2045 Phylogenetic studies on nuclear large subunit ribosomal DNA sequences of smut fungi and related taxar

Dominik Begerow, Robert Bauer, and Franz Oberwinkler

Abstract: To show phylogenetic relationships among the smut fungi and thcir relatives. we sequenced a part of the nuclear LSU rDNA from 43 different species of smut fungi and related taxa. Our data were combined with the existing sequences of seven further smut fungi and 17 other basidiomycetes. Two sets of sequences were analyzed. The first set with a representative number of simple septate basidiomycetes, complex scptate basidiomycetes, and smut fungi was analyzed with the neighbor-joining method to estimate the general topology of the basidiomycetes phylogeny and the positions of the smut fungi. The tripartite subclassification of the basidiomycetes into the Urediniomycetes, , and Hymcnomycetes was confirmed and two groups of smut fungi appeared. Thc smut genera Aurantiosporiurn, Microbotrl-unr, Fulvisporium, and Ustilentr-loma are members of the Urediniomycetes, whereas the other smut species tested are members of the Ustilaginomycetes wrth Entorrhiz.a as a basal taxon. The second set of 46 Ustilaginomycetes was analyzed using the neighbor-joining and the maximum parsimony methods to show the inner topology of the Ustilaginomycetes. The results indicated three major lineages among Ustilaginomycetes corresponding to the Entorrhizomycetidae, Exobasidiomycetidae, and Ustilaginomycetidae. The Entorrhizomycetidae are represented by Entorrhiza species. The Ustilaginomycctidae contain at least two groups, thc Urocystales and . The Exobasidiomycetidae include five orders, i.e., Doassansiales, Entylomatales, . Georgefischeriales. and Tilletiales, and phoenicis and Microstroma juglandis. Our results support a classification mainly based on ultrastructure. The description of the Glomosporiaceae is emended. The Doassansiopsaceae, Mclanotaeniaceae, and Urocystaceae are proposed as new taxa.

Key words'. basidiomycete systematics, LSU rDNA. Microbotryales, molecular phylogeny, smut fungi, Ustilaginomycetes.

Rdsumd : Afin de ddmontrer les relations phylogdndtiques parmi les champignons du charbon et espöces apparentdes, les auteurs ont s6quenc6 une partie de la grande sous-unitd nucl6aire de 1'ADN ribosomique (LSU rDNA), chez 43 espöces dilTdrentes de champignons du charbon et taxons apparentds. Les donn6es ont 6td combin6es avec les s6quences d6jä connues de sept champignons du charbon et 17 autres basidiomycötes. Ils ont analysd deux groupes de s6quences. Le premier comporte un nombre repr6sentatif de basidiomycötes ä septations simples, de basidiomycötes ä septations complexes et de champignons du charbon, en utilisant la mdthodc de jonction avec les voisins pour estimer la topologie gönd,rale de la phylogdnie des basidiomycötes et les positions des champignons du charbon. La subclassification tripartite des basidiomycötes en Ur6dinomycötes, Ustilagoinomycötes et Hymdnomycötes est confirm6e et on ddtecte deux groupes de champignons du charbon. Les champignons du charbon des genres Aurantiosporiunt, Microbotn'tnt, Fulvisporium et Ustilent,,-loma sont membres des Ur6dinomycötes, alors que les autres espöces examin6es sont membres des Ustilaginomycötes, les Entorrhiz,a 6tant le taxon de base. Le second groupc de 46 Ustilaginomycötes a 6t6 analysd cn utilisant les m6thodes de jonction avec les voisins et de parscimonie maximum pour illustrer la topologie interne des Ustilaginomycdtes. Les rdsultats montrent trois lign6es majeures parmi lcs Ustilaginomycötcs, lesquellcs corrcspondent aux Entorrhizomycetidae, Exobasidiomycetidae et Ustilaginomycetidae. Les Entorrhizomycetidae sont repr6sent6s par les espöces d'Entorrhizo. Les Ustilaginomycetidae contiennent au moins deux groupes, les Urocystales et les Ustilaginales. Les Exobasidiomycetidae comportent cinq ordres, i.e., Doassansiales, Entylomatales, Exobasidiales, Georgefischeriales et Tilletiales ainsi quc le Graphiola phoenicis et le Microstroma .juglandis. Les r6sultats des auteurs supportent la classification basde principalement sur les ultrastructures. La description des Glomosporiaceae est amendde et les auteurs proposent les Doassansiopsaceae, les Melanotaeniaceae et les Urocystaceae comme nouveaux taxons.

Mots c'lds : systdmatique des basidiomycötes, LSU rDNA, Microbotryales, phylogdnie mol6culaire. champignons du charbon, Ustilaginomycötes. [Traduit par la r6daction]

Rcceived February 3, 1997. D. Begerow,2 R. Bauer, and F. Oberwinkler. Universität Tübingen, Institut für Biologie I, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle I, D-'72076 Tübingen, Germany. I Part 141 of the series "studies in Heterobasidiomycetes". 2 Author to whom all correspondence should be addressed. e-mail: dominik.begerow@uni+uebingcn.de

