The opisthokont clade (Fungi, Metazoa and Choanoflagellida) was also recovered, as has been reported in other studies3,8,9. Two unexpected results were the placements of the endoparasitic, spizellomycetalean chytrids Olpidium brassicae and R. allomycis. Olpidium brassicae grouped with the Zygomycota as sister taxon to Basidiobolus ranarum, and R. allomycis grouped with the microsporidia as the earliest diverging branch of the Fungi. The phylum Chytridiomycota consists of true fungi that produce flagellated spores (zoospores). On the basis of ultrastructural studies, the chytrid zoospore is homologous to that of non-fungal opisthokonts10. The ultrastructural complexity of the opisthokont zoospore suggests that it has evolved only once. Because the zoospore is an ancestral trait, Chytridiomycota is solely defined on a shared ancestral trait (symplesiomorphy) rather than a shared derived trait (synapomorphy). Our phylogeny indicates that the Chytridiomycota is polyphyletic (Fig. 1), consisting of early diverging lineages that have retained the zoospore. However, one large clade of Chytridiomycota uniting the orders Chytridiales, Monoblepharidales, Neocallimastigales and some Spizellomycetales (which we call the ‘euchytrids’) is recovered with high support values in the combined analysis as well as in multiple, single-gene-based analyses (Fig. 1 and Supplementary Notes 3). In the present phylogeny (Fig. 1), six losses of the flagellum are inferred to have occurred during the evolution of the Fungi. Ancestral state reconstruction of the presence or absence of the flagellum along the phylogeny for each of the 58,611 credible trees demonstrated 4–6 losses (mean 5.86) of the flagellum within the Fungi. One well-supported loss took place along the branch leading to Hyaloraphidium curvatum, a unique fungus that grows superficially like a unicellular planktonic alga11. A second loss occurred in the lineage leading to the microsporidia and 2–4 losses occurred among Zygomycota. Variation in the number of losses of the flagellum is attributable, in part, to the uncertain placement of O. brassicae and members of the microsporidia. Rearrangement of the phylogenetic position of O. brassicae and microsporidia can create phylogenies requiring only two or three losses of the flagellum; however, each of these alternative phylogenies is rejected as statistically worse (in likelihood; P , 0.05) than that shown in Fig. 1. Most molecular phylogenies of the Fungi based on 18S rDNA have placed the zygomycete Basidiobolus among Chytridiomycota4,12. This placement indicated that Basidiobolus might have made the transition recently from a zoosporic state, and that an independent loss of a flagellum occurred in this lineage12. This argument was strengthened by the presence in two Basidiobolus species of a ring-shaped spindle pole body that contains 11–12 singlet microtubules similar to a centriole, but lacks centriolar ninefold symmetry13. Our phylogeny is the first to place Basidiobolus close to Entomophthorales, the order within which it has been classified traditionally and to which it is ecologically and morphologically allied14 (for additional phylogenetic support from a paralogous copy of EF1a, see Supplementary Notes 4). Unexpectedly, the phylogeny also suggests a relationship between B. ranarum and the chytrid O. brassicae (Fig. 1). A functional link between the two taxa is unclear: O. brassicae is an endoparasite of plant roots, whereas Basidiobolus is associated with insects, soil and amphibians. Phylogenetic position of the microsporidia Microsporidia are obligately endoparasitic, protist-like organisms with highly reduced morphology and genomes15. A defining characteristic of these parasites is the elaborate mechanism by which the spore contents are rapidly injected into the host’s cytoplasm through a thin polar tube. Placement of microsporidia in the tree of life has been problematic owing to their extremely accelerated rate of sequence evolution. The earliest phylogenetic analyses of 18S rRNA placed the microsporidia among the earliest diverging lineages of eukaryotes15; however, these analyses now seem to have been an artefact of ‘long branch attraction’ of microsporidia to the base of the phylogeny15. More recent results using RPB1, a- and b-tubulin, and other genes, have suggested a fungal origin of the microsporidia16–18, a placement consistent with their having the shared traits of closed mitosis and spores that contain chitin and trehalose 19. Only one study has placed the microsporidia with a specific fungal lineage, in which a relationship was demonstrated between members of the Zygomycota and microsporidia by using tubulin proteins18. However, tubulin proteins seem to have evolved at different rates in flagellated and non-flagellated fungi18,20. The microsporidia and R. allomycis are intracellular parasites of primarily animals and fungi, respectively. A similarity between microsporidia and R. allomycis is the absence of a cell wall when invading host cells, such that the plasma membrane of the parasite makes direct contact with the cytoplasm of the host cell19,21. Although R. allomycis does not seem to occupy a long phylogenetic branch, we tested whether the placement of microsporidia with R. allomycis was due to long branch attraction. Two different methods suggested that the relationship between microsporidia and R. allomycis is not due to long branch attraction (see Supplementary Notes 5). We also tested whether alternative placements for the microsporidia could be statistically rejected from the maximum likelihood phylogeny shown in Fig. 1 using the approximately unbiased test22. Alternative placements of microsporidia with Fungi that have been suggested 819 ©2006 Nature Publishing Group ARTICLES Phagotroph Phototroph Lichenized Mycorrhizal Plant pathogen Animal pathogen Mycoparasite Insect commensal Saprobe Uncertain ... to Ascomycota 0.05 substitutions per site Figure 1 | Phylogeny of the kingdom Fungi using bayesian analysis of the combined, six-gene data set. Each fungal species begins with a unique ‘Assembling the Fungal Tree of Life’ identifier, followed by genus and species. Indicated for each terminal taxon are: nutritional mode, whether they produce flagellated cells and if there is a genome sequence for the taxon completed or underway. Thickened branches indicate those that are supported both by heterogeneous bayesian analysis (BPP $95%) and by MLBS ($70%). Almost every branch was supported by BPP and thus values are not shown. Where indicated, support values (percentage of trees in Basidiomycota: Agaricomycotina Basidiomycota: Ustilaginomycotina Basidiomycota: Pucciniomycotina Glomeromycota ‘Zygomycota’: Mucormycotina ‘Zygomycota’ ‘Chytridiomycota’ ‘Zygomycota’: Entomophthorales ‘Chytridiomycota’: Blastocladiales ‘Chytridiomycota’: euchytrids microsporidia ‘Chytridiomycota’ Metazoa Choanoflagellida Mycetozoa Apicomplexa Stramenopiles Rhodophyta Viridiplantae Ascomycota: Eurotiomycetes 1087 Coprinopsis cinerea S 480 Lycoperdon pyriforme S 626 Coprinus comatus S 563 Clavaria zollingeri ? 673 Amanita brunnescens M 625 Pluteus romellii S 285 Cortinarius iodes M 564 Pleurotus ostreatus S 449 Armillaria mellea S P M 558 Flammulina velutipes S 556 Marasmius alliaceus S 542 Ampulloclitocybe clavipes ? Phagotroph 557 Collybia tuberosa S Phototroph Dikarya 468 Henningsomyces candidus S Mutualist 729 Hygrocybe aff. conica S Pathogen 439 Calostoma cinnabarinum M 100/71 Saprobe 713 Boletellus projectellus M 714 Hygrophoropsis aurantiaca S Uncertain 717 Suillus pictus M Genome 576 Fibulorhizoctonia sp. I sequenced 455 Echinodontium tinctorium S 682 Lactarius deceptivus M Motile cell stage 452 Bondarzewia montana S present 492 Stereum hirsutum S 447 Coltricia perennis M 688 Fomitiporia mediterranea S P 484 Phlebia radiata S 767 Climacodon septentrionalis S 776 Phanerochaete chrysosporium S 562 Grifola sordulenta S P 100/100 701 Grifola frondosa S P 770 Fomitopsis pinicola S P 518 Hyphoderma praetermissum S 100/92 700 Cotylidia sp. ? 466 Gautieria otthii M 100/73 724 Ramaria rubella M 471 Hydnum albomagnum M 438 Calocera cornea S 454 Dacryopinax spathularia S 1088 Cryptococcus neoformans A 505 Ustilago maydis P 867 Cintractia sorghi vulgaris P 870 Tilletiopsis sp. P 865 Tilletiaria anomala P 100/80 675 Agaricostilbum hyphaenes S 709 Colacogloea peniophorae Y 674 Rhodotorula hordea S P 456 Endocronartium harknessii P 1459 Puccinia graminis P 710 Platygloea disciformis Y 138 Scutellospora heterogama M 139 Glomus mosseae M 845 Glomus intraradices M 574 Geosiphon pyriformis M 844 Paraglomus occultum M 141 Mortierella verticillata S 144 Umbelopsis ramanniana S 184 Phycomyces blakesleeanus S 1241 Rhizopus oryzae S 539 Endogone pisiformis S 136 Dimargaris bacillispora Y 140 Coemansia reversa S 1062 Orphella aff. haysii I 29 Smittium culisetae I 185 Spiromyces aspiralis S 142 Rhopalomyces elegans A 145 Piptocephalis corymbifera Y --/85 301 Basidiobolus ranarum S A 633 Olpidium brassicae P 28 Entomophthora muscae A 137 Conidiobolus coronatus S A 19 Physoderma maydis P 18 Coelomomyces stegomyiae A 300 Allomyces arbusculus S 20 Rhizoclosmatium sp. S 24 Polychytrium aggregatum S 27 Cladochytrium replicatum S Fungi 21 Batrachochytrium dendrobatidis A 689 Rhizophydium macroporosum S 43 Rhizophlyctis rosea S 182 Spizellomyces punctatus S 635 Synchytrium macrosporum P 25 Monoblepharella sp. S 26 Hyaloraphidium curvatum S 638 Neocallimastix sp. S 1068 Encephalitozoon cuniculi A 1089 Antonospora locustae A 297 Rozella allomycis Y Caenorhabditis elegans H Ciona intestinalis H Homo sapiens H Drosophila melanogaster H Monosiga brevicollis H Dictyostelium discoideum H Cryptosporidium parvum A Toxoplasma gondii A Phytophthora sojae P Thalassiosira pseudonana O Cyanidioschyzon merolae O Arabidopsis thaliana O Populus trichocarpa O Oryza sativa O Chlamydomonas reinhardtii O 1078 Neurospora crassa S 216 Sordaria fimicola S 1085 Podospora anserina S 217 Chaetomium globosum S 1081 Magnaporthe grisea P 935 Diaporthe eres P 952 Gnomonia gnomon P 51 Xylaria hypoxylon S 63 Xylaria acuta S 100/100 1082 Fusarium graminearum P 161 Fusarium aff. solani P 186 Hydropisphaera erubescens S 52 Hypocrea citrina Y 914 Microascus trigonosporus S 413 Lindra thalassiae S 424 Lulworthia grandispora S 1 Leotia lubrica S 147 Coccomyces dentatus S 744 Potebniamyces pyri P 151 Chlorociboria aeruginosa S 76 Mollisia cinerea P 279 Monilinia fructicola P Ascomycota: 59 Botryotinia fuckeliana P Leotiomycetes 941 Dermea acerina S 166 Cudoniella clavus S 49 Lachnum virgineum S 56 Geoglossum nigritum S 64 Trichoglossum hirsutum S 1004 Pleopsidium chlorophanum L 1005 Acarospora schleicheri L 1007 Acarospora laqueata L 106 Echinoplaca strigulacea L 958 Diploschistes ocellatus L 78 Acarosporina microspora S 398 Stictis radiata S 296 Orceolina kerguelensis L 962 Trapelia placodioides L 224 Pertusaria dactylina L 358 Dibaeis baeomyces L Ascomycota: 645 Umbilicaria mammulata L Lecanoromycetes 687 Hypocenomyce scalaris L 134 Peltigera degenii L 196 Mycoblastus sanguinarius L 639 Lecanora hybocarpa L 6 Canoparmelia caroliniana L 3 Cladonia caroliniana L 642 Bacidia schweinitzii L 84 Physcia aipolia L 1079 Aspergillus fumigatus S A 1080 Aspergillus nidulans S 426 Monascus purpureus S 1083 Histoplasma capsulatum A 1084 Coccidioides immitis A 430 Spiromastix warcupii S 657 Capronia pilosella S 668 Exophiala dermatitidis S A 100/81 659 Ramichloridium anceps S 669 Exophiala pisciphila S A 684 Agonimia sp. L 91 Dermatocarpon miniatum L 661 Endocarpon pallidulum L 697 Staurothele frustulenta L 342 Pyrgillus javanicus L 387 Pyrenula pseudobufonia L 891 Peltula umbilicata L Ascomycota: 892 Peltula auriculata L Lichinomycetes 896 Lichinella iodopulchra L 101 Anisomeridium polypori L 1036 Trematosphaeria heterospora S 1037 Westerdykella cylindrica S 283 Pyrenophora phaeocomes P 54 Cochliobolus heterostrophus P 940 Pleospora herbarum S 110 Trypethelium sp. L Ascomycota: 274 Dothidea sambuci P 921 Dothidea insculpta S Dothideomycetes 939 Capnodium coffeae P 355 Dendrographa minor L Ascomycota: 126 Roccella fuciformis L Arthoniomycetes Pezizomycotina 80 Simonyella variegata L 148 Cheilymenia stercorea S 62 Scutellinia scutellata S 100/94 65 Aleuria aurantia S 949 Pyronema domesticum S 50 Sarcoscypha coccinea S 152 Caloscypha fulgens S Ascomycota: 176 Gyromitra californica S Pezizomycetes 179 Disciotis sp. S 60 Morchella aff. esculenta S 66 Helvella compressa S 181 Ascobolus crenulatus S 507 Peziza vesiculosa S 100/93 71 Peziza proteana S 905 Orbilia vinosa S Ascomycota: Orbiliomycetes 906 Orbilia auricolor S 1069 Saccharomyces cerevisiae S 1070 Saccharomyces castellii S 1073 Candida glabrata A 1071 Kluyveromyces waltii S 1072 Ashbya gossypii P 100/94 1075 Kluyveromyces lactis S Ascomycota: 1074 Candida albicans A Saccharomycotina 1269 Candida tropicalis A 1270 Candida guilliermondii A 1077 Debaryomyces hansenii S 1268 Candida lusitaniae A 1076 Yarrowia lipolytica S 1199 Schizosaccharomyces pombe S 265 Taphrina wiesneri P Ascomycota: 266 Protomyces inouyei P Taphrinomycotina 100/98 1192 Pneumocystis carinii A Ascomycota: Sordariomycetes H O L M P A Y I S ? NATURE | Vol 443 | 19 October 2006 ... agreement out of 58,611 trees) indicate BPP followed by MLBS. Branches are shaded according to reconstruction of nutritional mode. Microsporidia branches have been shortened three times (double black break) to increase readability. Red vertical ticks on branches indicate alternative placements of microsporidia that might be significantly rejected (P , 0.05) and green ticks indicate placements that cannot be rejected. Quotation marks indicate nonmonophyly of the taxon. The name ‘Mucormycotina’ will be validated in a manuscript that is in preparation. 