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SUPPLEMENTARY INFORMATION                                                                                         doi:10.1038/nature09984




   a            Basidiomycota           b
                Ascomycota
                Glomeromycota
                Zygomycota                                      Basidiomycota
                Olpidium
                Blastocladiomycota
                Chytridiomycota
                Neocallimastigomycota
                Microsporidia
                                                                Ascomycota
                Rozella                                                                                       Classic fungi that
                                                                                                              grow with a chitin-rich
                                                                                                              cell wall.
                                                                Glomeromycota

                                                                Zygomycota

                                                                Olpidium

                                                                Blastocladiomycota

                                                                Chytridiomycota

                                                                Neocallimastigomycota



                                                                Microsporidia


                                                                                                              Highly diverse novel
                                                                                                              basal group identified
                                                                                                              in this study.


                                                                                                             Cryptomycota:
                                                                                                             organisms that
                                                                                                             branch with the
    Key                                                                                                      fungi but putatively
           Branching relationship                                                                            grow and
           weakly supported                                                                                  develop without
                                                                Rozella                                      synthesizing
           Branching relationship
                                                                                                             a chitin-rich cell wall
           strongly supported                                   Opisthokont outgroup                         and therefore appear
           Group forms flagellated                                                                           to lack this classically
           zoospores                                                                                         defining fungal feature.




Supplementary Figure 1. Summary of the main findings of the paper. a, Standard working model of the fungal tree of life.
b, Identification of a very large uncultured basal clade in the kingdom Fungi, marked in red and, with Rozella, named here as
cryptomycota. This study therefore demonstrates that current models of fungal evolution and biodiversity, which are based on
cultured microbes, have missed a huge fraction of the kingdom (perhaps even approaching one half). Furthermore, this group of
microbes appears to grow without the defining fungal characteristics a chitin-rich cell wall (although this characteristic is also absent
during the flagellated zoospore stage of many other chytrid fungi). Therefore these data challenge existing ideas of fungal biodiversity
and current concepts regarding the adaptive features that drove the evolutionary diversification and ecological success of the fungal
kingdom.



                                                                                                    W W W. N A T U R E . C O M / N A T U R E | 1
doi:10.1038/nature09984                                                                                                                                     RESEARCH SUPPLEMENTARY INFORMATION
    a                                                                                                                        Batrachochytrium dendrobatidis
                                                                                                 Rhizophydium sphaerotheca
                                                                               Entophlyctis helioformis
                                                                                   Rhizophydium brooksianum
                                                                                                                   Rhizoclosmatium sp.
                                                                                                      Chytriomyces hyalinus
                                                                  Rhizophlyctis rosea
                                                                     Catenomyces sp.
                                                                       Powellomyces sp.
                                                                        Triparticalcar arcticum                                                                          ‘Chytrids’
                                                                                    Polychytrium aggregatum
                                                                                               Karlingiomyces sp.
                                                                                          Cladochytrium replicatum
                                                                                  Nowakowskiella sp.
                                                                                     Endochytrium sp.
                                                                                     Oedogoniomyces sp.
                                                                                       Monoblepharella sp.
                                                                                     Hyaloraphidium curvatum
                                                                            Cyllamyces aberensis
                                                                                                                    Piptocephalis corymbifera
                                                                                                                       Smittium culisetae                                Zygomycota
                                                                   Basidiobolus ranarum
                                                                                                                             Allomyces arbuscula
                                                                                                                                 Blastocladiella emersonii               Blastocladiomycota
                                                                            Physoderma maydis                                                                                                                       Fungi
                                                                                                                                   Neurospora crassa
                                                                                                                                       Magnaporthe grisea
                                                                                                                                  Hypocrea jecorina
                                                                                                           Monilinia fructicola
                                                                                                                           Monascus purpureus                            Ascomycota
                             0.93/44/--                                                              Dothidea sambuci
                                                                                                       Disciotis venosa
                                                                                                             Candida glabrata
                                                                                                    Debaryomyces hansenii
                                                                                         Schizosaccharomyces pombe
                                                                                                                                    Ustilago maydis
                                                                                                            Tilletiaria anomala
                                                                                                                                      Platygloea disciformis             Basidiomycota
                              1/95/99                                                                 Coprinopsis cinerea
                                                                                                     Calocera cornea
                                                                                                          Filobasidiella neoformans
                                                                                                                                             Phycomyces blakesleeanus    Zygomycota
                                                                                                                          Rhizopus oryzae
                                                                                                   Glomus mosseae
                                                                                            Paraglomus occultum                                                          Glomeromycota
                                                              1/100/100                                                               Lily Stem/Washington Singer CM2
                                                                                                  Washington Singer CM1                                                   Cryptomycota
                                           1/96/96                                           Rozella sp.              Rozella
                                                                                          Rozella allomycis
                                                                          Nuclearia simplex
                                                                                                      Suberites ficus
                                                                                                  Aurelia sp.                Outgroup
                                                                                 Amoebidium parasiticum
                                                                             Capsaspora owczarzaki                                                                         0.9/80/80             0.6/50/50                                0.07 subs/site


    b              Basidiobolus ranarum
                                       ‘Chytrids’