Can. J. Bot. 75:2045-2056 (19911 '9 1997 NRC Canada 2046 Can. J Bot Vol. 75,1997 lntroduction of 1000 replicates using SeqBoot and Consens of thc euvlte pack- age with defäult parameters (Felsenstcin 1993). Maxrmum parsi- A new system of smut fungi and allied taxa based on ultra- mony analysis was perforned by DNApars (urnble option: 100) structural studies was recently proposed by Bauer et al. (1997). and Consens. The alignments are available upon request. This system differs significantly from the traditional classifi- cation characterized by a basic dichotomy separating the Results phragmobasidiate Ustilaginaceae or Ustilaginales fionr the Sequence alignments holobasidiate Tilletiaceae or Tilletiales (e.g., Tulasne and Two sets of sequences were analyzed for phylogenetic dis- Tulasne 1847: Oberwinkler 1977, 1987). In the system of cussion of smut fungi and allied taxa. The flrst set with Bauer et al. (1997) six genera of smut fungi are grouped in sequences of 30 basidiomlicetes was analyzed to deterrnine the Microbotryales, whereas the other smut fungi are placed the general topology of the basidiomycetes, the positions of together with the Exobasidiales, Graphiolales. and Micro- smut fungi within this basidiomycetous topology and the root stromatales in the Ustila-einomycetcs. Owing to the profound of the Ustilaginomycetes. The second set with sequences of differences between traditional concepts and the systcm pro- 46 species was used to show the phylogenetic relationships posed Bauer et (1997), studies with independent by al. within the Ustilaginomycetes. The total consensus length of markers are required to test the classitications and to develop the first set was 518 bp, whereas the second set was 521 bp phylogenetic hypotheses. long. Although two small regions, positions 15-28 and Analyses of molecular sequence data have made impor- 465-413 of the alignment in the first set, 33-45 and462- tant contributions to the understanding of basidiomycetc 47 1 in the second set, respectively, included several ambigu- phylogeny (e.g., Berbee and Taylor 1993; Swann and Taylor ous positions owing to gap position. any manipulations were 1993, 1995; Berres et al. 1995; Boekhout et al. 1995). avoided to achieve highest reproducibility. Unfortunately. sequence data from smut fungi are available (Blanz only from a f'ew species and Gottschalk 1984; Swann Phylogenetic analysis of the first set and Taylor 1993, 1995; Boekhout et al. 1995; Berres et al. We analyzed a representative number of simple septate 1995). However. the previous results indicated the existence basidiomycetes, complex septate basidiomycetes. and smut of two distantly related groups of smut fungi. Because the fungi with allied taxa. Results of neighbor-joining analysis existing molecular sequence data base is too small to identify are shown in Fig 1. The three previously recognized classes natural groups among the smut fungi, we sequenced the 5' of basidiomycetes were confirmed. It was not the aim of this (LSU end of the large subunit of ribosomai DNA rDNA) of paper to study the origin of basidiomycetes. Analyses with presumptiveiy related species as a 38 smut fungi and five severai ascomycetes and zygorttycetes as outgroups led to (Qu Bruns et 1991) to semiconserved region et al. 1988; al. different arrangements of the three groups a-nd Entorrhi«t validate the system proposed by Bauer et al. (1997). Data was not always included in the Ustilaginomycetes (data not sets were completed with the sequences of seven further smut shown). Thus. the origin of basidiomycetes must be placed literature. fungi and l7 other basidiomycetes from somewhere close to the basal trifurcation. The first group, representing the Hymenomycetes, contains the heterobasid- Materials and methods iomycetes Tremella mesentericcr, Calocera liscosa, and Aurictrlaria auricula-jutlae, and the homobasidiomycetes Species studied for molecular analysis are listed in Tablc 1. DNA Ga nod e rm a mi c r o sp onon, Rus sul a mai r ei, B ol e t Lr s ntbine I I trs . was isolated from cultures or herbarium specimens fbllowing a Cortinarius stuntzii, Marasmius delectans. and Ag(tricLts modified version of the SDS method of Edwards ct al. (1991) and Henrion ct al. (1992): sanrples were ground in liquid nitrogen and arvensis. The second lineage representing the Urediniomy- incubated in 500 prl cxtraction buffer, tbr I h at 65"C. Thc extracts cetes is composed of the rusts (Melctmpsora lini, Put:c'inio werc centrifuged at 13.793 x g for 15 min and the supernatant was gruminis, and Puccinia recondita) together with some related transferred to a new Eppendorf tubc. RNA was digested with 10 U fungi (.Helicobasidium mompa, Eocronartium muscic:ola, RNAse at 37'C tbr I h. DNA was prccipitated by addition of and Septobusiditun corestianLrm), and Kriegeria eriophori at 400 pL isopropanol and l0 pL 3-M sodium acetate before centrifu- the base of the smut fwgi (.Microbonl'um vioLaceum, Ftrlvi- gation. After a washing step with 70% ethanol. the pellet was dried sporiunt restifaciens, Aurcmtiosporium subniteni. and Usrl- and dissolved in stcrile water. at room temperature lenryloma fluirans). The third lineage, representing the The the rDNA was amplified using thc 5' region of nuclear LSU Ustilaginomycetes, contains Erobasidium vaccinii and the polymerase chain reaction (PCR) with NL1 and NL4 as primers smut fungi Entorrhiza aschersoniana, Entonhi«t casp(tt)'(tna, (Boekhout et al. 1995). The PCR product was purified using thc ranunculi. Thecaphora seminis-convolt'uli, Ustilago QlAquick"' Kit (QIAGEN) according to the manutäcturer's pro- tocol. This dsDNA was scqucnccd directly using the ABI PRISM" horclei, Enn^loma microsporum, Georgefischeria riveae. Dye Terminator Cycle Sequencing Kit (Perkin Elmer) and an auto- Tilletia caries. and Doassansia epilobii. Whereas the flrst mated sequencer (ABI 373, Perkin Elmer). lineage contains only complex septate basidiomycetes. the An alignment was produced using the ClustalW program (Thomp- other two lineages both contain smut fungi. Bootstrap values son et al. 1994). The sequence data were analyzed with the psYup support the Hymenomycetes and Urediniomycetes but not packagc version 3.5r: (Felsenstein 1993). We used neighbor-joining the Ustilaginomycetes. thus in this analysis the monophyly of phylogenetic and maximum parsimony methods to reconstruct trees the Ustilaginomycetes is indicated but remains unccrtain. from LSU rDNA sequences. Neighbor-joining analysis (Saitou and Nei 1987) of a distance matrix produced by DNAdist (Kimura 2-parameter model. transi- Phylogenetic analyses of the second set tion to transvcrsion rate: 2.0) was applied with the detäult param- This set contained sequences of,16 species. The l8 most eters of the prograrn. Bootstrapping (Felsenstcin 1985) was made parsimonious trees required 1189 steps and their strict con-

icl 1997 NRC Canada Begerow et al. 2047

Table 1. List of species studied in molecular analysis

GcnBank Species Host accession no Source"