820 ©2006 Nature Publishing Group ARTICLES NATURE | Vol 443 | 19 October 2006 include: a sister relationship to the dikarya23; sister to the zygomycete order Entomophthorales18; and among the harpellid Trichomycetes19, represented here by Smittium culisetae. We were able to reject (P , 0.05) nine alternative placements of the microsporidia (red vertical ticks in Fig. 1), including early divergences among eukaryotes. However, we were unable to reject a placement of microsporidia as sister to Entomophthorales, as sister to the blastocladialean chytrids, as sister to the zygomycete Dimargaris, as sister to dikarya and as sister to the Fungi (green vertical ticks in Fig. 1). Taken together, our results suggest that the relationship between the microsporidia and R. allomycis is a result of true phylogenetic signal. The present phylogeny provides an alternative hypothesis for the placement of microsporidia, specifically on the earliest diverging fungal branch with the chytrid R. allomycis. However, support for this relationship is derived only from the RPB1 and RPB2 gene partitions and is not supported by rDNA (see Supplementary Notes 3); alternative hypotheses in which the microsporidia diverge among early fungi cannot be rejected. The ultimate resolution of the placement of microsporidia will require sampling of additional genes from basal fungal taxa. Dikarya The majority (,98%) of described fungal species are members of the dikarya clade, which includes the two phyla Ascomycota and Basidiomycota. Ascomycota is the largest phylum within the Fungi and is characterized by the production of meiospores (ascospores) in specialized sac-shaped meiosporangia (asci), which may or may not be produced within a sporocarp (ascoma). Ascomycota is divided into three monophyletic subphyla: Taphrinomycotina, Saccharomycotina and Pezizomycotina (each of which is well supported as monophyletic in the phylogeny; Fig. 1). Taphrinomycotina is resolved as the earliest diverging clade; it includes a diverse group of species that exhibit yeast-like (for example, Pneumocystis) and dimorphic—that is, yeast-like and filamentous (for example, Taphrina)—growth forms. The subphylum Saccharomycotina consists of the ‘true yeasts’, including bakers’ yeast (Saccharomyces cerevisiae) and Candida albicans, the most frequently encountered fungal pathogen of humans. Pezizomycotina is the largest subphylum of Ascomycota and includes the vast majority of filamentous, fruit-body-producing species. Data presented here resolved the Orbiliomycetes and Pezizomycetes as the early-diverging lineages of the Pezizomycotina, with the remaining seven classes sampled forming a well-supported crown clade. Reduced ascomatal morphologies, whereby asci are contained within fruit bodies that are enclosed partially (Dothideomycetes, Eurotiomycetes and some Sordariomycetes) or completely (Eurotiomycetes, Leotiomycetes and some Sordariomycetes), are restricted to the crown clade of Pezizomycotina. The Basidiomycota includes about 30,000 species of rusts, smuts, yeasts, and mushroom fungi24. Most are characterized by meiospores (basidiospores) on the exterior of typically club-shaped meiosporangia (basidia). Phylogenetic relationships among the three subphyla of Basidiomycota are uncertain. The subphylum Pucciniomycotina is primarily distinguished by containing the rust fungi (7,000 species), which are primarily pathogens of land plants. Cytological and biochemical data25 are consistent with a sister group relationship between the subphyla Ustilaginomycotina and Agaricomycotina, as shown in Fig. 1. The Ustilaginomycotina includes 1,500 species of true smut fungi and yeasts, most of which cause systemic infections of angiosperm hosts. The Agaricomycotina includes almost two-thirds of known basidiomycetes, including the vast majority of mushroomforming fungi. Much of the morphological diversity exemplified in mushroom fruiting bodies is the result of radiations of certain lineages within the Agaricomycotina, and recovering their relationships with confidence has proven difficult26,27. Early-diverging lineages in the Agaricomycotina, which are strongly supported in Fig. 1, also include parasitic and/or saprotrophic fungi capable of dimorphism or yeast-like phases. The mycorrhizal basidiomycetes seem to have multiple, independent evolutionary origins from saprotrophic ancestors as previously suggested28. Characteristics of early fungi We reconstructed ancestral states for major nutritional modes in the Fungi using maximum likelihood (Fig. 1). Most of the ancestral character states of deep nodes are equivocal, with the exception of the common ancestor of members of the Basidiomycota, for which a parasitic ancestor is suggested. The phylogeny suggests that numerous transitions from a pathogenic to a saprophytic nutritional mode have occurred, as well as the reverse (Fig. 1). Although the nutritional mode of the common ancestor of Fungi is ambiguous, the earliest diverging branch in the Fungi contains parasitic species (R. allomycis and microsporidia). Recent studies9,29 showed that the closest known relative to Fungi is the amoeboid protist Nuclearia, which grows phagotrophically on algae and bacteria. Amoeboid phases are also observed in basal fungi: Rozella seems to phagocytose the organelles of its host30 and many chytrid zoospores undergo an amoeboid, motile phase before encysting. After the divergence of the Rozella and microsporidia lineage, the remaining fungi evolved filamentous growth (for example, hyphae and rhizoids), which aids in substrate attachment and absorptive nutrition involving extracellular digestion. Within the Basidiomycota and Ascomycota, a reversion to a unicellular, yeast-like growth form is observed among the earliest diverging lineages, perhaps implicating a prior advantage for this growth form in the early history of the Fungi. It is unclear whether the common ancestor of Fungi was marine. Most zoosporic true fungi, including all of the chytrids sampled in this study, grow in freshwater or soil habitats. Therefore, the diversification of the major lineages (phyla) within the kingdom Fungi probably occurred in a terrestrial environment but before the emergence of land plants31,32. Mycorrhiza-like symbioses of the phylum Glomeromycota are suggested to have been crucial in the colonization of land by plants33. Extant members of the Glomeromycota live exclusively as obligate symbionts of photoautotrophs, including not only vascular plants and bryophytes, but also cyanobacteria. This raises the hypothesis that terrestrial members of the Glomeromycota living symbiotically with cyanobacteria or algae, in semiaquatic and humid habitats later became the symbiotic partners of early land plants34. The present multilocus phylogeny explains the possible morphology and ecology of early fungi. The early-diverging lineages consist of a grade of zoosporic fungi, suggesting that the earliest fungi were primarily aquatic and lacked aerial spore dispersal. The loss of flagellated spores is inferred to have occurred at least four times. Each loss seems to have coincided with novel innovations in spore production and dispersal: microscopic wind-dispersed spores in terrestrial fungi; forcibly discharged conidia in the Entomophthorales; non-flagellated, mitotically produced spores in the planktonic Hyaloraphidium curvatum; and a complex polar tube apparatus in microsporidia. The sister kingdom to the Fungi (Animalia) evolved diverse body plans capable of feeding by ingestion, whereas the fungal branch developed a myriad of unicellular and filamentous forms optimized for absorptive nutrition. With a well-resolved phylogeny, fungal biologists can now study the evolution of complexity and multicellularity, and compare the evolution of these traits in fungi with their evolution in plants and animals. METHODS Molecular techniques. Sequence data were generated from 170 fungal species, primarily using pure cultures and herbarium material (Supplementary Notes 1). We used standard polymerase chain reaction (PCR) protocols25 for amplification and sequencing of six gene regions: the 18S ribosomal RNA gene (nearly full length), the 28S ribosomal RNA gene (primers LR0R and LR7), the internal transcribed spacer (ITS) RNA gene region (full length), EF1a (mostly primers EF1-983F and EF1-2218R), RNA polymerase II largest subunit (RPB1, mostly primers RPB1-Af and RPB1-G2R) and RNA polymerase II second largest subunit (RPB2, primers RPB2-5F and RPB2-11bR). Information on the PCR primers can be found at http://www.aftol.org/primers.php. In a number of basal 821 ©2006 Nature Publishing Group ARTICLES NATURE | Vol 443 | 19 October 2006 fungal taxa, the EF1a gene was not detected, but a paralogous copy of the gene was recovered (EFL, or the EF1a-like gene35; see Supplementary Notes 4). We also obtained sequences from fungal and eukaryotic genomes by retrieving sequences from GenBank and genome servers. Although our data set contains both partial sequences and missing data points, in the case of only one taxon (the choanoflagellate Monosiga brevicollis) were fewer than four genes sampled. Phylogenetic reconstruction. The data set consisted of 214 taxa, 199 of which were fungi. Sequences were aligned and ambiguous regions excluded in MacClade36. Conflict among the six genes was assessed by separate MLBS of each data partition using 250 bootstrap replicates in PHYML37. We ignored two conflicts, one including microsporidian 18S sequences (known to be subject to long branch attraction) and the other involving marginally conflicting signal of the Pyrenulales (Ascomycota). Data were combined into one matrix with EF1a, RPB1 and RPB2 translated into amino acids and 18S, 28S and 5.8S as nucleotides. We applied a heterogeneous maximum likelihood model to the data set with six unlinked partitions, one for each gene. The 18S and 28S genes were fitted to a general-time-reversible model with a proportion of invariant sites and gamma distributed rates (GTR1I1C), the 5.8S data used GTR1C and proteins used the JTT1I1C fixed rate model. The gamma distribution was approximated using four rate classes. We used MrBayes 3.1.1 (ref. 38) for phylogenetic estimation. Five independent runs were conducted (each with four chains) for 9.5 3 106 generations, sampling every 500 generations. Runs were discarded if they failed to reach the same likelihood plateau observed in other independent runs. We computed the consensus of the sampled trees, the posterior probabilities of clades, and average branch lengths from runs that converged to the same likelihood plateau (58,611 trees). For the analysis of the combined super-matrix we also tested for convergence of runs by analysing frequencies of splits using the software AWTY39 and found that the consensus topology constructed using this criterion trivially differed from that based on log likelihood scores. We also assessed support for nodes on the nucleotide data (third codon positions excluded) by MLBS (500 replicates) using PHYML with a GTR1I1C model. Tests for statistical differences in likelihoods of alternative topologies were assessed using the approximately unbiased test22 on the nucleotide data with site-wise, log-likelihood values calculated using TREE-PUZZLE v5.2 (ref. 40). Ancestral character state reconstruction of nutritional mode was conducted using the maximum likelihood model Mk1 in Mesquite 1.0 (ref. 41). Taxa were assigned to ecological character states on the basis of published literature, resolving ambiguous assignments when possible. Reconstructions are reported for only those branches significantly assigned an unequivocal character state in a majority of 1,000 trees randomly drawn from the sample of credible trees. The number of losses of the flagellum within the Fungi was also estimated for all 58,611 credible trees using Dollo parsimony as implemented in MacClade. Received 4 May; accepted 25 July 2006. 