                        Piptocephalis corymbifera          Zygomycota
                                    Smittium culisetae
                                      Blastocladiomycota
                             Lily Stem/Washington Singer CM2
                        Washington Singer CM1                          Cryptomycota
                        Rozella sp. JEL347
                                           Rozella
                       Rozella allomycis
                                                      Ascomycota
                             Glomeromycota
                                                                                                                                                                                                             Encephalitozoon cuniculi
                                                                                                                                                                                    Heterosporis anguillarum
                                                                                                                                                                                                                                          microsporidia
                                                                                                                                                                                                          Brachiola algerae
                                                                                                                                                                        Antonospora locustae
                               Filobasidiella neoformans
                                      Platygloea disciformis
                         Coprinopsis cinerea
                                                                  Basidiomycota
                         Calocera cornea
                                      Ustilago maydis
                           Tilletiaria anomala
                                                  Zygomycota
                       Oedogoniomyces sp. CR84
                                                         ‘Chytrids’
                      Monoblepharella sp. M15
                Nuclearia simplex
                    Outgroup


    c
                                                                                                                                                                           0.9/80/80           0.6/50/50                                  0.02 subs/site
        21/71                   Ascomycota
                         Glomeromycota
                             Basidiomycota
                                                                                                                                                                  Antonospora locustae
                                                                                                                                                                                                             Brachiola algerae
                                                                                                                                                                                                                                        microsporidia
                                                                                                                                                                                  Heterosporis anguillarum
                   63/75                                                                                                                                                                               Encephalitozoon cuniculi
                                    Rhizopus oryzae
                                                                      Zygomycota
                                      Phycomyces blakesleeanus
                       Washington Singer CM1
                          Lily Stem/Washington Singer CM2
                                                                      Cryptomycota
                     Rozella allomycis
                     Rozella sp. JEL347
                                                  Rozella
                           Chytridiomycota
                               Smittium culisetae
                                                         Zygomycota
                        Piptocephalis corymbifera
                         Batrachochytrium dendrobatidis
                        Chytriomyces hyalinus                  ‘Chytrids’
                             Rhizoclosmatium sp. JEL347
                  Basidiobolus ranarum       Zygomycota
                     Monoblepharella sp. M15
                      Oedogoniomyces sp. CR84            ‘Chytrids’
                    Hyaloraphidium curvatum
                              Blastocladiomycota
                Nuclearia simplex

                   Outgroup                                                                                                                                                80/80         50/50                           0.09 subs/site

Supplementary Figure 2. Phylogenetic analysis of the SSU-5.8S-LSU alignment demonstrating the branching position of the
cryptomycota. a, The phylogeny was calculated from an alignment of 54 sequences and 1,877 nucleotide alignment characters
and demonstrates strong support for the placement of the novel clade as a sister group to Rozella. Node support values are labelled
using the same convention as Fig. 1. b, Bayesian phylogeny based on SSU-LSU alignment with four microsporidia sequences added
(58 sequences and 1,072 DNA characters – character number is reduced from a because of alignment of divergent microsporidia
sequences). c, PhyML (fast Maximum Likelihood) tree topology with 1000 bootstrap values using PhyML and Log-Det distance methods
calculated from the same alignments as b. These phylogenetic analyses followed the same procedure as described for the analyses
shown in a and described in the methods section. Key topology support values are shown, however all other bootstrap values are notated
with symbols: nodes supported by 0.9 posterior probability or greater and 80% bootstrap support or greater by both methods are marked
with a black dot; nodes supported by 0.6 posterior probability or greater and 50% bootstrap support or greater by both methods are marked
with a ringed circle.

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Supplementary information 1 & 2