Attrantiosporium subnitens (Schröter & Henn.) Scleria ntelalerca Reichb. ex Schlecht AF 009846 MP 1173 M. Piepenbr.. K. Vänky & Oberw. & Cham. Cintraoia axicola (Berk.) Cornu Fimbristy'lis tetragona R. Br. AF 009847 H.U.V. 17460 Conidiosporomlces ayresii (Berk.) K. Vänky Panicum maximum lacq. AF 009848 H.U.V. 15197 Doassansia epilobii Farlow Epilobiurn montanwn L. AF 007523 FO 38252

Doa.s san s ia ltr grophila e Thirum. HygrophiLa spinosa T. Anders AF 007524 H.U.V. 15474 deJbrmans (Setch.) Dietel Sagitturia lattceolata L. ssp. lancifolia AF 009849 MP 2066 Doassansiopsis limnocharidis (Cif.) K. Vänky Limnocharis flava (L.) Buchenau AF 009850 H.U.V. 15198 Entorrhiza aschersoniana (Magnus) Lagerh. Juncus bufonius L. AF 009851 H.U.V. 15899 Entorrhiztr casparyana (Magnus) Lagerh. Juncus articulatus L. AF 009852 H.U.V. 17623 Enn'loma callitrichis Liro Callit riche sragnali.r Scop. AF 007525 RB 1079 Ennktma dattylidis (Pass.) Cif. Agrosti.r .ttolottift' ra L. AF 009853 RB 915 Entl-loma holv,at'i H. & P. Sydow Cosnos caudatus H.B.K. AF 009854 MP 1769 Entt,loma microsporum (Unger) Schröter Ranunculus repens L. AF 007530 FO 37329 Entl,ktma polysponun (Peck) Farlow Ambrosia artemisiifolia L. AF 007529 H.U.V. 2960 Erratom,-ces patelii (Pavgi & Thirum.) Phaseolus vulgaris L. AF 009855 MP l99l M. Piepenbr. & R. Bauer Exobasidium rhododendri (Fuck.) Cram. Rltodod end ron fe r rug i ne um L. AF 009856 RB 2050 E-robasidium rzstrrpii Nannf. Vaccinium oxvcot:cos L. AF 009857 RB 949 Exobosidium vaccinii (Fuck.) Woronin Vac'cinium vitis-idaea L. AF 009858 RR 945 Farysi a chttrdoniana Zundel Carex polystacft,ya Swartz ex Wahlenb. AF 009859 MP 2062 Fulvisporitun restifociens (D.E. Shaw) K. Vänky Stipa stuposa Hughes AF 009860 H.U.V. 17637 Georgefischeria riveae Thirum. & Naras. Rive a hy p o c ra t e r ifo r m i s Chois AF 00986r H.U.V. 15614

Graphiola phoenicis (Moug.) Poiteau P h oe n i.r cana ri ens i s Chatb. AF 009862 FO 29350

M e lanop s i c'hium pennsy Ltanicrzn Hirschh. Polygonum hispidu m H.B.K. AF 009863 MP 8OI

M e I anot ae ni um b ra c'h ia r i ae Y iegas Brachiaria distachya (L.) Stapf AF 009864 H.U.V. 17510 Melanotaenium euphorbiae (Lenz) M.D. Euphorbia genicuhta (Kl. & Garke) AF 009865 H.U.V. 17733 Whitehead & Thirum. Ortega Mit'robotn'unt violaceum (Pers.:Pers.) Deml & Gt'psophila repens L. AF 009866 FO 38227 C)herw. Microstroma juglandi s (Bereng.) Sacc. Juglans regia L. AF 009867 FO 39211 MoesT.iomyces bullatus (Schröter) K. Vänky Paspalurn distichum L. AF 009868 H.U.V. 15514 MundkurelLa kalopanacis K. Vänky Kaktparuu piclls (Thumb.) Nakai AF 009869 H.U.V. 16732 Rhamphospora nynphaeae D.D. Cunn. Ny-mphaea alba L. AF 007526 RB 862 Schizonello melmtogramma (DC.) Schröter Cure.r piLulifera L. AF 009870 FO 3'7174

Het e roto I yp o sp orium p i lulifo rme (Berk. ) Juncus planifollls R. Br. AF 009871 H.U.V. 15732 K. Vänky Sporisorium sorg&i Ehrenb. ex Link Sorghum bicolor (L.) Moench AF 009872 MP 2036a Thecaphora amaranthi (Hirsch.) K. Vänky Amaranthus hybridus L. AF 009873 H.U.V. 15882

The cap ho ra s e mi n i s - c ottv o lr.,u1i (Desm. ) Ito Co nv o lvu I us a n- e n s i s L. AF 009874 GD l39l Tol,-posporello brunkii (Ell. & Gall.) Clinton Andropogon saccharoides Swarrz AF 009875 H.U.V. 17816 Tol,v-posporium jrrc'i (Schröter) Woronin ex Juncus brfonius L. AF 009876 H.U.V. 17169 Schröter Trichocintractiu utriculicola (Hcnn.) Rhync'hospora crtrymbostt (L.) Britmn AF 009877 MP 2075 M. Piepenbr. Urocystis colchici (Schlecht.) Rabenh. Colchicum outumnctle L. AF 009878 RB 2041 Urocvstis ranunculi (Lib.) Moesz. Ranunculus repens L. AF 009879 RB 609 [Jstatl'stis waldsteiniae (C.H. Beck) Zundel Waldsreinia geoides W ild. AF 009880 FO 38439 Ustilago c,*nodontis (Henn.) Henn. Cynodon dactylon (L.) Pers. AF 00988r MP 1838 Ustilenn'ktma fluitans (Liro) K. Vänky Glycerio plicata (.Fr.) Fr. AF 009882 RB 9OO Species sequenced from other authors Agcricus arvensis (authors not cited) u l t9t0 Chapela et al. i994 Auriculuria auricula-.judae (Bull. :Fr.) Schröter L 202'78 Berres et al. 1995 Boletus rubinellus Peck L 20279 Berres et al. 1995 Cttktcera llsco.sa (Pers. ex Fr.) Fr. AF 011569 M. Weiß (unpublished data)

O 1997 NRC Canada 2048 Can. J. Bot. Vol. 75,1997

Table 1 (.concluded)