1. Karol, K. G., McCourt, R. M., Cimino, M. T. & Delwiche, C. F. The closest living relatives of land plants. Science 294, 2351–-2353 (2001). 2. Groth-Malonek, M., Pruchner, D., Grewe, F. & Knoop, V. Ancestors of transsplicing mitochondrial introns support serial sister group relationships of hornworts and mosses with vascular plants. Mol. Biol. Evol. 22, 117–-125 (2005). 3. Lang, B. F., O’Kelly, C., Nerad, T., Gray, M. W. & Burger, G. The closest unicellular relatives of animals. Curr. Biol. 12, 1773–-1778 (2002). 4. James, T. Y., Porter, D., Leander, C. A., Vilgalys, R. & Longcore, J. E. 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Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes. Nature 407, 506–-508 (2000). 29. Medina, M. et al. Phylogeny of Opisthokonta and the evolution of multicellularity and complexity in Fungi and Metazoa. Int. J. Astrobiol. 2, 203–-211 (2003). 30. Powell, M. J. Fine structure of the unwalled thallus of Rozella polyphagi in its host Polyphagus euglenae. Mycologia 76, 1039–-1048 (1984). 31. Berbee, M. L. & Taylor, J. W. in The Mycota (eds McLaughlin, D. J., McLaughlin, E. G. & Lemke, P. A.) 229–-245 (Springer, New York, 2001). 32. Heckman, D. S. et al. Molecular evidence for the early colonization of land by fungi and plants. Science 293, 1129–-1133 (2001). 33. Pirozynski, K. A. & Malloch, D. W. The origin of land plants: a matter of mycotrophism. Biosystems 5, 153–-164 (1975). 34. Schüßler, A. Molecular phylogeny, taxonomy, and evolution of arbuscular mycorrhiza fungi and Geosiphon pyriformis. Plant Soil 244, 75–-83 (2002). 35. Keeling, P. J. & Inagaki, Y. A class of eukaryotic GTPase with a punctuate distribution suggesting multiple functional replacements of translation elongation factor 1a. Proc. Natl Acad. Sci. USA 101, 15380–-15384 (2004). 36. Maddison, D. & Maddison, W. MacClade Version 4.05: Analysis of Phylogeny and Character Evolution (Sinauer Associates, Sunderland, Massachusetts, USA, 2002). 37. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–-704 (2003). 38. Huelsenbeck, J. P. & Ronquist, F. MrBayes: bayesian inference of phylogenetic trees. Bioinformatics 17, 754–-755 (2001). 39. Wilgenbusch, J. C., Warren, D. L. & Swofford, D. L. AWTY: A system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. http://ceb.csit.fsu.edu/awty (2004). 40. Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–-504 (2002). 41. Maddison, W. P. & Maddison, D. R. Mesquite: a modular system for evolutionary analysis. Version 1.0. http://mesquiteproject.org (2003). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements Funding for the project was provided by the National Science Foundation’s ‘Assembling the Tree of Life’ and ‘Research Coordination Network’ programs. For technical assistance we thank L. Bukovnik, C. Roberts and H. Matthews. We also thank the following individuals for sharing research materials: M. C. Aime, W. R. Buck, M. S. Cole, P. Crane, Y. Dalpe, D. M. Hillis, S. L. Joneson, R. Petersen, C. Printzen, E. Vellinga, H. Whisler and A. Zavarzin. We are very thankful to B. Mueller, J. Harer, B. Rankin, J. Pormann and S. Dilda for providing access to the Duke CSEM computer cluster. P. Keeling provided unpublished information used to analyse EFL in Fungi. Author Information Data for this project have been deposited in GenBank (see Supplementary Notes 1 for accession numbers), and the alignments can be accessed on the Assembling the Fungal Tree of Life website at http://www.aftol.org/. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to T.Y.J. ([email protected]) or R.V. ([email protected]). 822 ©2006 Nature Publishing Group NEWS & VIEWS been the key to their success is the use of highthroughput experiments to assess various zeolite compositions: the window of compositions that yielded ITQ-33 is narrow, and outside the common range usually used to prepare zeolites. This demonstration that new materials can be discovered within such a narrow compositional window should lead to the wider use of high-throughput technology in the search for further zeolites. The role of hexamethonium in the formation of ITQ-33 is intriguing. To date, the general strategy has been to prepare increasingly large organic molecules possessing the rigidity, solubility and stability needed to ‘direct’ the crystallization of new materials. Typically, the size and shape of the resulting pores corresponds to the size and shape of the organic molecule. ITQ-33, however, is different: hexamethonium is small and flexible, and there is no obvious fit between it and the resulting pore structure. It could be that the hexamethonium molecules pack in such a way as to provide an exact fit for the voids; this is the case, for instance, with VPI-5, which is stabilized by a chain of water molecules that perfectly fit the interior of the pores6. Hexamethonium is a simple and relatively inexpensive reagent, and its use bodes well for making ITQ-33 viable for practical application. Other zeolites prepared with organic compounds of similar complexity are used in the petrochemical industry and as additives in catalytic converters. Another exciting aspect of the latest work1 is that the structure of ITQ-33 was ‘predicted’ by algorithms that generate framework structures consistent with the geometrical requirements of a zeolite7. In the past year, roughly half of the reported zeolite structures have been previously ‘discovered’ by these algorithms. It is possible to search the large structural databases generated by these programs for structures with hitherto unavailable properties. An example is given in Figure 2, in which a computer-generated framework with 18- and 24ring pores is compared with ITQ-33 and other known zeolites. The advent of these powerful algorithms will help in solving the structure of microporous materials, and can make the synthesis of zeolites more ‘directed’ and perhaps more successful. Although ITQ-33 has all the characteristics of a good acid catalyst, much work remains to be done to make it practical. The amount of germanium and fluoride required must be minimized or eliminated to reduce manufacturing costs. Better ways of recovering the organic director and recycling it could further increase its potential. Substitution of other atoms in the framework, such as titanium or tin, could expand the range of properties to catalytic reactions such as oxidation and Lewisacid catalysis. More generally, ITQ-33 may help us to gain a better understanding of the adsorption processes that occur at the interface between the microporous (pore diameter less than 2 nm) 758 NATURE|Vol 443|19 October 2006 and mesoporous (pore diameter 2–100 nm) scales. It is at this length scale that the transition between monolayer and multilayer adsorption occurs and where the assumptions of classical adsorption theories can break down. The problem can be approached from the other side, and there are, indeed, mesoporous silicas with ordered and highly uniform pore sizes in the 2-nm range8. These materials are, however, difficult to prepare with uniform pores below 2 nm. ITQ-33 bridges these two length scales; and because it is crystalline, and all its pores are — except for defects — identical, one should be able to relate atomic structure precisely to the adsorption isotherms of simple gases. This information could help in the future to interpret adsorption isotherms of other non-crystalline materials that have substantial porosity at the micro–meso transition. Finally, the discovery of ITQ-33 raises the question of whether we need materials with even larger cavities. Some of the unique properties of zeolites arise from the large curvature of their pores. As the pores get larger, the interaction of adsorbates with the pore walls increasingly resembles the interaction with a flat surface. At some point, the zeolite pore will start to look like the surface of layered aluminosilicates such as clays (albeit without their characteristic hydroxyl groups). Yet perhaps it is not catalysis or separations where the large-pore materials of the future will find use. Instead, it may be in such niches as sensors or photonics9, or where the low-dielectric constant of such materials, arising from their porosity, can be exploited in the manufacture of improved microelectronic devices. The challenge remains to make structures with less and less in them. ■ Raul F. Lobo is at the Center for Catalytic Science and Technology, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA. e-mail: [email protected] 1. Corma, A., Díaz-Cabañas, M. J., Jordá, J. L., Martínez, C. & Moliner, M. Nature 443, 842–845 (2006). 2. Davis, M. E., Saldarriaga, C., Montes, C., Garces, J. & Crowder, C. Nature 331, 698–699 (1988). 3. Burton, A. et al. Chem. Eur. J. 9, 5737–5748 (2003). 4. Strohmaier, K. G. & Vaughan, D. E. W. J. Am. Chem. Soc. 125, 16035–16039 (2003). 5. Corma, A. & Davis, M. E. ChemPhysChem 5, 304–313 (2004). 6. McCusker, L. B., Baerlocher, C., Jahn, E. & Bulow, M. Zeolites 11, 308–313 (1991). 7. Treacy, M. M. J., Rivin, I., Balkovsky, E., Randall, K. H. & Foster, M. D. Micropor. Mesopor. Mater. 74, 121–132 (2004). 8. Kresge, C. T., Leonowicz, M. E., Roth, W. J., Vartuli, J. C. & Beck, J. S. Nature 359, 710–712 (1992). 9. Ruiz, A. Z., Li, H. R. & Calzaferri, G. Angew. Chem. Int. Edn 45, 5282–5287 (2006). 10. www.hypotheticalzeolites.net EVOLUTIONARY BIOLOGY A kingdom revised Tom Bruns An international consortium of researchers has produced an impressive new tree of life for the kingdom Fungi. The results are a testament to cooperation between systematists with different expertise. On page 818 of this issue, James and colleagues1 provide a landmark study in fungal evolution. Before now, the only broadly sampled phylogenetic trees of the fungi were based on sequences of a single gene — that encoding the small-subunit (18S) ribosomal RNA. Broad sampling of species is essential, because under-sampling is known to adversely affect the construction of evolutionary trees. However, the quantity and quality of data are equally important, and the 18S data were insufficient to provide strong statistical support for many key branches in the evolutionary trees. In any case, a single-gene tree is always questionable, because different genes can give different views of evolutionary history. James et al.1 addressed these problems by collecting sequence data from two additional ribosomal RNA genes, and three proteincoding genetic loci, for a carefully selected sample of 199 species. The results of the combined analyses, outlined in Figure 1, are quite similar to those seen with the earlier 18S data, but ©2006 Nature Publishing Group statistical support for some key branches in the tree has improved. This will be a relief to those who have followed the 18S data closely; it means that the new data have produced incremental shifts, not major alterations, in our understanding of fungal evolution. The fungi, animals and plants are thought to have diverged from each other roughly a billion years ago. They are the only three eukaryotic kingdoms of life that developed multicellularity in terrestrial environments. Like plants and animals, the fungi had to adapt to terrestrial environments from ancestors that were aquatic. But the fossil record for fungi is much the worst; most of them are microscopic with relatively simple morphologies. For these reasons the evolutionary patterns within the fungi were poorly understood before the advent of nucleotide sequence data. It was known that most fungi lacked zoospores, motile cells that are propelled by flagella in water. Therefore the Chytridiomycota, the one aquatic group of fungi that contains flagella, was assumed Basidiomycota Dikarya Ascomycota Glomeromycota Mucormycotina (Zygomycota) Entomophthorales (Zygomycota) Olpidium (Chytridiomycota) Blastocladiales (Chytridiomycota) Euchytrids (Chytridiomycota) Microsporidia Rozella (Chytridiomycota) Animals Figure 1 | The main branches of the kingdom Fungi. This highly simplified evolutionary tree shows the traditional phyla — Ascomycota, Basidiomycota, Glomeromycota, Zygomycota and Chytridiomycota. The Ascomycota and Basidiomycota are united as the dikarya, fungi in which part of the life cycle is characterized by cells with paired nuclei. Their closest relatives seem to be the Glomeromycota, a group that was previously included within the Zygomycota. Neither the Zygomycota nor the Chytridiomycota are monophyletic groups; instead they seem to be ‘paraphyletic grades’ that are grouped only by shared primitive morphologies. Also shown are the microsporidia and Rozella branches, which seem to be basal to the all other fungi. (Note that all of these branches are still in need of stronger statistical support. James and colleagues’ much more detailed tree1 appears on page 820.) to be primitive. This has turned out to be correct but the details of the relationship are complicated. Both the 18S data and the new multigene analyses show that the Chytridiomycota is paraphyletic — that is, it does not include all the descendants of its most recent common ancestor. But James et al.1 show that a minimum of four independent losses of flagella has occurred; thus one of the key adaptations to the terrestrial environment has actually happened multiple times. Surprisingly, they show that one chytrid, Olpidium brassicae (Fig.1), may lie within the Entomophthorales, a group that includes insect parasites that lack flagella and that is usually considered a subgroup of the Zygomycota. Basidiobolus, traditionally a member of the Entomophthorales, had been placed within the Chytridiomycota by 18S data, but is now moved back by the multigene analysis to its more traditional place. An interesting example of multigene support concerns the placement of the Glomeromycota. These fungi form mutualisms called mycorrhizae with the roots of most plants, and they had been considered to be members of the Zygomycota. The 18S data consistently depicted them as a distinct group closely related to the Ascomycota and Basidiomycota, but there was no statistical support for this placement. The multigene data, however, provide at least bayesian statistical support for the latter relationship (Fig. 1). The most surprising result concerns Rozella (Fig. 2), an obscure genus that is parasitic on other Chytridiomycota. Together with the microsporidia, an enigmatic group of animal parasites, Rozella seems to be basal to all other sampled fungi (Fig. 1). There was no reason to expect this, and in this sense the result is reminiscent of the finding by plant systematists that an obscure tropical genus, Amborella, is the sister group to all other flowering plants2. These types of result again underline the importance of which species are sampled. The placement of the microsporidia themselves is another notable result. On the one hand, the analyses with 18S sequences originally put them at the base of the eukaryotic tree, distant from fungi, animals and plants. But this conclusion turned out to be erroneous owing to a confounding factor known as long-branch attraction. Other studies using protein-coding genes had previously placed them in the fungi, but the exact relationship was unclear because of limited sampling within the kingdom3,4. James and colleagues1 have now improved the sampling dramatically, and show that the microsporidia must be either at the base of the fungal tree, within Figure 2 | Rozella allomycis. This parasite of other members of the same phylum, the Chytridiomycota, seems to be one of the most primitive fungi. Its resting sporangia (sporeproducing bodies) are approximately 18 µm across and are shown within a hypha of its host chytrid, Allomyces. ©2006 Nature Publishing Group the Chytridiomycota, or within the Entomophthorales; in addition they were able to reject eight previously theorized placements within the fungi or outside the kingdom. There is still room for improvement in two key areas: branch support and taxon sample. Even with six gene loci, many branches remain unsupported or supported only by bayesian statistics, which may give overly optimistic assessments. For many branches it may be possible to increase support by adding additional data, and genomics will be a major contributor. Data from 29 complete fungal genomes were included in the analysis, but this sample is highly biased towards serious pathogens and model genetic systems. With the cost of sequence acquisition dropping, the number of sequenced fungal genomes will increase, and it may be possible to distribute this effort more evenly across the kingdom to provide a better evolutionary sample. As to the second area for improvement, greater effort needs to be focused on sampling the environment for unknown fungal groups. It is estimated that the kingdom contains 1.5 million species, fewer than 5% of which have been described5. If most of the unknown species are members of well-known groups, then the current phylogenetic estimates should be largely unaffected by additional discoveries. However, some entirely new lineages have been recovered by sequence analysis of common but previously unsampled environments6,7: we can’t predict how such discoveries will affect our perception of fungal evolution. The cooperation among researchers that has resulted in the new paper1 is almost as impressive as the product itself. Systematics can be a fairly balkanized field, with specialists defending their turf or their analytical methods against perceived competitors8. However, cooperation has always been common among fungal researchers because the field is woefully underpopulated. The James group included both traditional, morphologically based systematists, who contributed a wealth of knowledge on the organisms, and molecular systematists, who supplied the methodological and analytical techniques. Even Ralph Emerson, who died in 1979, made a notable posthumous contribution: it was his culture of Rozella, isolated in 1947, that made the sequence acquisition for this critical branch possible. This fusion of talents was essential to ensure that the broadest possible sample of fungi was selected, and that the data were collected and analysed rigorously. The results represent a proud moment for the field, and will be in the textbooks for some time to come. ■ Tom Bruns is in the Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102, USA. e-mail: [email protected] 1. James, T. Y. et al. Nature 443, 818–822 (2006). 2. Qiu, Y.-L. et al. Nature 402, 404–407 (1999). 759 R. EMERSON NEWS & VIEWS NATURE|Vol 443|19 October 2006 NEWS & VIEWS NATURE|Vol 443|19 October 2006 3. Hirt, R. P. et al. Proc. Natl Acad. Sci. USA 96, 580–585 (1999). 4. Keeling, P. J. & McFadden, G. I. Trends Microbiol. 6, 19–23 (1998). 5. Hawksworth, D. L. Mycol. Res. 105, 1422–1432 (2001). 6. Schadt, C. W., Martin, A. P., Lipson, D. A. & Schmidt, S. K. Science 301, 1359–1361 (2003). 7. Suh, S. O., McHugh, J. V., Pollock, D. D. & Blackwell, M. Mycol. Res. 109, 261–265 (2005). 8. Hull, D. L. Science as a Process (Univ. Chicago Press, 1988). STRUCTURAL BIOLOGY Enzyme target to latch on to Malcolm A. Leissring and Dennis J. Selkoe Insulin-degrading enzyme is implicated in diabetes and Alzheimer’s disease, but few molecular tools exist that can probe its function. A study now reveals its unusual structure and may lead to an expanded toolbox. Proteases are vital enzymes that have been targeted for the treatment of many diseases. One such protease, insulin-degrading enzyme (IDE), has strong links to diabetes and Alzheimer’s disease but has nonetheless proved to be an elusive drug target, despite more than 50 years of intensive research. On page 823 of this issue, Shen and colleagues1 reveal high-resolution crystal structures of IDE that open the door to the rational design of pharmacological modulators of this protease*. Crucially, the authors show that it might be possible to develop not just inhibitors, but activators as well. IDE was discovered in 1949 by the physician and biochemist I. Arthur Mirsky 2. Mirsky reasoned that inhibitors of IDE would be an ideal anti-diabetic therapy, as they would slow the degradation of insulin. In support of this approach, Mirsky found that liver extracts containing an inhibitor of IDE enhance the action of insulin when injected into rabbits3. Thereafter, Mirsky and many others sought to develop potent inhibitors of IDE as potential drugs. Despite these efforts, very few compounds that specifically inhibit IDE are available today, apart from substrates of IDE such as insulin itself. By revealing IDE’s active site in unprecedented detail, the crystal structures provided by Shen et al.1 may hold the key to realizing a potent and selective IDE inhibitor. Recent discoveries, however, raise concerns about the wisdom of inhibiting IDE. Chief among them is the finding that IDE naturally degrades the amyloid-β protein that accumulates abnormally in Alzheimer’s disease4. Here, it would be desirable to activate rather than inhibit IDE, a strategy that has already proven effective in mouse models of the disease5. Moreover, results from different animal models cast doubt on the concept of treating diabetes by chronically inhibiting IDE. A wellestablished rat model of diabetes was found to harbour mutations in IDE that reduce its ability to degrade both insulin and amyloid-β protein6,7. More recently, genetically modified mice that lack the gene for IDE were created. These mice had elevated insulin levels upon fasting, *This article and the paper concerned1 were published online on 11 October 2006. as predicted, but they also developed glucose intolerance, and they showed increased levels of cerebral amyloid-β protein8. These and other findings suggest that in some cases of diabetes (and perhaps also in some cases of Alzheimer’s disease), there might be too little IDE activity rather than too much, with chronically elevated insulin levels perhaps leading to insulin resistance. If this is true, IDE activators seem to be the logical therapeutic approach, especially for Alzheimer’s disease. Current thinking suggests that activators would be difficult to achieve in practice, for the same reason that it is easier to break a machine than to improve its performance. But the work of Shen et al.1 shows that IDE has unorthodox enzymatic properties that might permit activators to be developed after all. The authors’ crystal structures1 reveal that IDE resembles a clam shell, with two bowl-shaped halves connected by a flexible hinge (Fig. 1). This configuration allows the protease to switch between ‘open’ and ‘closed’ states. Shen et al. show that extended hydrogen bonding between the two halves of IDE creates a ‘latch’ that tends to keep the protease closed (Fig. 1a). Notably, by introducing mutations to the enzyme that destabilize the hydrogen-bond latch, the authors were able to increase the protease’s efficiency in cleaving a test substrate by as much as 40-fold (Fig. 1b). This improved efficiency was also seen in the degradation of insulin and amyloid-β protein. So what is the mechanistic basis of the profound enzyme activation seen in the mutant IDE? This can be understood by considering a simple, two-step model9 of the enzyme reaction. First, the enzyme and the substrate bind to each other in a reversible process to form an enzyme–substrate complex. Second, catalytic cleavage of the substrate occurs with concomitant release of the reaction products. Mutations that promote the open state of the protease — thus allowing it to bind substrate — could improve the efficiency of the reaction by accelerating the rate of the enzyme–substrate complex formation. However, there is a second way that these mutations could activate the protease. In our simple model of the enzyme reaction, the second step actually includes at least two discrete processes: catalysis (that is, substrate cleavage) and dissociation of the products from the enzyme. This complication is usually ignored by assuming that the rate of product dissociation is rapid compared with that of catalysis, making catalysis the rate-limiting step. Although this assumption holds for many proteases, the new work suggests that IDE probably conforms to a more complex kinetic model, where catalysis does not lead automatically to product release. Instead, an additional step is required in which the Insulin-degrading enzyme a Cleavage products Substrate Latch Slow Closed formation Slow Open formation Substrate cleavage within enzyme b Fast Fast Figure 1 | Enzyme activation. a, Insulin-degrading enzyme (IDE) cleaves molecules implicated in diabetes and Alzheimer’s disease. The crystal structures of IDE