articles - Genetics

Transcription

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
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
?
...
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
ARTICLES
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
ARTICLES
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.
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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. (tyj2@duke.edu) or R.V. (fungi@duke.edu).
822
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: lobo@udel.edu
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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
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.
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: pogon@berkeley.edu
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
NEWS & VIEWS
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.
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
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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)
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.
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.
http://www.nature.com/nature/journal/v443/n7113/suppinfo/nature05110.html
26.10.2006
This file contains a phylogeny of a paralogous copy of elongation factor 1-Î± (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.
This file contains the methods and results from tests of long branch attraction between
Rozella allomycis and microsporidia.
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GenBank and strain numbers for the taxa used in this study with Supplementary Table 1.
Supplementary Table 1. Strain/voucher listed as “GenBank” are composite taxa utilizing data from multiple sources (GenBank,
genome projects, AFTOL data). 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
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.
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
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.
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
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
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

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