GenBank Species Hosl accession no Source"

Cortinarius stuntull (authors not cited) u ll9l7 Chapela et al. 1994 Entyloma calendulae (Oudem.) De Bary Calendulo fficinalis L not submitted Boekhout et al. 1995 Entyloma Jicariae (Cornu & Rose) Fischer v. Ficaria uerrrz Huds. not submitted Boekhout et al. 1995 Waldh. Eocronartium muscicola (Fr.) Fitz. (host not cited) L 20280 Bcrrcs et al. 1995 Ganoderma microsporum Hsel x 18179 Moncalvo et al. 199-5 Helicobasidium mompa Tanaka L 20281 Berres et al. 1995 Krie gerio eriophori Bres. (host not cited) L 20288 Berres et al. 1995 Marasmius delectans (authors not cited) u 11922 Chapela et al. 1994 MeLampsora /Inl (Ehrenb.) Desm. (host not cited) L 20283 Berres et at. 1995 Melanotaenium endogenum (Unger) De Bary Galium molLugo L. not submitted Boekhout ct al. 1995 Pachnoc.vbe Jbrrugirca (Sow. : Fr.) Berk. L 20284 Berres ct al. 1995 Puccinia g,raminis Pers. :Pers. (host not cited) L 08728 Bcrrcs et al. 1995 Puccinia recondita f .sp. tritici Huerta-Espino Triticum spp. L 08'729 Zambino and Szabo 1993 Russula mairei (atthors not cited) u I 1926 Chapela et al. 1994

Sept obas i d ium care st i0num Bres. L 20289 Berres et al. 1995 Tilletia caries (DC.) Tul. Triticum lestivum L. not subnritted Bockhout et al. 1995 Tilletiaria anonnla Bandoni & Johri not submitted Boekhout et al. 1995 Tremella ntesenterica Retz. in Hook. AF 0r 1570 M. Weill (unpublished data) Ustilago hordei (Pers.) Lagerh. (host not cited) L 20286 Berres et al. 1995 Ustilag,o rna-r'dls (DC.) Corda (host not cited) L 20281 Berres et al. 199-5

'H.U.V., Herbarium Ustilaginales Vänky: collection numbers: FO. F. Oberwinkler; GD. G. Dernlt MP. Meike Piepenbring: RB. R. Bauer sensus tree is shown in Fig 2. The topology resulting from joining analysis lower than 50% leave the phylogeny between neighbor-joining analysis is illustrated in Fig. 3. The results the orders of Exobasidiomycetidae unresolved. The grouping of the two analyses were similar and differed mainly in the of Melanotaeniwn endogenum and Melanotaenium euphor- position of the order Georgefischeriales. However, the posi- biae, and possible arrangements within the Georgefischeri- tion of Georgefischeriales is not supported with an approp- ales that are different between the two analyses will be riate bootstrap value in the neighbor-joining tree. In the discussed below. following, the principal topology of the two analyses is described in more details. Discussion Based on the topology in Fig. l, the trees were rooted with Entoruhlza; therefore Entonhiza forms a separate line- The three lineages shown in Fig. 1 are consistent with previ- age. Two lineages were found within the inner group. The ous molecular sequence analyses of basidiomycetes (Berbee first lineage representing the Ustilaginomycetidae is well and Taylor 1993; Swann and Taylor 1993, 1995 Berres supported by a bootstrap value of 90% (Fig. 3) and includes et al. 1995; Suh and Nakase 1995). The topology within the four groups of smut fungi. The first group in this lineage is Ustilaginomycetes illustrated in Fig. I differs in some details composed of 12 species of the Ustilaginales. The second from the results illustrated in Figs. 2 and 3. These differ- group includes Melanotaenium endogenum and Melano- ences may be founded in the different numbers of taxa used taenium euphorbiae. The third group is composed of Theca- in the respective analyses. Thus, in the analysis illustrated in phora amaranthi and Thecaphora seminis-convolvuli. The Fig. 1, the number of taxa is too small, or the amount of fourth group includes species ofthe Urocystaies. The second signal in the data set is too low, to interpret natural relation- iineage representing the Exobasidiornycetidae contains species ships among the Ustilaginomycetes. Within the Hymenomy- of Exobasidium, Graphiola phoenicis, Microstroma ju7- cetes, the three representatives of the heterobasidiomycetous landis, the presumptive smut Tilletiaria anom(tla and orders Tremellales, Dacrymycetales, and Auriculariales are an assortment of smut fungi. The groups that appear in the basal to the representatives of homobasidiomycetes. Except maximum parsimony analysis and that are well supported by for the common branch of Tremella mesenterica and bootstrap values in neighbor-joining analysis correspond to Calocera viscosa, the results illustrated for the Hymeno- the orders of the Exobasidiomycetidae recognized by Bauer mycetes in Fig. I are in agreement with the morphological et al. (1997). These are the Georgefischeriales, Entylo- and ultrastructural data (Oberwinkler 1985). However, the matales, Tilletiales, Microstromatales, Doassansiales, Graphi- differences between the Tremellales and Dacrymycetales. olales, and Exobasidiales. Except for the Graphiolales and e.g., in the septal pore apparatus (Oberwinkler 1985), as the Microstromatales, they are represented by three to five well as a bootstrap value lower than 50%, do not support a species. The differences in relationship ofthe orders between common origin of these two orders. Therefore, further the two analyses and the bootstrap values for the neighbor- studies with more taxa and longer sequences are necessary to

O 1997 NRC Canada Begerow et al. 2049

Fig. 1. Topology obtained by neighbor-joining analysis of LSU rDNA sequences of 30 basidiomycetes. Percentage bootstrap values of 1000 replicates are given at each furcation. Values smaller than 50% have not been taken into consideration.