reported by Shen et al.1 reveal a ‘latch’ mechanism (green) that holds the enzyme in a closed state, delaying entry of the substrate or exit of the cleavage products. b, Mutations (red) that disrupt the latch promote the open conformation of the enzyme. Such mutants accept substrates and release products more readily than naturally occurring IDE, and so are more active. ©2006 Nature Publishing Group 761 Supplementary information : Reconstructing the early evolution of Fungi using a six... Seite 1 von 3 Supplementary information From the following article: Reconstructing the early evolution of Fungi using a six-gene phylogeny Timothy Y. James, Frank Kauff, Conrad L. Schoch, P. Brandon Matheny, Valérie Hofstetter, Cymon J. Cox, Gail Celio, Cécile Gueidan, Emily Fraker, Jolanta Miadlikowska, H. Thorsten Lumbsch, Alexandra Rauhut, Valérie Reeb, A. Elizabeth Arnold, Anja Amtoft, Jason E. Stajich, Kentaro Hosaka, Gi-Ho Sung, Desiree Johnson, Ben O'Rourke, Michael Crockett, Manfred Binder, Judd M. Curtis, Jason C. Slot, Zheng Wang, Andrew W. Wilson, Arthur Schü ler, Joyce E. Longcore, Kerry O'Donnell, Sharon Mozley-Standridge, David Porter, Peter M. Letcher, Martha J. Powell, John W. Taylor, Merlin M. White, Gareth W. Griffith, David R. Davies, Richard A. Humber, Joseph B. Morton, Junta Sugiyama, Amy Y. Rossman, Jack D. Rogers, Don H. Pfister, David Hewitt, Karen Hansen, Sarah Hambleton, Robert A. Shoemaker, Jan Kohlmeyer, Brigitte Volkmann-Kohlmeyer, Robert A. Spotts, Maryna Serdani, Pedro W. Crous, Karen W. Hughes, Kenji Matsuura, Ewald Langer, Gitta Langer, Wendy A. Untereiner, Robert Lücking, Burkhard Büdel, David M. Geiser, André Aptroot, Paul Diederich, Imke Schmitt, Matthias Schultz, Rebecca Yahr, David S. Hibbett, François Lutzoni, David J. McLaughlin, Joseph W. Spatafora and Rytas Vilgalys Nature 443, 818-822(19 October 2006) doi:10.1038/nature05110 Download plugins and applications Supplementary Notes 1 A table of the strains and species used and the sources and GenBank numbers for the gene sequences used in this study. Supplementary Notes 1 - Download PDF file (618KB) Supplementary Notes 2 These file shows the results of a Bayesian phylogenetic analysis of the six gene super-matrix using only nucleotide data. Note: Figure 1 of the manuscript shows a phylogeny based on a heterogeneous nucleotide-amino acid model, whereas this phylogeny uses nucleotides divided into genes and codons. Supplementary Notes 2 - Download PDF file (120KB) Supplementary Notes 3 This file contains the results of analysis of individual gene partitions (and relevant combinations) and the Bayesian posterior probabilities and maximum likelihood bootstrap support for nodes of interest. Supplementary Notes 3 - Download PDF file (66KB) http://www.nature.com/nature/journal/v443/n7113/suppinfo/nature05110.html 26.10.2006 Supplementary information : Reconstructing the early evolution of Fungi using a six... Seite 2 von 3 Supplementary Notes 4 This file contains a phylogeny of a paralogous copy of elongation factor 1-α (EFL). It shows the species of basal fungi from which this gene copy has been sequenced, and demonstrates a relationship between Basidiobolus and the Entomophthorales. Supplementary Notes 4 - Download PDF file (115KB) Supplementary Notes 5 This file contains the methods and results from tests of long branch attraction between Rozella allomycis and microsporidia. Supplementary Notes 5 - Download PDF file (67KB) DOWNLOAD BROWSER PLUGINS AND OTHER APPLICATIONS Movie files z z z QuickTime Player (PC or Mac) Realplayer (PC or Mac) Windows Media player (PC only) PDF douments z Adobe Acrobat Reader (PC or Mac) Text documents z z Textpad (PC only) SimpleText (Mac only) PostScript documents z GhostView (Mac and PC) Flash movies z Macromedia Flash Player Audio files z z z z Apple iTunes (PC or Mac) QuickTime Player (PC or Mac) Realplayer (PC or Mac) Windows Media player (PC only) Chemical structures z MDL Chime Microarray http://www.nature.com/nature/journal/v443/n7113/suppinfo/nature05110.html 26.10.2006 Supplementary information : Reconstructing the early evolution of Fungi using a six... 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Species containing only the EFL (EF1α-like) gene and not the homologue of EF1α are listed as “paralog.” AFTOLID Phylum Order Ascomycota Acarosporales Genus+species Pleopsidium chlorophanum Ascomycota Acarosporales Acarospora schleicheri 1005 Ascomycota Acarosporales Acarospora laqueata Ascomycota Agyriales Orceolina kerguelensis Ascomycota Agyriales Trapelia placodioides 962 Ascomycota Arthoniales Roccella fuciformis Ascomycota Arthoniales Ascomycota Arthoniales Ascomycota Ascomycota Strain/ voucher VR 8-VIII02/8 18S 28S RPB1 RPB2 EF1A nucITS DQ525541 DQ842017 DQ782858 DQ525442 DQ782920 DQ525474 AY640986 AY640945 DQ782859 AY641026 -- DQ525529 1007 23865 VR 6-VII98/14 AY640984 AY640943 DQ782860 AY641024 -- DQ842014 296 20964 DQ366257 AY212830 -- DQ366256 DQ366254 AY212814 AF119500 AF274103 DQ366259 DQ366260 DQ366258 AF274081 126 15572 AY584678 AY584654 DQ782825 DQ782866 -- DQ782840 Dendrographa minor 355 10,070 AF279381 AF279382 -- AY641034 DQ842007 DQ842015 Simonyella variegata 80 14310a AY584669 AY584645 DQ782819 DQ782861 DQ782891 DQ782835 Capnodiales Capnodium coffeae 939 CBS 147.52 DQ247808 DQ247800 DQ471162 DQ247788 DQ471089 DQ491515 Chaetothyriales Capronia pilosella 657 WUC28 DQ823106 DQ823099 DQ840554 DQ840561 DQ840565 DQ826737 Ascomycota Chaetothyriales Ramichloridium anceps 659 WUC375 DQ823109 DQ823102 DQ840557 DQ840564 DQ840568 DQ826740 Ascomycota Chaetothyriales Exophiala dermatitidis 668 WUC176 DQ823107 DQ823100 DQ840555 DQ840562 DQ840566 DQ826738 Ascomycota Chaetothyriales Exophiala pisciphila 669 WUC137 DQ823108 DQ823101 DQ840556 DQ840563 DQ840567 DQ826739 Ascomycota Diaporthales Diaporthe eres 935 CBS 109767 DQ471015 AF408350 -- DQ470919 DQ479931 DQ491514 Ascomycota Diaporthales Gnomonia gnomon 952 DQ471019 AF408361 DQ471167 DQ470922 DQ471094 DQ491518 Ascomycota Dothideales Dothidea sambuci 274 CBS 199.53 DAOM 231303 AY544722 AY544681 -- DQ522854 DQ497606 DQ491505 Ascomycota Dothideales Dothidea insculpta 921 CBS 189.58 DQ247810 -- Eurotiales Aspergillus fumigatus 1079 GenBank AB008401 AY660917 Ascomycota Eurotiales Aspergillus nidulans 1080 GenBank ENU77377 AF454167 DQ247792 XM_74164 7 XM_67729 7 DQ471081 XM_74529 5 XM_65673 0 AF027764 Ascomycota DQ471154 XM_74774 4 XM_65332 1 Ascomycota Eurotiales Monascus purpureuss 426 CBS 109.07 DQ782881 DQ782908 DQ842012 -- -- DQ782847 Ascomycota Helotiales Leotia lubrica 1 OSC 100001 AY544687 AY544644 DQ471113 DQ470876 DQ471041 DQ491484 1004 AY373851 AY373888 1 Strain/ voucher 18S 28S RPB1 RPB2 EF1A nucITS 151 OSC 100056 AY544713 AY544669 DQ471125 DQ470886 DQ471053 DQ491501 166 DQ470992 DQ470944 DQ471128 DQ470888 DQ471056 DQ491502 279 OSC 100054 DAOM 231119 AY544714 AY544670 -- DQ470889 DQ471057 DQ491506 49 OSC 100002 AY544688 AY544646 -- DQ470877 DQ497602 DQ491485 Geoglossum nigritum 56 OSC 100009 AY544694 AY544650 DQ471115 DQ470879 DQ471044 DQ491490 Helotiales Botryotinia fuckeliana 59 OSC 100012 AY544695 AY544651 DQ471116 DQ247786 DQ471045 DQ491491 Helotiales Trichoglossum hirsutum 64 OSC 100017 Y544697 AY544653 DQ471119 DQ470881 DQ471049 DQ491494 Ascomycota Helotiales Mollisia cinerea 76 OSC 100029 DQ470990 DQ470942 DQ471122 DQ470883 DQ471051 DQ491498 Ascomycota Helotiales Dermea acerina 941 CBS 161.38 DQ247809 DQ247801 DQ471164 DQ471091 AF141164 Ascomycota Hypocreales Fusarium graminearum 1082 GenBank -- AY188924 XM_38109 2 DQ247791 AACM010 00132 REGION: 154552.. 157390 XM_38898 7 AF132798 Ascomycota Hypocreales 161 U00748 AY489666 DQ518180 AF543785 DQ518177 Hypocreales GJS 89-70 ATCC 36093 U32412 Ascomycota Fusarium aff. solani Hydropisphaera erubescens AY545722 AY545726 DQ518182 AY545731 DQ518174 -- Ascomycota Hypocreales Hypocrea citrina 52 OSC 100005 AY544693 AY544649 DQ522853 -- DQ471043 DQ491488 Ascomycota Lecanorales Mycoblastus sanguinarius 196 07.01.03-3 DQ782879 DQ782915 DQ782827 DQ782867 DQ782898 DQ782842 Ascomycota Lecanorales Cladonia caroliniana 3 01-26-03.2 AY584664 AY584640 DQ782822 AY584684 DQ782888 DQ782832 Ascomycota Lecanorales Canoparmelia caroliniana 6 01-26-03.20 AY584658 AY584634 DQ782817 AY584683 DQ782889 DQ782833 Ascomycota Lecanorales Lecanora unknown 639 03.07.04-2 DQ782883 DQ782910 DQ782829 DQ782871 DQ782901 DQ782849 Ascomycota Lecanorales Bacidia schweinitzii 642 03.07.04-3 DQ782884 DQ782911 DQ782830 DQ782872 DQ782902 DQ782850 Ascomycota Lecanorales Hypocenomyce scalaris 687 2058 DQ782886 DQ782914 DQ782854 DQ782875 DQ782918 DQ782852 Ascomycota Lecanorales Physcia aipolia 84 DQ782876 DQ782904 DQ782820 DQ782862 DQ782892 DQ782836 Ascomycota Lichinales Peltula umbilicata 891 14901a-1 DQ782887 DQ832334 DQ782855 DQ832335 DQ782919 DQ832333 Ascomycota Lichinales Peltula auriculata 892 24901 DQ832332 DQ832330 DQ782856 DQ832331 -- DQ832329 Ascomycota Lichinales Lichinella iodopulchra 896 16319a -- -- DQ782857 DQ832328 DQ832327 DQ842016 Ascomycota Lulworthiales Lindra thalassiae 413 JK 5090A DQ470994 DQ470947 -- DQ470897 DQ471065 DQ491508 Ascomycota Lulworthiales Lulworthia grandispora 424 JK 4686 DQ522855 DQ522856 -- DQ518181 DQ497608 -- Ascomycota Microascales Microascus trigonosporus 914 CBS 218.31 DQ471006 DQ470958 DQ471150 DQ470908 DQ471077 DQ491513 Ascomycota Onygenales Histoplasma capsulatum 1083 GenBank A A A A A AB055230 Ascomycota Onygenales Coccidioides immitis 1084 RS AAEC0200 AAEC0200 AAEC0200 AAEC0200 AAEC0200 -- Phylum Order Genus+species Ascomycota Helotiales Chlorociboria aeruginosa Ascomycota Helotiales Cudoniella clavus Ascomycota Helotiales Monilinia fructicola Ascomycota Helotiales Lachnum virgineum Ascomycota Helotiales Ascomycota Ascomycota AFTOLID 186 2 AFTOLID Strain/ voucher Phylum Order Genus+species 18S 0046 REGION: 75557.. 77144 28S 0027 REGION: 29738 ..31122 RPB1 0017 REGION: 34058 ..37220 RPB2 0035 REGION: 62079.. 64829 EF1A 0020 REGION: 35520..370 37 nucITS Ascomycota Onygenales Spiromastix warcupii 430 CBS 576.63 DQ782882 DQ782909 -- DQ782870 DQ782900 DQ782848 Ascomycota Orbiliales Orbilia vinosa Ascomycota Orbiliales Orbilia auricolor 905 CBS 917.72 DQ471000 DQ470952 DQ471145 -- DQ471071 DQ491511 906 CBS 547.63 DQ471001 DQ470953 -- DQ470903 DQ471072 DQ491512 Ascomycota Ostropales Ascomycota Ostropales Echinoplaca strigulacea 106 16001d DQ782878 DQ782905 DQ782823 DQ782865 DQ782895 -- Stictis radiata 398 JP222 U20610 AF356663 -- AY641079 -- DQ782846 Ascomycota Ostropales Acarosporina microspora 78 CBS 338.39 AY584667 AY584643 DQ782818 AY584682 DQ782890 DQ782834 Ascomycota Ostropales Diploschistes ocellatus 958 E-995 AF038877 AY605077 DQ366252 DQ366253 DQ366251 AF098411 Ascomycota Peltigerales Peltigera degenii 134 19/02 AY584681 AY584657 DQ782826 AY584688 DQ782897 DQ782841 Ascomycota Pertusariales Pertusaria dactylina 224 DQ782880 DQ782907 DQ782828 DQ782868 DQ782899 DQ782843 Ascomycota Pertusariales Dibaeis baeomyces 358 07.01.03-13 93.08.20-1 1/1 AF113712 AF279385 DQ842011 AY641037 DQ842008 DQ782844 Ascomycota Pezizales Cheilymenia stercorea 148 OSC 100034 AY544705 AY544661 DQ471123 AY544733 DQ471052 DQ491500 Ascomycota Pezizales Caloscypha fulgens 152 OSC 100062 DQ247807 DQ247799 DQ471126 DQ247787 DQ471054 DQ491483 Ascomycota Pezizales Gyromitra californica 176 AY544717 AY544673 DQ471130 DQ470891 DQ471059 -- Ascomycota Pezizales Disciotis sp. 179 OSC 100068 NRRL 22213 AY544711 AY544667 DQ471131 DQ470892 DQ471060 DQ491503 Ascomycota Pezizales Ascobolus crenulatus 181 KH.02.005 AY544721 AY544678 DQ471132 DQ470893 DQ471061 DQ491504 Ascomycota Pezizales Sarcoscypha coccinea 50 OSC 100003 AY544691 AY544647 -- DQ497612 -- DQ491486 Ascomycota Pezizales Peziza vesiculosa 507 TL-6398 DQ470995 DQ470948 -- DQ470898 DQ471066 DQ491509 Ascomycota Pezizales Morchella aff. esculenta 60 MV3 AY544708 AY544664 DQ471117 DQ470880 DQ471046 -- Ascomycota Pezizales Scutellinia scutellata 62 OSC 100015 DQ247814 DQ247806 -- DQ247796 DQ471047 DQ491492 Ascomycota Pezizales Aleuria aurantia 65 OSC 100018 AY544698 AY544654 DQ471120 DQ247785 DQ466085 DQ491495 Ascomycota Pezizales 66 OSC 100019 AY544699 AY544655 -- DQ497613 DQ497604 DQ491496 Ascomycota Pezizales Helvella compressa Peziza proteana f. sparassoides 71 OSC 100024 AY544703 AY544659 DQ518184 -- -- DQ491497 Ascomycota Pezizales 949 OSC 100503 DQ247813 DQ247805 DQ471166 DQ247795 DQ471093 DQ491517 Ascomycota Pleosporales Pyronema domesticum Trematosphaeria heterospora 1036 CBS 644.86 AY016354 AY016369 -- DQ497615 DQ497609 -- Ascomycota Pleosporales Westerdykella cylindrica 1037 AY016355 AY779322 DQ471168 DQ470925 DQ497610 DQ491519 Ascomycota Pleosporales Pyrenophora phaeocomes 283 CBS 454.72 DAOM 222769 DQ499595 DQ499596 -- DQ497614 DQ497607 DQ491507 3 Strain/ voucher Order Ascomycota Pleosporales 54 CBS 134.39 AY544727 AY544645 DQ518183 DQ247790 DQ497603 DQ491489 Ascomycota Pleosporales Pneumocystidiales Pleospora herbarum 940 CBS 191.86 DQ247812 DQ247804 DQ471163 DQ247794 DQ471090 DQ491516 Pneumocystis carinii 1192 GenBank S83267.1 AF047831 B AY485631 -- M86760.1 Ascomycota Protomycetales Protomyces inouyei 266 IAM 14512 AY548295 AY548294 -- AY548299 DQ497611 DQ497617 Ascomycota Pyrenulales Anisomeridium polypori 101 4237a DQ782877 DQ782906 DQ782822 DQ782864 DQ782894 DQ782838 Ascomycota Pyrenulales Trypethelium sp. 110 15322b AY584676 AY584652 DQ782824 AY584690 DQ782896 DQ782839 Ascomycota Pyrenulales Pyrgillus javanicus 342 DQ823110 DQ823103 DQ842010 DQ842009 -- DQ826741 Ascomycota Pyrenulales Pyrenula pseudobufonia 387 03.24.03-9 VR 14-VI02/5 AY641001 AY640962 DQ840558 AY641068 -- DQ782845 Ascomycota Rhytismatales Coccomyces dentatus 147 OSC 100021 AY544701 AY544657 -- DQ247789 DQ497605 DQ491499 Ascomycota Rhytismatales Potebniamyces pyri 744 OSC 100199 DQ470997 DQ470949 -- DQ471068 DQ491510 Ascomycota Saccharomyceta les Debaryomyces hansenii 1077 GenBank DHA50827 3 XM_45692 1 DQ470900 CR382139 REGION: 645109.. 