  • 1. SUPPLEMENTARY INFORMATION doi:10.1038/nature09984 a Basidiomycota b Ascomycota Glomeromycota Zygomycota Basidiomycota Olpidium Blastocladiomycota Chytridiomycota Neocallimastigomycota Microsporidia Ascomycota Rozella Classic fungi that grow with a chitin-rich cell wall. Glomeromycota Zygomycota Olpidium Blastocladiomycota Chytridiomycota Neocallimastigomycota Microsporidia Highly diverse novel basal group identified in this study. Cryptomycota: organisms that branch with the Key fungi but putatively Branching relationship grow and weakly supported develop without Rozella synthesizing Branching relationship a chitin-rich cell wall strongly supported Opisthokont outgroup and therefore appear Group forms flagellated to lack this classically zoospores defining fungal feature. Supplementary Figure 1. Summary of the main findings of the paper. a, Standard working model of the fungal tree of life. b, Identification of a very large uncultured basal clade in the kingdom Fungi, marked in red and, with Rozella, named here as cryptomycota. This study therefore demonstrates that current models of fungal evolution and biodiversity, which are based on cultured microbes, have missed a huge fraction of the kingdom (perhaps even approaching one half). Furthermore, this group of microbes appears to grow without the defining fungal characteristics a chitin-rich cell wall (although this characteristic is also absent during the flagellated zoospore stage of many other chytrid fungi). Therefore these data challenge existing ideas of fungal biodiversity and current concepts regarding the adaptive features that drove the evolutionary diversification and ecological success of the fungal kingdom. W W W. N A T U R E . C O M / N A T U R E | 1
  • 2. doi:10.1038/nature09984 RESEARCH SUPPLEMENTARY INFORMATION a Batrachochytrium dendrobatidis Rhizophydium sphaerotheca Entophlyctis helioformis Rhizophydium brooksianum Rhizoclosmatium sp. Chytriomyces hyalinus Rhizophlyctis rosea Catenomyces sp. Powellomyces sp. Triparticalcar arcticum ‘Chytrids’ Polychytrium aggregatum Karlingiomyces sp. Cladochytrium replicatum Nowakowskiella sp. Endochytrium sp. Oedogoniomyces sp. Monoblepharella sp. Hyaloraphidium curvatum Cyllamyces aberensis Piptocephalis corymbifera Smittium culisetae Zygomycota Basidiobolus ranarum Allomyces arbuscula Blastocladiella emersonii Blastocladiomycota Physoderma maydis Fungi Neurospora crassa Magnaporthe grisea Hypocrea jecorina Monilinia fructicola Monascus purpureus Ascomycota 0.93/44/-- Dothidea sambuci Disciotis venosa Candida glabrata Debaryomyces hansenii Schizosaccharomyces pombe Ustilago maydis Tilletiaria anomala Platygloea disciformis Basidiomycota 1/95/99 Coprinopsis cinerea Calocera cornea Filobasidiella neoformans Phycomyces blakesleeanus Zygomycota Rhizopus oryzae Glomus mosseae Paraglomus occultum Glomeromycota 1/100/100 Lily Stem/Washington Singer CM2 Washington Singer CM1 Cryptomycota 1/96/96 Rozella sp. Rozella Rozella allomycis Nuclearia simplex Suberites ficus Aurelia sp. Outgroup Amoebidium parasiticum Capsaspora owczarzaki 0.9/80/80 0.6/50/50 0.07 subs/site b Basidiobolus ranarum ‘Chytrids’ Piptocephalis corymbifera Zygomycota Smittium culisetae Blastocladiomycota Lily Stem/Washington Singer CM2 Washington Singer CM1 Cryptomycota Rozella sp. JEL347 Rozella Rozella allomycis Ascomycota Glomeromycota Encephalitozoon cuniculi Heterosporis anguillarum microsporidia Brachiola algerae Antonospora locustae Filobasidiella neoformans Platygloea disciformis Coprinopsis cinerea Basidiomycota Calocera cornea Ustilago maydis Tilletiaria anomala Zygomycota Oedogoniomyces sp. CR84 ‘Chytrids’ Monoblepharella sp. M15 Nuclearia simplex Outgroup c 0.9/80/80 0.6/50/50 0.02 subs/site 21/71 Ascomycota Glomeromycota Basidiomycota Antonospora locustae Brachiola algerae microsporidia Heterosporis anguillarum 63/75 Encephalitozoon cuniculi Rhizopus oryzae Zygomycota Phycomyces blakesleeanus Washington Singer CM1 Lily Stem/Washington Singer CM2 Cryptomycota Rozella allomycis Rozella sp. JEL347 Rozella Chytridiomycota Smittium culisetae Zygomycota Piptocephalis corymbifera Batrachochytrium dendrobatidis Chytriomyces hyalinus ‘Chytrids’ Rhizoclosmatium sp. JEL347 Basidiobolus ranarum Zygomycota Monoblepharella sp. M15 Oedogoniomyces sp. CR84 ‘Chytrids’ Hyaloraphidium curvatum Blastocladiomycota Nuclearia simplex Outgroup 80/80 50/50 0.09 subs/site Supplementary Figure 2. Phylogenetic analysis of the SSU-5.8S-LSU alignment demonstrating the branching position of the cryptomycota. a, The phylogeny was calculated from an alignment of 54 sequences and 1,877 nucleotide alignment characters and demonstrates strong support for the placement of the novel clade as a sister group to Rozella. Node support values are labelled using the same convention as Fig. 1. b, Bayesian phylogeny based on SSU-LSU alignment with four microsporidia sequences added (58 sequences and 1,072 DNA characters – character number is reduced from a because of alignment of divergent microsporidia sequences). c, PhyML (fast Maximum Likelihood) tree topology with 1000 bootstrap values using PhyML and Log-Det distance methods calculated from the same alignments as b. These phylogenetic analyses followed the same procedure as described for the analyses shown in a and described in the methods section. Key topology support values are shown, however all other bootstrap values are notated with symbols: nodes supported by 0.9 posterior probability or greater and 80% bootstrap support or greater by both methods are marked with a black dot; nodes supported by 0.6 posterior probability or greater and 50% bootstrap support or greater by both methods are marked with a ringed circle.