Tremella mesenter

Georgelischeria riveae

Entyloma microsporum

Urocystis ranunculi

Cortinarius stuntzii - ,4 1

10K \., nn Kriegeria eriophorr

72 \ \ -- \ /i,/icrobotryum violaceum

Fulvisporium restifaciens septobasidium carestianum/ \ \\ \\

Eocronartium muscicola \] 00 Puccinia recoÄdita Melampsora lini

U rediniomycetes interpret the phylogeny of the Hymenomycetes. Within the rDNA are discussed predominantly with respect to the sys- Urediniomycetes, the previously studied taxa show the same tem proposed by Bauer et al. (1997). topology as presented in Berres et al. (1995). Depending on sampling and outgroup, any of the three Microbotryales and Urediniomycetes lineages may be regarded as basal group of basidiomycetes. This order is represented here by Aurantiosporium subnitens, This situation parallels results of other sequencing projects. Fulvisporium restifaciens (Vänky et al. 1997), since all three possible topologies have been published (Swann violac eum, and U sti I entyl oma Jlui t ans. The Microbotryales and Taylor 1993,19951' Berres et al. 1995). Thus. it is possi- are phytoparasitic, teliosporic, and dimorphic. Hosts are ble that the relationships among the three lineages cannot be monocots as well as dicots. They lack the interaction charac- resolved by this kind of sequence analysis. Certainly, ultra- ters of the Ustilaginomycetes (Bauer el aL 1991). In contrast structural data of septal pores and spindle pole body mor- to the phragmobasidiate members of the Ustilaginomycetes, phology and ontogeny suggest that the Urediniomycetes are they do not produce intracellular hyphae or haustoria (Bauer the basal group of the basidiomycetes and that the Ustilagino- et al. 1991). Blanz and Gottschalk (1984) and Deml (1987) mycetes represent the sister group of the Hymenomycetes suggested that the phragmobasidiate smut fungi occurring (e.g., see Bauer and Oberwinkler 1994; Wells 1994, and either on dicots or monocots constitute two phylogenetic references therein). Because of the unclear molecular situa- lines. Interestingly, the ultrastructural results of Bauer et al. tion, however, we have not used a simple septate or complex (1997) suggest a dichotomy of the Microbotryales separating septate fungus as outgroup for the analysis of the Ustilagino- the members occurring on dicots from those on monocots. mycetes. Entoruhiza is very distant to other basidiomycetes Thus, the members occurring on monocots have simple sep- in sequence analysis, but in Fig. l, Entorhiza represents the tal pores, whereas those occurring on dicots have poreless sister group of the rest of smut fungi. Therefore, this taxon septa. To verify this dichotomy with molecular data, further was used as a root tbr the analysis of the Ustilaginomycetes. studies on additional taxa are required. However, the mono- In the following sections, the results obtained with LSU phyly is supported by a bootstrap value of 100%.

(c) 1997 NRC Canada 2050 Can J. Bot. Vol. 15,1997

Fig. 2. Strict consensus tree of 18 most parsimonious trees (1 189 steps) of LSU rDNA sequences of 46 Ustilaginomvcetes. Topology was rooted with Entorrhiza spp. m f Entorrhiza aschersoniana entorrnizates o Entorrhiza casparyana I Exobasidium rhododendri Exobasidium rostrupii Exobasidium vaccinii lexooasioiares

Graphiola phoenicis lGraphiolales Doassansia hygrophilae m Doassansia epilobii I oor..rn.,r,". x o Entyloma callitrichis o) Rhamphospora nymphaeae I o9.. icrostromatales Microstroma juglandis lM 6' 3 Tilletia caries o Conidiosporomyces ayresii o d. Erratomyces patelii o- 1,,,,",,,,", §) Entyloma microsporum o Entyloma calendulae Entyloma polysporum Entylomatales Entyloma holwayi Entyloma ficariae Tolyposporella brunkii Entyloma dactylidis Georgef ischeria riveae Melanotaenium brachiariae Tilletiaria anomala Sporisorium sorghi Ustilago maydis Melanopsichium pennsylvanicum Ustilago cynodontis c U) Ustilago hordei +. §) Moesziomyces bullatus ginales e. Schizonella melanogramma o= Farysia chardoniana 3 c) Cintractia axicola o =.o_ Trichocintractia utriculicola g) Tolyposporium junci o Heterotolyposporium piluliforme Melanotaenium endogenum Me lanotaeniaceae Melanotaenium euphorbiae Mundkurella kalopanacis Urocystis colchici Urocystis ranunculi Urocystales Ustacystis waldsteiniae Doassansiopsis limnocharidis Doassansiopsis def ormans Thecaphora amaranthi Glomosporiaceae Thecaphora seminis-convolvuli

O 1997 NRC Canada Begerow et al. 2051

Fig. 3. Topology obtained by neighbor-joining analysis of LSU rDNA sequences of 46 Ustilaginomycetes. Topology was rootcd with Entorrhiza spp. Percentage bootstrap values of 1000 replicates are given at each furcation. Values smallcr than 50% have not been taken into consideration. m Entorrhiza aschersoniana f Entorrhiza casparyana lentorrnizates -o Exobasidium rhododendri f Exobasidium rostrupii lE*ooasioiat"s

Exobasidium vaccinii I

Graphiola phoenicis lGraphiolales Doassansia hygrophilae

I oo""."n.,","" Doassansia epilobii m Entyloma x callitrichis o Rhamphospora nymphaeae I 9) 9.. Microstroma juglandis lNlicrostromatales o- Entyloma microsporum ö' 3 Entyloma calendulae 7 Entylomatales o Entyloma holwayi o r00 -. polysporum o- Entyloma §) Entyloma ficariae o Tilletia caries Conidiosporomyces ayresii Erratomyces patelii 1,,,,",,,,". Tolyposporella brunkii Entyloma dactylidis Georgef ischeriales Georgef ischeria riveae Tilletiaria anomala Melanotaenium brachiariae Sporisorium sorghi Ustilago maydis Melanopsichium pennsylvanicum Moesziomyces bullatus Ustilago cynodontis (,C Ustilaginales Ustilago hordei g,=. Trichocintractia utriculicola e. Cintractia axicola o= Tolyposporium junci 3 o Heterotolyposporium pilulif orme o Schizonella melanogramma o_=. 9) Farysia chardoniana o Melanotaenium endogenum Melanotaenium euphorbiae Urocystis ranunculi Urocystis colchici