647847 XM_46053 3 AF210327 Ascomycota Saccharomycetales Saccharomyces cerevisiae 1069 GenBank Z75578 X96876 AACF0100 0092 REGION: 14761.. 17730 AADM010 00294 REGION: 25792.. 28761 NM_20953 5 XM_44741 5 Z75059 AACF0100 0010 REGION: 85223.. 87931 AADM010 00162 REGION: 31572.. 34283 NM_21130 6 XM_44895 9 M10992 AACF0100 0123 REGION: 6264 .. 7490 AADM010 00010 REGION: 45684.. 46913 NM_20907 9 XM_44856 1 AY198398 XM_71432 1 XM_71334 6 XM_70680 6 AF455531 Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Saccharomycetales Saccharomycetales Saccharomycetales Saccharomycetales Saccharomycetales Genus+species Cochliobolus heterostrophus AFTOLID Phylum Saccharomyces castellii 1070 18S 28S AF485980 U53879 REGION: 24144.. 25525 GenBank Z75577 AACF0100 0279 REGION: 1595..2975 AADM010 00465 REGION: 1022 ..2407 Kluyveromyces waltii 1071 GenBank AADM010 00401 REGION: 1935.. 3527 Ashbya gossypii 1072 GenBank AF113137 AF113137 Candida glabrata 1073 GenBank AY198398 GenBank AACQ010 00290 REGION: 7072.. 8646 AY198398 AACQ010 00290 REGION: 9209.. 10587 Candida albicans 1074 RPB1 RPB2 EF1A nucITS Z73326 AY046180 AY046208 AF113137 4 Phylum Order Ascomycota Saccharomycetales Saccharomycetales Ascomycota Saccharomycetales Ascomycota AFTOLID Strain/ voucher Kluyveromyces lactis 1075 Yarrowia lipolytica Candida lusitaniae Genus+species GenBank 18S NC_00604 0 REGION: 1514508..1 516092 28S NC_00604 0 REGION: 1494981..1 496333 1076 GenBank AB018158 1268 GenBank M55526 RPB1 RPB2 EF1A nucITS XM_45531 0 XM_50190 9 AAFT0100 0002 REGION: 415878.. 418490 XM_45178 4 XM_50237 6 AAFT0100 0003 REGION: 85594.. 88339 AAFN0100 0119 REGION: 2828.. 5441 AY485615 C AY485613 XM_45192 9 XM_50162 8 AAFT0100 0062 REGION: 135156..13 6436 AAFN0100 0051 REGION: 10387 .. 11574 AAFM010 00070 REGION: 128541.. 129769 D13337 XM_95201 3 U42189 XM_95977 5 AF388914 AY338969 -- Saccharomycetales Candida tropicalis 1269 GenBank M55527 Candida guilliermondii Schizosaccharomyces pombe 1270 GenBank AB013587 Ascomycota Saccharomycetales Schizosaccharomycetales AJ616903 AAFT0100 0087 REGION: 56020.. 56838 AAFN0100 0124 REGION: 191890..19 3271 AAFM010 00051 REGION: 270781.. 272161 1199 GenBank X54866 Z19136 Ascomycota Sordariales Neurospora crassa 1078 GenBank X04971 AF286411 X56564 XM_95900 4 Ascomycota Sordariales Podospora anserina 1085 GenBank D -- D D X74799 AF388930 Ascomycota Sordariales Sordaria fimicola 216 AY545724 AY545728 DQ518178 Sordariales Sordariomycetes incertae sedis Chaetomium globosum 217 GenBank AY545725 AY545729 DQ518176 DQ518179 Magnaporthe grisea 1081 GenBank AB026819 AB026819 -AAFU0100 1128 REGION: 238 .. 2979 XM_36226 9 DQ518175 Ascomycota -AAFU0100 0188 REGION: 73498 ..76125 XM_36220 7 AY849694 AB026819 Ascomycota Taphrinales Taphrina wiesneri 265 IAM 14515 AY548293 AY548292 -- AY548298 DQ479936 DQ497616 Ascomycota Umbilicariales Umbilicaria mammulata 645 03.07.04-5 AY648114 DQ782912 DQ782831 DQ782873 DQ782903 DQ782851 Ascomycota Verrucariales Endocarpon pallidulum 661 4028 DQ823104 DQ823097 DQ840552 DQ840559 -- DQ826735 Ascomycota Verrucariales Agonimia sp. 684 45172 DQ782885 DQ782913 DQ782853 DQ782874 DQ782917 DQ826742 Ascomycota Verrucariales Staurothele frustulenta 697 53935 DQ823105 DQ823098 DQ840553 DQ840560 -- DQ826736 Ascomycota Ascomycota Ascomycota AY321475 AF321539 AB054109 Z19578 5 AFTOLID Strain/ voucher 18S 28S RPB1 91 9702 AY584668 AY584644 DQ782821 DQ782863 DQ782893 DQ782837 51 OSC 100004 ATCC 56487 AY544692 AY544648 DQ471114 DQ470878 DQ471042 DQ491487 AY544719 AY544676 GenBank M92991 AF041494 DQ247797 AACS0100 0026 REGION: 389547.. 392387 DQ471048 AACS0100 0024 REGION: 71702.. 73198 DQ491493 1087 DQ471118 AACS0100 0015 REGION: 48684.. 52695 AF345819 285 PBM 2426 AY771605 AY702013 AY857984 AY536285 AY881027 AF389133 Armillaria mellea 449 AY787217 AY700194 AY788849 AY780938 AY881023 AY789081 Agaricales Echinodontium tinctorium 455 PBM 2470 DAOM 16666 AF026578 AF393056 AY864882 AY218482 AY885157 AY854088 Agaricales Henningsomyces candidus 468 Thorn 156 AF334916 AF287864 AY218513 AY883424 AY571043 Basidiomycota Agaricales Lycoperdon pyriforme 480 DSH 96-054 AF026619 AF287873 AY860521 AY860523 -4 AY218495 AY883426 AY854075 Basidiomycota Agaricales Ampulloclitocybe clavipes 542 AY771612 AY639881 Marasmius alliaceus 556 AY787214 AY635776 AY786060 AY881022 AY883430 -1 AY789080 Agaricales AY788848 AY8605256 AY780937 Basidiomycota AY854076 Basidiomycota Agaricales Collybia tuberosa 557 AY771606 AY639884 AY857982 AY787219 AY881025 AY854072 Basidiomycota Agaricales Flammulina velutipes 558 AY665781 AY639883 AY858966 AY786055 AY883423 AY854073 Basidiomycota Agaricales Pleurotus ostreatus 564 PBM 2474 TENN 55620 TENN 53540 TENN 52002 TENN 53662 AY657015 AY645052 AY862186 AY786062 AY883432 AY854077 Basidiomycota Agaricales Pluteus romellii 625 ECV 3201 AY657014 AY634279 AY862187 AY786063 AY883433 AY854065 Basidiomycota Agaricales Coprinus comatus 626 ECV 3198 AY665772 AY635772 AY857983 AY780934 AY881026 AY854066 Basidiomycota Agaricales Amanita brunnescens 673 PBM 2429 AY707096 AY631902 AY788847 AY780936 AY881021 AY789079 Basidiomycota Agaricales Hygrocybe aff. conica 729 PBM 918 AY752965 AY684167 AY860522 AY803747 AY883425 AY854074 Basidiomycota Agaricostilbales Agaricostilbum hyphaenes 675 CBS 7811 AY665775 AY634278 AY788845 AY780933 AY879114 AY789077 Basidiomycota Aphyllophorales Bondarzewia montana 452 DAOM 415 U59063 DQ234539 DQ256049 AY218474 DQ059044 DQ200923 Basidiomycota Aphyllophorales 484 FPL6140 AF026606 AF287885 AY864881 AY218502 AY885155 AY854087 Basidiomycota Aphyllophorales Phlebia radiata Hyphoderma praetermissum 518 AY707094 AY700185 AY885150 AY854081 Aphyllophorales Grifola sordulenta 562 AY665780 AY645050 AY864871 AY8648779 AY787221 Basidiomycota GEL 2182 TENN 55054 AY786058 AY885154 AY854085 Basidiomycota Aphyllophorales Fibulorhizoctonia sp. 576 LA082103L AY654887 AY635779 AY857985 AY885161 AY879115 AY854062 Phylum Order Genus+species Ascomycota Verrucariales Dermatocarpon miniatum Ascomycota Xylariales Xylaria hypoxylon Ascomycota Xylariales Xylaria acuta 63 Basidiomycota Agaricales Coprinopsis cinerea Basidiomycota Agaricales Cortinarius iodes Basidiomycota Agaricales Basidiomycota Basidiomycota RPB2 EF1A nucITS 6 AFTOLID Strain/ voucher 18S 28S RPB1 -6 RPB2 EF1A nucITS Grifola frondosa 701 DSH s.n. AY705960 AY629318 AY864876 AY786057 AY885153 AY854084 Aphyllophorales Ramaria rubella 724 PBM 2408 AY707095 AY645057 AY786064 AY883435 AY854078 Basidiomycota Aphyllophorales Fomitopsis pinicola 770 MB 03-036 AY705967 AY684164 AY864866 AY864874 -5 AY885152 AY854083 Basidiomycota Aphyllophorales Phanerochaete chrysosporium 776 GenBank AF026593 AF287883 AY864880 AY786056 AADS0100 0047 REGION: 96253.. 99302 AY885155 AY854086 Basidiomycota Boletales Calostoma cinnabarinum 439 AW 136 AY665773 AY645054 AY857979 AY780939 AY879117 AY854064 Basidiomycota Boletales 713 MB 03-118 AY662660 AY684158 AY879116 AY789082 Boletales 714 MB 03-127 AY662663 AY684156 AY788850 AY858961 -2 AY787218 Basidiomycota Boletellus projectellus Hygrophoropsis aurantiaca AY786059 AY883427 AY854067 Basidiomycota Boletales Suillus pictus 717 MB 03-002 AY662659 AY684154 AY858965 AY786066 AY883429 AY854069 Basidiomycota Cantharellales Hydnum albomagnum 471 AY665777 AY700199 DQ234568 DQ218305 Cantharellales Clavaria zollingeri 563 AY657008 AY639882 DQ234570 AY8579878 DQ234553 Basidiomycota PBM 2512 TENN 58652 AY780940 AY881024 AY854071 Basidiomycota Dacrymycetales Calocera cornea 438 GEL 5359 AY771610 AY701526 AY857980 AY536286 AY881019 AY789083 Basidiomycota Dacrymycetales Dacryopinax spathularia 454 GEL 5052 AY771603 AY701525 AY857981 AY786054 AY881020 AY854070 Basidiomycota Entylomatales Entyloma holwayi 870 CBS 111593 DQ234562 -- DQ234552 DQ028593 DQ206984 Basidiomycota Filobasidiales Cryptococcus neoformans ser. D 1088 GenBank L05428 AY745721 AE017342 REGION: 277938.. 279341 XM_57020 4 XM_56846 2 AB034643 Basidiomycota Gautieriales Georgefischeriales Hymenochaetales Hymenochaetales Gautieria otthii 466 REG 636 AF393043 AF393058 XM_57094 3 AY864864 -5 AY218486 AY883434 AF377072 Tilletiaria anomala 865 CBS 436.72 AY803752 AY745715 DQ234571 AY803750 -- DQ234558 Coltricia perennis 447 DSH 93-198 AF026583 AF287854 AY218526 AY885147 -- Fomitiporia mediterranea 688 3/22.7 AY662664 AY684157 AY864867 AY864869 -70 AY803748 AY885149 AY854080 Rhodotorula hordea 674 CBS 6403 AY657013 AY631901 -- DQ234555 -- DQ234557 Basidiomycota Microbotryales Microbotryomycetidae incertae sedis Colacogloea peniophorae 709 CBS 684.93 DQ234564 -5 AY629313 DQ234569 DQ234550 DQ234566 DQ202270 Basidiomycota Platygloeales Platygloea disciformis 710 IFO 32431 DQ234563 AY629314 -- DQ234554 DQ056288 DQ234556 Phylum Order Genus+species Basidiomycota Aphyllophorales Basidiomycota Basidiomycota Basidiomycota Basidiomycota Basidiomycota 7 AFTOLID Strain/ voucher 18S 28S 492 FPL8805 AF026588 AF393078 Phylum Order Genus+species Basidiomycota Russulales Stereum hirsutum Basidiomycota Russulales Lactarius deceptivus 682 PBM 2462 AY707093 AY631899 Basidiomycota Stereales Cotylidia sp. Climacodon septentrionalis 700 MB-5 AY705958 AY629317 767 ZW s.n. AY705964 1459 GenBank AY125409 Basidiomycota Stereales Basidiomycota Uredinales RPB1 AY864885 -6 AY8648834 RPB2 EF1A nucITS AY218520 AY885159 AY854063 AY803749 AY885158 AY854089 AY883422 AY885148 AY854079 AY684165 AY864868 AY864872 -3 AY780941 AY885151 AY854082 AF522177 -- -- X73529 AF468044 Basidiomycota Uredinales Puccinia graminis Endocronartium harknessii 456 CFB 22250 AY665785 AY700193 -- DQ234551 DQ234567 DQ206982 Basidiomycota Ustilaginales Ustilago maydis 505 GenBank X62396 AF453938 XM401478 AY485636 AY885160 AY854090 Basidiomycota Ustilaginales 867 AY745726 -- DQ234549 DQ028590 DQ200931 Blastocladiales AF322406 DQ273767 DQ294579 DQ302766 paralog AY997038 Chytridiomycota Blastocladiales Physoderma maydis 19 CBS 104.17 DUH000892 5 DUH000793 2 DQ234548 Chytridiomycota Cintractia sorghi-vulgaris Coelomomyces stegomyiae AY601708 DQ273767 DQ294580 DQ302767 DQ282600 AY997072 Chytridiomycota Blastocladiales Allomyces arbusculus 300 Brazil 2 AY552524 AY552525 DQ294578 DQ302765 paralog AY997028 Chytridiomycota Chytridiales 20 JEL347-h AY601709 DQ273769 DQ294581 DQ302768 DQ282601 AY997076 Chytridiomycota Chytridiales Rhizoclosmatium sp. Batrachochytrium dendrobatidis 21 JEL197 AF051932 AY546693 DQ294583 DQ302769 DQ282602 AY997031 Chytridiomycota Chytridiales Polychytrium aggregatum 24 JEL109 AY601711 AY546686 DQ294584 DQ302770 DQ282604 AY997074 Chytridiomycota Chytridiales 27 AY546688 DQ294587 DQ302774 DQ282607 AY997037 Chytridiales 635 JEL180 DUH000936 3 AY546683 Chytridiomycota DQ322623 DQ273820 DQ294605 DQ302792 DQ282622 AY997095 Chytridiomycota Chytridiales Monoblepharidales Monoblepharidales Neocallimastigales Spizellomycetales Spizellomycetales Spizellomycetales Cladochytrium replicatum Synchytrium macrosporum Rhizophydium macroporosum 689 PL AUS 21 DQ322622 DQ273823 DQ294600 DQ302793 DQ282603 AY997084 Monoblepharella sp. 25 M15 AY546682 AY546687 DQ294608 DQ302771 DQ282605 AY997060 Hyaloraphidium curvatum 26 SAG 235-1 Y17504 DQ273771 DQ294585 DQ302772 DQ282606 AY997055 Neocallimastix sp. 638 GE13 DQ322625 DQ273822 DQ294611 -- DQ282608 AY997064 Spizellomyces punctatus 182 ATCC48900 AY546684 AY546692 DQ294586 DQ302773 paralog AY997092 Rozella allomycis 297 UCB 47-54 AY635838 