Ustacystis waldsteiniae Urocystales Mundkurella kalopanacis Doassansiopsis limnocharidis Doassansiopsis def ormans Thecaphora amaranthi Glomosporiaceae Thecaphora seminis-convolvuli

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Separation of the Microbotryales from the Ustilagino- (Fineran 1982). The great genetic distance implicating a mycetes and their position in the Urediniomycetes (Fig. 1) separated evolution over a long period may reflect the iso- are also supported by 55 rRNA, 18S rDNA, ultrastructural lated ecological niche of Entoruhiza species. features, and biochemical data (Blanz and Gottschalk 1984; Müller 1989; Prillinger et al. 1993; Swann and Taylor 1993, Ustilaginomycetidae 1995; Celerin et al. 1995; Bauer et al. 1997). Moreover, the A bootstrap value of 90% strongly supports the Ustilagino- Microbotryales share a specific cell wall carbohydrate com- mycetidae sensu Bauer et al. (1997). In contrast with the position, i . e. , dominance of mannose and presence of fucose, Entorrhizomycetidae and Exobasidiomycetidae, the mem- with other members of the Urediniomycetes (Prillinger et al. bers of the Ustilaginomycetidae interact with their respective 1993). In contrast with the Ustilaginomycetes and Hymeno- hosts by enlarged interaction zones (Bauer et al. 1991). mycetes, the 55 rRNA secondary structure of the Uredinio- Basidial morphology is variable within the Ustilaginomyceti- mycetes is of type A (Gottschalk and Blanz 1985; Müller dae. Hosts are monocots and dicots. Al1 species of Ustilagi- 1 989) . As discussed by Bauer et al. (1991) , within the Uredi- nomycetidae form teliospores. The results obtained from the niomycetes, the Microbotryales are closely related to a group LSU rDNA with the two different analyses differ in the posi- of potential or real mycoparasites possessing colacosomes. tion of Melanotaeniaceae. The members of this group are predominantly dimorphic and produce rhodotorulic as siderochrome (Deml 1987). acid Urocystales'. The Urocystales are represented by Doassansi- Analyses of 55 rRNA and 18S rDNA sequences support this op s i s d efo rman s, D o a s s an s i op s i s li mn o c hari di s, M undku r e I I a (Gottschalk 1987; line and Blanz 1985; Wolters Müller kalopanacis, Urocystis colchici, Urocystis ranuculi, and 1995). Our data indicate that the 1989; Swann and Taylor Ustacystis waldsteiniae. Mundkurella kalopanacis was not phytoparasite Kriegeria eriophori also belongs to unusual studied by Bauer et al. (1997); however, all other species are group (Fig. 1). this characterized by simple pores with membrane caps and non- membranous bands (Bauer et al. 1995a) and haustoria. The Ustilaginomycetes interaction of Ustacystis waldsteiniae was described in detail In contrast with the Urediniomycetes and Hymenomycetes, by Bauer et al. (1995b) and the same type was shown for almost members of the Ustilaginomycetes are known as all Doas sansiop sis defo rmans, Doas s ansi op sis limnocharidi s, phytoparasites. Because Tilletiaria anomala was discovered (Jrocystis ranunculi, and Urocltstis colchici (Bauer et ai. in culture (Bandoni and Johri 1972), its life strategy in nature 1997). Our analysis indicates a dichotomy within the Uro- phragmo- is still unknown. The Ustilaginomycetes contain cystales separating Doassansiopsis from the other members basidiate as weli as holobasidiate species. The members are (Figs. 2 and 3). Doassansictpsis spp. differ from the other present They usually dimorphic. Teliospores are or absent. members of the Urocystales in the presence of complex spore share type B of 55 rRNA secondary structure with the balls with a central mass of pseudoparenchymateous cells Hymenomycetes (Gottschalk and Blanz 1985; Müller 1989). surrounded by a layer of firmly adhered light colored telio- are charac- In terms of ultrastructure, the Ustilaginomycetes spores and an external cortex of sterile cells, and their occur- terized by a unique interaction type designated by Bauer rence on paludal or aquatic plants (Vänky 1987). The sister et al. (1991) as interaction with primary interactive vesicles. group is composed of diverse species. Mundkurella keilo- Glucose is the major cell wall carbohydrate component and panacis has several-celled spores (Vänky 1990) and develops xylose is absent (Prillinger et al. 1993). The short basal phragmobasidia (R. Bauer, unpublished data). The species of distances within the Ustilaginomycetes (Fig. 1) and the low (Jroc1'stis have spore balls usually composed of several dark short bootstrap values (Fig. 3) suggest radiation within a colored teliospores surrounded by sterile cells (Vänky 1987). time. But further analyses with more taxa and more sequence Ustacr-stis v,aldsteiniae differs from the species of Uroc,-vsrls necessary final conclusions are information are befbre any in the presence of phragmobasidia {Zundel 1945) and the parsimony analysis supports drawn. However, the maximum absence of sterile cells in the spore balls. Accordingly, LSU the subdivision of the Ustilaginomycetes into three lineages rDNA analysis shows the close relationship of phragmo- (Fig. 2) corresponding to the Entorrhizomycetidae, Ustilagi- basidiate and holobasidiate taxa. (Bauer et 1997). nomycetidae, and Exobasidiomycetidae al. Doassansiopsaceae and Urocystaceae can be distinguished The topology and arrangement of these groups (Fig. 2) as follows: matches the results obtained by ultrastructural analysis (Bauer et al. 1997). Doassansiopsaceae Begerow, Bauer et Oberrvinkler. fam.nov. Entorrhizomycetidae Fungi Urocystalium hypharum septis simpliciter perforatis, Entorrhizales: This order is represented by Entoruhiza poris fasciculis nonmembranaceis galeisque membranaceis aschersoniana and Entorrhiza casparl'ana. The basal posi- indutis, teliosporis incoloratis. tion of the species of Entonhiza in relation to the other Members of the Urocystales having simple septal pores members of the Ustilaginomycetes is well supported by with nonmembranous bands and membrane caps and color- genetic distances (Fig. 1), ultrastructure, morphology, and less teliospores. ecology (Bauer et al. 1997). Species of Entonhiza cause rypus FAMILrap.'. Doassansiopsis (Setchell) Dietel. in galls on roots of Juncaceae and Cyperaceae; the teliospores Engler and Prantl 1891:21. are produced in living host cells (Deml and Oberwinkler Urocystaceae Begerow, Bauer et Oberwinkler, fam.nov. 1981). and germinate internally by becoming four-celled Fungi Urocystalium hypharum septis simpliciter perforatis,

'O 1997 NRC Canada Begerow et al. 2053 poris fasciculis nonmembranaceis galeisque membranaceis (1994), Thecaphora is related to Glomosporium and Soro- indutis. teliosporis pigmentosis. sporium. Among the Ustilaginomycetes, these taxa are Members of the Urocystales having simple septal pores characterized by light brown spore balls containing only with nonmembranous bands and membrane caps and pig- teliospores. Glomosporium leptideum (H. & P. Sydow) mented teliospores. Kochman produces holobasidia (Kochman 1939). Spore rypus FAMTLT»p.: Uroct'stis Rabenhorst ex Fuckel. 1870: germination among the species of Thecaphora is variable, 41 (nomen conserv.). ranging from true holobasidia to aseptate or septate hyphae that sometimes bear "conidia" (Nagler 1986; Piepenbring Melanotaenium euphorbiae was not studied by Bauer and Bauer 1995). The spores of Sorosporium saponariae et al. (1997), but the monophyly of Melanotaenlun is sup- Rudolphi usually germinate with long hyphae in which the ported by a bootstrap value of 100% (Fig. 3). Melano- cytoplasm is located at the apex, where "conidia" develop germi- taenium endogenum shares with the Urocystales the presence occasionally (Ingold 1987). We interpret the hyphal of simple pores with membrane caps and haustoria (Bauer nation of these taxa as atypical germinations resulting tiom et al. 1991). Therefore, this species was ascribed to the nonoptimal environmental conditions. It is known that envi- Urocystales by Bauer et al. (1997). It differs from other ronmental conditions influence the germination pattern of members of the Urocystales in the lack of inner nonmem- smut fungi. For example, for Obenuinkleria annulata K. & C. branous bands in the pores. This type of pore apparatus was Vänky (Vänky and Bauer 1995) and Thecaphora haumanii interpreted by Bauer et al. (1997) as plesiomorphic for the Spegazzini (Piepenbring and Bauer 1995) both germination Ustilaginomycetidae. Melanotaenium endogenum was inter- types have been reported. Thus, Thecaphora, Sorosporium, preted by Bauer et al. (1997) as the basal taxon of the Uro- and Glomosporium can be interpreted as holobasidiate taxa. cystales and appears in the maximum parsimony analysis in Among the Ustilaginaceae sensu Bauer et al. (1997 ), exactly that position (Fig. 2), but not in the neighbor-joining analysis these three genera are characterized by holobasidia, whereas (Fig. 3). The position illustrated in Fig. 3 would mean that the other members are phragmobasidiate. As discussed above, the haustoria of Melanotaenium endogenum and the Uro- the LSU rDNA analysis indicates that Thecaphora stands cystales are a result of convergent evolution. The haustoria apart from the other members of the Ustilaginaceae. In inter- of M. endogenum are clearly of the Urocystales type (Bauer preting the morphological, ultrastructural, and LSU rDNA et al. 1997). Therefore, additional studies are necessary to situation, we use the described Glomosporiaceae in an clarify the discrepancy between the ultrastructural and emended form to separate Thecaphora, Glomosporium, and neighbor-joining analysis concerning MeLanotaeniurt species Sorosporium from the Ustilaginaceae. The Glomosporiaceae on dicots. Yet, both analyses reveal that Melemotaenium is a are tentatively placed in the Ustilaginales, More studies are polyphyletic and that a higher taxon for Melano- necessary to draw final conclusions about the affiliation. taenium spp. on dicots is justified. The family rank is there- fore proposed here. This family is placed in the Urocystales. Glomosporiaceae Ciferri 1963, emended Members of the Ustilaginomycetidae having poreless septa, Melanotaeniaceae Begerow, Bauer et Oberwinkler, fam. nov. intracellular hyphae, and light brown spore balls without Fungi Ustilaginomycetidarum hypharum septis simpliciter sterile elements. Spore germination results in holobasidia or perforatis, poris galeis membranaceis neque fasciculis indutis. in septate or aseptate hyphae. Members of the Ustilaginomycetidae having simple septal lf Glomosporium, Thecaphora, and Sorosporium are pores with membrane caps and without nonmernbranous excluded from the Ustilaginaceae, the morphological and bands. ecological characters of the Ustilaginaceae as defined by rypus FAMILtdE'. Melanotaenium de Bary, 1874: 105. Bauer et al. (1997) are more uniform. Thus, the members of the Glomosporiaceae parasitize dicots, whereas those of the predominantly occur on monocots. In addi- Ustilaginales: In the emended classification (Bauer et al. Ustilaginaceae the Glomosporiaceae are holobasidiate, whereas the 1997), the Ustilaginales are characterized by enlarged inter- tion, are phragmobasidiate. action zones and poreless septa. While this order is well Ustilaginaceae supported by a bootstrap value of 100% (Fig. 3), the rela- tionships among the members of the Ustilaginales remain Exobasidioml'cetidae unclear in the ultrastructural analysis (Bauer et al. 1997) as While the respective groups within the Exobasidiomycetidae well as in the LSU rDNA analysis (Fig. 3). Therefore, it is are well supported by high bootstrap values (88 - 100%), the obvious that the phylogenetic analysis of the Ustilaginales relationships among them are poorly resolved and inconsis- requires large scale DNA investigations in the future. tent in some details (compare Figs. 2 and 3). The unclear sit- Thecaphora was interpreted by Bauer et al. (1997) as one uation obtained fiom LSU rDNA analyses reflects the results of several basal taxa of the Ustilaginales. The position of of ultrastructural analysis (Bauer et al. 1997). The short Thecaphora obtained from LSU rDNA analysis (Figs. 2 and internal distances (Fig. 1) and the small bootstrap values 3) is different. The position of Thecaphora seminis-convoLvuli (Fig. 3) suggest that after their split from the Ustilaginomy- and Thecaphora amaranthi at the base of Urocystaceae and cetidae, the Exobasidiomycetidae separated rapidly into the Doassansiopsaceae was stable in every analysis. This posi- several groups. In contrast with maximum parsimony analy- tion suggests that intracellular hyphae and poreless septa of sis (Fig. 2), the Exobasidiomycetidae are not supported by Thecaphora and the Ustilaginaceae sensu Bauer et al. (1997) neighbor-joining analysis (Fig. 3). However, there is no are results of convergent evolution. According to Vänky statistical significance for the position of Georgefischeriales