DQ273803 DQ294582 DQ302791 paralog AY997087 Rhizophlyctis rosea 43 JEL318 AY635829 DQ273787 DQ294597 DQ302786 DQ282617 AY997062 Chytridiomycota Chytridiomycota Chytridiomycota Chytridiomycota Chytridiomycota Chytridiomycota 18 8 Genus+species Chytridiomycota Order Spizellomycetales Olpidium brassicae 633 Strain/ voucher DUH000936 1 Glomeromycota Archaeosporales Geosiphon pyriformis 574 W4756 Glomeromycota Diversisporales Scutellospora heterogama 138 FL225 AY635832 DQ273792 DQ294604 DQ302780 DQ282612 AY997088 Glomeromycota Glomerales Glomus mosseae 139 AY635833 DQ273793 DQ294592 DQ302781 DQ282613 AY997053 Glomeromycota Glomerales Glomus intraradices 845 UT101 4695rac11G2 DQ322630 DQ273828 DQ294603 DQ302794 DQ282611 AY997054 Glomeromycota Paraglomerales Paraglomus occultum 844 IA702 DQ322629 DQ273827 DQ294602 DQ282614 AY997069 Encephalitozoon cuniculi 1068 genome AJ005581 AJ005581 AL590443 -AL590449 REGION: 26605..300 30 NC_00323 1 AJ005581 Phylum Microsporidia Microsporidia AFTOLID 18S 28S RPB1 RPB2 EF1A nucITS DQ322624 DQ273818 DQ294609 -- paralog AY997067 AM183923 AM183920 AM183921 -- AM183922 -- Antonospora locustae 1089 genome AY376351 -- AF061288 E AY452734 -- Zygomycota Dimargaritales Dimargaris bacillispora 136 AB016020 DQ273791 DQ294588 DQ302775 DQ282609 AY997043 Zygomycota Endogone pisiformis 539 DQ322628 DQ273811 DQ294601 DQ302776 DQ282618 AY997046 Conidiobolus coronatus 137 NRRL28638 AF113418 AY546691 DQ294591 DQ302779 paralog AY997041 Entomophthora muscae 28 ARSEF3074 AY635820 DQ273772 DQ294590 DQ302778 paralog AY997047 Zygomycota Endogonales Entomophthorales Entomophthorales Entomophthorales NRRL 2808 DAOM 233144 Basidiobolus ranarum 301 NRRL34594 AY635841 DQ273807 DQ294589 DQ302777 DQ282610 AY997030 Zygomycota Harpellales Orphella aff. haysii 1062 NS-35-W16 DQ322626 DQ273830 DQ294606 -- -- AY997068 Zygomycota Harpellales Smittium culisetae 29 COL-18-3 AF007540 DQ273773 DQ294593 DQ302782 AB077104 AY997089 Zygomycota Kickxellales Coemansia reversa 140 NRRL1564 AF007533 AY546689 DQ294594 DQ302783 DQ282615 AY997039 Zygomycota Kickxellales Spiromyces aspiralis 185 NRRL22631 AF007543 DQ273801 DQ294599 DQ302790 DQ282621 AY997090 Zygomycota Mortierellales Mortierella verticillata 141 NRRL6337 AF157145 DQ273794 DQ294595 Mucorales Rhizopus oryzae 1241 GenBank AF113440 AY213626 C AF157262 AACW020 00199 REGION: 121681.. 122907 AY997063 Zygomycota DQ302784 AACW020 00219 REGION: 138893.. 141577 AB097334 Zygomycota Mucorales 144 NRRL5844 DQ322627 DQ273797 DQ294598 DQ302787 AF157258 AY997097 Zygomycota Mucorales Umbelopsis ramanniana Phycomyces blakesleeanus 184 AY635837 DQ273800 DQ294607 DQ302789 DQ282620 AY997071 Zygomycota Zoopagales Rhopalomyces elegans 142 NRRL1555 NRRL A10835 AY635834 DQ273795 DQ294596 DQ302785 DQ282616 -- Zygomycota Zoopagales Piptocephalis corymbifera 145 NRRL2385 AB016023 AY546690 DQ294610 DQ302788 DQ282619 AY997073 Zygomycota Zygomycota 9 Phylum Order Genus+species AFTOLID Strain/ voucher 18S 28S RPB1 RPB2 EF1A nucITS NM_06812 2 AABS0100 0823 REGION: 9183.. 18948 NM_07856 9 NM_06564 6 AABS0100 0442 REGION: 13491 ..23574 NM_05735 8 NM_07692 2 X03680 X63563 XM_62853 3 Outgroup taxa Animalia Caenorhabditis elegans GenBank X03680 X03680 Animalia Ciona intestinalis GenBank AB013017 AF212177 Animalia Drosophila melanogaster GenBank M21017 M21017 Animalia Homo sapiens GenBank U13369 U13369 Apicomplexa Cryptosporidium parvum GenBank AF161859 AF040725 X63564 XM_62682 2 U71180 AF040725 Apicomplexa Toxoplasma gondii Chlamydomonas reinhardtii GenBank X75429 X75429 F F F X75429 GenBank M32703.1 AF183463 G G paralog U66954 Monosiga brevicollis GenBank AY026374 AF315821 -- paralog -- Chromista Phytophthora sojae GenBank AF100940 AY742749. 1 -- Thalassiosira pseudonana GenBank AF374481 -- G AAFD0100 1599 REGION: 5653 .. 7347 Mycetozoa Dictyostelium discoideum GenBank X00601.1 X00601.1 X55972.1 X00601.1 Plantae Arabidopsis thaliana GenBank X52322 X52322 AY133532 X52322 Plantae Oryza sativa GenBank AF069218 M11585 AF058710 NM_11974 6 AP008214 REGION: 3222137 .. 3226410 G AAFD0100 0966 REGION: 31046.. 33994 XM_63172 0 NM_11829 1 AL731878 REGION: 36606.. 41631 AF266769 Chromista G AAFD0100 0071 REGION: 25606.. 28760 Plantae Populus trichocarpa GenBank -- -- G Rhodophyta Cyanidioschyzon merolae GenBank AB158485 AB158485 AB095187 Chlorophyta Choanoflagellida G AP006490 REGION: 284244 .. 286886 AB070230 NM_16585 0 NM_00140 2 -M21017 U13369 -- AK073196 AF169230 G AJ006440 AB095182 AB158485 10 A. Histoplasma capsulatum sequence was obtained from the Washington University at St. Louis Genome Sequencing Center website funded by the National Institute of for Allergy and Infectious Diseases (http://genomeold.wustl.edu/blast/histo_client.cgi). B. The data for Pneumocystis carinii were obtained from the Pneumocystis Genome Project, University of Cincinnati, Cincinnati, OH. http://pgp.cchmc.org/ C. The Candida guilliermondii and Rhizopus oryzae genomes were accessed through the Genbank submissions from the Fungal Genome Initiative at the Broad Institute (http://www.broad.mit.edu/annotation/fungi/fgi). D. Podospora anserina release 1 from complete sequence was established through fundings from CNRS / Ministère de la recherche, "Séquençage à grande échelle 2002" and Génoscope. Bio-informatic tools were funded by IFR Génome. E. Antonospora locustae Genome Project, Marine Biological Laboratory at Woods Hole, funded by NSF award # 0135272. F. Toxoplasma gondii preliminary genomic and/or cDNA sequence data were accessed via http://ToxoDB.org and/or http://www.tigr.org/tdb/t_gondii/. Genomic data were provided by The Institute for Genomic Research (supported by the NIH grant #AI-05093), and by the Welcome Trust Sanger Centre. G. The data for Chlamydomonas reinhardtii, Phytophthora sojae, and Populus trichocarpa were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/. 11 Supplementary Notes 2 Phylogenetic analysis of super-matrix using nucleotide-only analysis with Supplementary Figure 1. We also analysed the data using a heterogenous Bayesian model based on 9 data partitions: 18S, 28S, 5.8S, EF1α (1st codon position), EF1α (2nd codon position), RPB1 (1st codon position), RPB1 (2nd codon position), RPB2 (1st codon position), and RPB2 (2nd codon position). We did not use 3rd codon positions because these appeared to introduce homoplasy and decrease support by Bayesian posterior probability (BPP) for many nodes. Models for each partition were chosen based on ModelTest1. Each partition utilized a general-time-reversible model with a proportion of invariant sites and gamma distributed rates (GTR+I+Γ) except the 5.8S partition which used a GTR+ Γ model. Five independent MCMC runs were used to search model and tree space using the software MrBayes 3.1.12. The analyses were run for 1.65x107 generations sampling every 500 generations. From each run 1.2x107 trees were discarded and the rest were used to compute a consensus topology (Suppl. Fig. 1). Maximum likelihood bootstrap support was assessed with 500 bootstrap replicates using the software PHYML3. References 1. 2. 3. Posada, D. & Crandall, K. A. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818 (1998). Huelsenbeck, J. P. & Ronquist, F. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-755 (2001). Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696-704 (2003). 1 Basidiomycota: Agaricomycotina Basidiomycota: Ustilaginomycotina Glomeromycota “Zygomycota”: Mucormycotina “Chytridiomycota” “Zygomycota”: Entomophthorales “Chytridiomycota”: euchytrids “Chytridiomycota”: Blastocladiales microsporidia Chytridiomycota Metazoa Apicomplexa Stramenopiles Viridiplantae 0.05 substitutions/site Ascomycota: Eurotiomycetes “Zygomycota” Ascomycota: Dothideomycetes Basidiomycota: Urediniomycotina 1087 Coprinopsis cinerea 468 Henningsomyces candidus 285 Cortinarius iodes 564 Pleurotus ostreatus 449 Armillaria mellea 558 Flammulina velutipes 556 Marasmius alliaceus 480 Lycoperdon pyriforme 626 Coprinus comatus 563 Clavaria zollingeri 673 Amanita brunnescens 625 Pluteus romellii 542 Ampulloclitocybe clavipes 729 Hygrocybe aff. conica 557 Collybia tuberosa 439 Calostoma cinnabarinum 713 Boletellus projectellus 714 Hygrophoropsis aurantiaca 717 Suillus pictus 576 Fibulorhizoctonia sp. 484 Phlebia radiata 767 Climacodon septentrionalis 776 Phanerochaete chrysosporium 562 Grifola sordulenta 701 Grifola frondosa 770 Fomitopsis pinicola 455 Echinodontium tinctorium 682 Lactarius deceptivus 452 Bondarzewia montana 492 Stereum hirsutum 447 Coltricia perennis 688 Fomitiporia mediterranea 518 Hyphoderma praetermissum 700 Cotylidia sp. 466 Gautieria otthii 724 Ramaria rubella 471 Hydnum albomagnum 438 Calocera cornea 454 Dacryopinax spathularia 1088 Cryptococcus neoformans 505 Ustilago maydis 867 Cintractia sorghi vulgaris 870 Tilletiopsis sp. 865 Tilletiaria anomala 675 Agaricostilbum hyphaenes 709 Colacogloea peniophorae 674 Rhodotorula hordea 456 Endocronartium harknessii 1459 Puccinia graminis 710 Platygloea disciformis 138 Scutellospora heterogama 139 Glomus mosseae 845 Glomus intraradices 844 Paraglomus occultum 574 Geosiphon pyriformis 141 Mortierella verticillata 144 Umbelopsis ramanniana 184 Phycomyces blakesleeanus 1241 Rhizopus oryzae 539 Endogone pisiformis 136 Dimargaris bacillispora 140 Coemansia reversa 1062 Orphella aff. haysii 29 Smittium culisetae 185 Spiromyces aspiralis 142 Rhopalomyces elegans 145 Piptocephalis corymbifera 301 Basidiobolus ranarum 633 Olpidium brassicae 28 Entomophthora muscae 137 Conidiobolus coronatus 20 Rhizoclosmatium sp. 43 Rhizophlyctis rosea 182 Spizellomyces punctatus 24 Polychytrium aggregatum 27 Cladochytrium replicatum 21 Batrachochytrium dendrobatidis 689 Rhizophydium macroporosum 635 Synchytrium macrosporum 25 Monoblepharella sp. 26 Hyaloraphidium curvatum 638 Neocallimastix sp. 19 Physoderma maydis 18 Coelomomyces stegomyiae 300 Allomyces arbusculus 1068 Encephalitozoon cuniculi 1089 Antonospora locustae 297 Rozella allomycis Caenorhabditis elegans Ciona intestinalis Homo sapiens Drosophila melanogaster Monosiga brevicollis (Choanoflagellida) Dictyostelium discoideum (Mycetozoa) Cryptosporidium parvum Toxoplasma gondii Phytophthora sojae Thalassiosira pseudonana Cyanidioschyzon merolae (Rhodophyta) Arabidopsis thaliana Populus trichocarpa Oryza sativa Chlamydomonas reinhardtii 1078 Neurospora crassa 216 Sordaria fimicola 1085 Podospora anserina 217 Chaetomium globosum 1081 Magnaporthe grisea 935 Diaporthe eres 952 Gnomonia gnomon 51 Xylaria hypoxylon 63 Xylaria acuta 1082 Fusarium graminearum 161 Fusarium aff. solani 186 Hydropisphaera erubescens 52 Hypocrea citrina 914 Microascus trigonosporus 413 Lindra thalassiae 424 Lulworthia grandispora 1 Leotia lubrica 166 Cudoniella clavus 49 Lachnum virgineum 151 Chlorociboria aeruginosa Ascomycota: 76 Mollisia cinerea Leotiomycetes 279 Monilinia fructicola 59 Botryotinia fuckeliana 941 Dermea acerina 147 Coccomyces dentatus 744 Potebniamyces pyri 1004 Pleopsidium chlorophanum 1005 Acarospora schleicheri 1007 Acarospora laqueata 106 Echinoplaca strigulacea 958 Diploschistes ocellatus 78 Acarosporina microspora 398 Stictis radiata 296 Orceolina kerguelensis 962 Trapelia placodioides 224 Pertusaria dactylina Ascomycota: 358 Dibaeis baeomyces Lecanoromycetes 645 Umbilicaria mammulata 687 Hypocenomyce scalaris 134 Peltigera degenii 196 Mycoblastus sanguinarius 639 Lecanora hybocarpa 6 Canoparmelia caroliniana 3 Cladonia caroliniana 642 Bacidia schweinitzii 84 Physcia aipolia Ascomycota: 56 Geoglossum nigritum Leotiomycetes 64 Trichoglossum hirsutum 891 Peltula umbilicata Ascomycota: 892 Peltula auriculata Lichinomycetes 896 Lichinella iodopulchra 101 Anisomeridium polypori 1036 Trematosphaeria heterospora 1037 Westerdykella cylindrica 283 Pyrenophora phaeocomes 54 Cochliobolus heterostrophus 940 Pleospora herbarum 110 Trypethelium sp. 274 Dothidea sambuci 921 Dothidea insculpta 939 Capnodium coffeae 355 Dendrographa minor Ascomycota: 126 Roccella fuciformis 80 Simonyella variegata Arthoniomycetes 1079 Aspergillus fumigatus 1080 Aspergillus nidulans 426 Monascus purpureus 1083 Histoplasma capsulatum 1084 Coccidioides immitis 430 Spiromastix warcupii 657 Capronia pilosella 668 Exophiala dermatitidis 659 Ramichloridium anceps 669 Exophiala pisciphila 684 Agonimia sp. 91 Dermatocarpon miniatum 661 Endocarpon pallidulum 697 Staurothele frustulenta 342 Pyrgillus javanicus 387 Pyrenula pseudobufonia 148 Cheilymenia stercorea 62 Scutellinia scutellata 65 Aleuria aurantia 949 Pyronema domesticum 50 Sarcoscypha coccinea 152 Caloscypha fulgens Ascomycota: 176 Gyromitra californica Pezizomycetes 179 Disciotis sp. 60 Morchella aff. esculenta 66 Helvella compressa 181 Ascobolus crenulatus 507 Peziza vesiculosa 71 Peziza proteana 905 Orbilia vinosa Ascomycota: Orbiliomycetes 906 Orbilia auricolor 1069 Saccharomyces cerevisiae 1070 Saccharomyces castellii 1073 Candida glabrata 1071 Kluyveromyces waltii 1072 Ashbya gossypii 1075 Kluyveromyces lactis Ascomycota: 1074 Candida albicans Saccharomycotina 1269 Candida tropicalis 1270 Candida guilliermondii 1077 Debaryomyces hansenii 1268 Candida lusitaniae 1076 Yarrowia lipolytica 1199 Schizosaccharomyces pombe 1192 Pneumocystis carinii Ascomycota: 265 Taphrina wiesneri Taphrinomycotina 266 Protomyces inouyei Ascomycota: Sordariomycetes ... to Ascomycota ... Supplementary Figure 1. Phylogeny of Fungi based on nucleotide analysis of six gene regions. Fungal taxa begin with AFTOL ID (see http://www.aftol.org) followed by genus and species. Thickened branches indicate nodes that are supported by both Bayesian posterior probability (BPP≥95%) and maximum likelihood bootstrap (MLBS≥70%). Almost every branch was supported by BPP and thus values are not shown. Microsporidia branches have been shortened by five times to increase readability. 3 Supplementary Notes 3 Analysis of individual gene partitions with Supplementary Table 2. The consistency of support for nodes was further assessed by separate analyses of the individual gene partitions as well as subsets of the combined data (Suppl. Table 2). These were analyzed using MrBayes 3.1.11 . The MCMC sampling was accomplished using 4 independent runs of sampling every 500 generations. We ran the MCMC sampler for 20 x 106 generations for nucleotides, 12 x 106 generations for RPB1 and RPB2, 20 x 106 for EF1α, and 5 x 106 for combined RPB1 and RPB2. We also performed maximum likelihood bootstrapping (MLBS) using PHYML2 on amino acids or nucleotides (500 replicates). Low MLBS values appeared to be the result of including partial data. For example, the use of only three of the six genes for the choanoflagellate (Monosiga brevicollis) appeared to substantially reduce MLBS. When M. brevicollis is pruned from the RPB1+2 and combined six locus bootstrap trees, MLBS support for dikarya increases to 32.6% and 100.0%, respectively. Similarly, the support for microsporidia + Fungi increases to 77.6% and 54.2% after pruning M. brevicollis from the RPB1+2 and combined trees, respectively. References 1. 2. Huelsenbeck, J. P. & Ronquist, F. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-755 (2001). Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696-704 (2003). 1 Supplementary Table 2. Consensus of support among data partitions. rDNA is the combined data set of 18S+28S+5.8S. Combined refers to the data set of all six loci. Euchytrids, Mucormycotina, and Entomophthorales are as indicated in Figure 1. Microsporidia+Fungi, indicates microsporidia grouping anywhere in the Fungi and is compatible with microsporidia+Rozella. Values that are not shown show analyses that are not possible due to missing data. BPP= Bayesian posterior probability; MLBS= maximum likelihood bootstrap. Data Partition Clade 18S 28S rDNA EF1a RPB1 RPB2 dikarya BPP=20.1 MLBS=20.4 BPP=0.0 MLBS=15.4 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.6 BPP=3.6 MLBS=33.0 BPP=0.5 MLBS=3.6 BPP=0.0 MLBS=0.0 BPP=0.4 MLBS=4.2 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.4 BPP=93.3 MLBS=46.2 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.0 BPP=85.3 MLBS=54.0 BPP=2.1 MLBS=12.8 BPP=0.0 MLBS=6.6 BPP=0.0 MLBS=0.6 BPP=0.0 MLBS=0.0 BPP=42.9 MLBS=36.0 BPP=100.0 MLBS=53.6 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.0 BPP=8.5 MLBS=65.8 BPP=99.7 MLBS=63.6 BPP=0.1 MLBS=2.2 BPP=43.0 MLBS=34.8 BPP=0.0 MLBS= BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=6.4 BPP=100.0 MLBS=5.6 BPP=100.0 MLBS=67.0 BPP=100.0 MLBS=11.2 BPP=0.0 MLBS=0.0 BPP=90.5 MLBS=35.4 BPP=22.1 MLBS=6.8 BPP=91.0 MLBS=16.4 BPP=0.0 MLBS=0.2 BPP=95.9 MLBS=11.0 BPP=99.0 MLBS=40.6 BPP=100.0 MLBS=50.0 BPP=98.2 MLBS=20.6 BPP=99.1 MLBS=19.4 microsporidia+Fungi microsporidia+Rozella Basidiobolus+ Entomophthorales Basidiobolus+Olpidium euchytrids Mucormycotina Glomeromycota+dikarya Glomeromycota+ Mucormycotina BPP=0.0 MLBS=0.4 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.0 BPP=0.0 MLBS=0.0 RPB1+2 BPP=100.0 MLBS=0.0 BPP=100.0 MLBS=0.0 BPP=100.0 MLBS=38.2 BPP=0.0 MLBS=3.6 BPP=98.8 MLBS=28.2 BPP=90.9 BPP=100.0 MLBS=4.8 MLBS=31.4 BPP=97.8 BPP=99.9 MLBS=48.8 MLBS=56.4 BPP=1.3 BPP=0.0 MLBS=0.6 MLBS=0.0 BPP=15.7 BPP=91.8 MLBS=17.0 MLBS=32.2 Combined BPP=100.0 MLBS=71.4 BPP=100.0 MLBS=8.0 BPP=99.6 MLBS=21.2 BPP=9.8 MLBS=1.4 BPP=87.2 MLBS=85.2 BPP=100.0 MLBS=79.4 BPP=99.9 MLBS=44.2 BPP=100.0 MLBS=1.6 BPP=0.0 MLBS=50.8 2 Supplementary Notes 4 Analysis of an EF-like gene in Fungi with Supplementary Figure 2. We were unable to obtain sequences for EF1α from a large number of basal fungal lineages using PCR. However, we were able to amplify and sequence a copy of an EF-like (EFL) gene from all of the basal fungal isolates from which we could not amplify EF1α using primers EF1-983F+EF1aZ-1R (http://www.aftol.org/primers.php). Other eukaryotic lineages (e.g., choanoflagellates, green algae, dinoflagellates) have also been reported to lack EF1α but encode an EFL gene that is a GTPase paralogous to the normal EF1α gene found in most organisms1. We have detected a homologue of EFL in the following lineages: Blastocladiales, Entomophthorales, Spizellomycetales, Olpidium, and Rozella. However, one spizellomycetalean (Rhizophlyctis rosea) and one blastocladialean chytrid (Physoderma maydis) only have the normal EF1α homologue and not EFL. With only one exception, Basidiobolus ranarum, did we detect both EF1α and EFL homologues in a single strain, and this is the first species ever reported to encode both genes. Phylogenetic analyses of the EFL protein sequences confirms the relationship between B. ranarum and Entomophthorales and demonstrate the monophyly of the fungal sequences, though the fungal clade is not supported (Suppl. Figure 2). The monophyly of the fungal EFL sequences supports the hypothesis that the ancestor of Fungi had both EF1α and EFL genes and that selective loss of one of the two paralogues has happened quite often in the early evolution of the Fungi. However, the forces that drive the selective replacement of one paralog of EF1α versus EFL are completely unknown, as is the role of horizontal gene transfer. One speculation is that the evolutionary trend of early fungi to become endoparasites (particularly of other Opisthokonts) could have driven rapid fixation of one or the other paralogue to distinguish host and parasite translation apparati. 1. Keeling, P. J. & Inagaki, Y. A class of eukaryotic GTPase with a punctuate distribution suggesting multiple functional replacements of translation elongation factor 1α. Proc. Natl. Acad. Sci. USA 101, 15380-15384 (2004). 1 --/70 FUNGI 100/98 182 Spizellomyces punctatus 637 Spizellomycete JEL355 Spizellomycetales 696 Triparticalcar arcticum 34 Gaertneriomyces semiglobiferus 32 Powellomyces sp. 302 Blastocladiella emersonii 36 Catenophlyctis sp. 299 Microallomyces sp. Blastocladiales 300 Allomyces arbusculus 18 Coelomomyces stegomyiae 633 Olpidium brassicae 16 Rozella sp. 96/-- 297 Rozella allomycis 301 Basidiobolus ranarum 28 Entomophthora muscae 96/-- 137 Conidiobolus coronatus ** Pleodorina sp. * Chlamydomonas reinhardtii * Prototheca wickerhamii 95/-- Chlorophyta Scenedesmus obliquus Helicosporidium sp. * Pavlova lutheri 100/-- * 100/93 Entomophthorales Haptophyceae Isochrysis galbana ** Heterocapsa triquetra Lingulodinium polyedrum ** Amphidinium carterae Dinophyceae Monosiga brevicollis Choanoflagellida Bigelowiella natans Cercozoa Saccharomyces cerevisiae EF1α 0.1 substitutions/site Supplementary Figure 2. Consensus phylogram of EF1α-like (EFL) sequences from Fungi and eukaryotes. Tree is rooted using the EF1α gene from Saccharomyces cerevisiae (GenBank accession # M10992). Tree is the consensus of sampled credible trees (2 x 106 generations). Numbers above nodes supported in the phylogeny are posterior probabilities followed by MLBS estimated using 1,000 replicates in PHYML. Both analyses used the JTT+Γ amino acid model. Fungal sequences have been deposited to GenBank under accession numbers (DQ275334DQ275349). Sequences of other eukaryotes are from Keeling and Inagaki1. Short internodes in non-fungal eukaryotes are labeled with * to indicate support ≥ 95% BPP, and ** to indicate ≥ 95% BPP plus ≥ 70% MLBS. 2 Supplementary Notes 5 Analyses of long branch attraction with Supplementary Table 3. Two methods were used to test whether the placement of microsporidia with R. allomycis was due to long branch attraction (LBA): “fast site removal” (FSR1) and long branch extraction (LBE2). FSR was accomplished by estimating the rate class (gamma model with eight rate classes) for each nucleotide position using the entire unpartitioned nucleotide data with the software Tree-Puzzle v5.2. We then sequentially and cumulatively deleted the fastest evolving sites from rate class 8 (the fastest) down to rate class 5 (Supplementary Table 3). Each pruned data set was analyzed using a heterogeneous nucleotide model with six partitions (divided into genes but not codon positions) using MrBayes 3.1.1 sampled using four independent runs for 20 x 106 generations each. This analysis differs from the analysis presented in Figure 1 of the paper and the nucleotide-only analysis presented as Supplementary Notes 2 in that the protein-coding gene regions are analyzed as nucleotides but not divided into codon positions. The concept of FSR is that the fastest evolving sites are those that are more subject to homoplasy and are contributing most to the LBA. The FSR test discredits the hypothesis that R. allomycis plus microsporidia is an artifact of LBA: following deletion of the fastest evolving sites (as many as ~30% of all sites) the grouping of R. allomycis and microsporidia remains the most likely (Supplementary Table 3). In fact, with the Bayesian model used to analyze the dataset, only after deleting the very fastest sites (rate class 8) is the relationship between microsporidia and R. allomycis recovered. LBE was performed by separately deleting three taxa: R. allomycis, microsporidia, and the outgroup. The data were then reanalyzed under the heterogeneous protein + nucleotide model using MrBayes 3.1.1 for 5 x 106 generations. The concept of LBE is that if two taxa group together because of LBA, removing one of the two branches and re-estimating the phylogeny will allow the other long branch to find the right place in the tree3. If the taxa group together because of a true relationship, removal of one or the other suspect taxon should not change the relationship of the remaining taxa. The LBE tests also confirmed that the grouping of microsporidia with R. allomycis is not likely due to LBA. When the microsporidia were removed from the data set and the phylogeny re-estimated, R. allomycis appeared as the earliest diverging lineage in the Fungi with 100% BPP. When R. allomycis was removed from the data set and the phylogeny re-estimated, microsporidia are significantly supported as being among the earliest diverging lineages of Fungi, but without support for an exact placement. Finally, by removing all outgroup taxa and re-estimating the phylogeny, the microsporidia still grouped with R. allomycis with 100% BPP among the Chytridiomycota. These data specifically suggest a relationship between R. allomycis and microsporidia such that, in the absence of R. allomycis in the dataset, the phylogenetic position of microsporidia is unresolved. References 1. Dacks, J. B., Marinets, A., Doolittle, W. F., Cavalier-Smith, T. & Logsdon, J. M., Jr. Analyses of RNA polymerase II genes from free-living protists: phylogeny, 1 2. 3. long branch attraction and the eukaryotic big bang. Mol. Biol. Evol. 19, 830-840 (2002). Siddall, M. E. & Whiting, M. F. Long-branch abstractions. Cladistics 15, 9-24 (1999). Bergsten, J. A review of long-branch attraction. Cladistics 21, 163-193 (2005). Supplementary Table 3. Bayesian posterior probabilities (BPP) supporting the R. allomycis + microsporidia clade using sequential “fast site removal.” Rate categories excluded % of sites removed none 8 7-8 6-8 5-8 0 8.1 17.7 29.3 40.6 % BPP for R. allomycis + microsporidia 0 100 100 88 49 Rate categories correspond to the 8 classes estimated using a discrete approximation to a gamma distribution of rates (α shape parameter equal to 0.33), with 8 being the fastest class and 1 the slowest class. 2