-^ 1997 NRC Canada 2054 can. J Bot. Vol. 75.1997 illustrated in Fig. 3. In addition, in most analyses with dif- phylogenetic groups or the species with sori in leaves and ferent assortments of taxa, the Exobasidiomycetidae was those with sori in ovaries constitute two phylogenetic groups. consistent (not illustrated). In contrast with the Ustilagino- Sequence analyses of additional species are required before mycetidae, the Exobasidiomycetidae are characterized by any conclusion concerning the subclassification of the local interaction zones (Bauer et al. 1997'l. The local inter- Tilletiales may be drawn. action type was thought to be plesiomorphic for the Ustilagi- nomycetes by Bauer et al. (1997). Except for Tilletiaria Entylomatales: This order, represented by Entl'loma calen- anomala (Bandoni and Johri 1972), all analyzed members of dulae, E. ficariae, E. holwayi, E. microsporum, and E. pol.t- the Exobasidiomycetidae for which basidial morphology has sporum, comprises species with light colored teliospores of been studied possess holobasidia. In addition, among the Ent.yloma on dicots. A bootstrap value of 100% strongly species of the Exobasidiomycetidae, the hilar appendices of supports the Entylomatales (Fig. 3). Among the Exobasidio- forcibly discharged basidiospores are oriented adaxially. mycetidae, presence of simple interaction apparatus charac- Those of all other holobasidiate basidiomycetes forming bal- terizes the order (Bauer etal. 1997). The topology illustrated listosporic basidiospores are oriented abaxially (Bauer et al. in Fig. 3 with a long genetic distance of the group and short 1997). Teliospores are present or absent arnong the species distances between its species implies that a common ancestor of the Exobasidiomycetidae. of the Entylomatales separated recently into the several species. In other words, the group seems to be relatively old, Georgefischeruales: This order is represented by En4nloma whereas the several species seem to be relatively young. This dactylidis, Georg,efischeria riveae. Melanotaenium brachiar- may be reflected in the morphological situation. The mem- iae, Tilletiaria anornala, and Tol-tposporella brunkil and is bers of Entylomatales are holobasidiate, and light colored well supported by bootstrapping (Fig. 3). Among the sori appear generally in vegetative parts of the hosts. In fact, Exobasidiomycetidae, presence of poreless septa character- the species are difficult to distinguish from each other izes the Georgefischeriales (Bauer et al. 1997), while general (Vänky 1994). morphology and ecology are variable. Tilletiaria anomaltt forms phragmobasidia and was discovered in culture (Bandoni Microstromatales'. This order is represented by Microstroma and Johri 1972). The other species currently representing juglandis. It differs from other studied species in most this order possess holobasidia. Enty161*o dacrylidis, George- characters (Oberwinkler 1982). Among the Exobasidio- fischeria riveae, and Tilletiaria anomala form ballistosporic mycetidae, presence of simple pores and lack of interaction basidiospores, whereas the other taxa are gastroid. In addi- apparatus characterize the order (Bauer et al. 1997). The tion, Melanotaenium brachiariae develops a true yeast phase known species of Microstroma are holobasidiate and gastroid. in the haploid state, whereas the other taxa form a They lack teliospores like the Exobasidiales (Oberwinkler Tilletiopsis-l1ke pseudohyphal anamorph with ballistoconidia 1982) and have traditionally been placed in the Exobasidiales (see Bauer et al. 1997). Except for Georgefischeria riveae, (Hennings 1900). Mic:rostroma juglandis, however, differs they parasitize grasses and the sori appear in leaves. The two totally from the Exobasidiales in the mode of parasitism species of Georg,eJischeria occtr on Convolvuiaceae and (Bauer et al. 1997). There is no statistical significance for cause hypertrophy and development of witches' brooms the positions of Microstroma juglandis illustrated in Figs. 2 (Narasimhan et al. 1963). The inner topology of the George- and 3. fischeriales deduced from neighbor-joining analysis (Fig. 3) correlates with the family concept proposed by Bauer et al. Doassansiale.s: This order is represented by Entylomo calli- (1991), while the topology from the maximum parsimony tricltis, Doassansia epilobii, Doassansia hygrophilue, and analysis is not obviously related to morphological characters. Rhamphospora n-\mphaeae and is well supported by boot- LSU rDNA analyses (Figs. 2 and 3) as well as ultrastructural strapping (Fig. 3). A complex interaction apparatus includ- studies reveal that Erulloma and Melanotaenium arc poly- ing cytoplasmic compartments characterizes this order phyletic genera. (Bauer et al. 1997). The known species ofthe Doassansiales differ morphologically but have similar ecology. Rhampho- Tilletiales : This order is represented by Conidiosporom)'ces spora n.n-mphaeae and Entl-loma callitrichis produce single ayresii, Erratomyces patelii, and Tilletia caries . A bootstrap teliospores, whereas Doassetnsia species form complex value of 88% supports this order. Among the Exobasidio- structured spore balls (Vänky 1994). Hosts are of different mycetidae, the Tilletiales are characterized by the presence systematic positions. Ecologically, however, the species of of dolipores (Bauer et al. 1991). The known species of the the Doassansiales share occurrence on paludal or aquatic Tilletiales are similar, morphologically and with the excep- plants. They apparently evolved in the ecological niche of tion of Erratom\ces patelii (Piepenbring and Bauer 1997) aquatic plants. The inner topology of the Doassansiaies also ecologically. All species of the Tilletiales form holo- shows a dichotomy separating Rhamphospora n-\mphaeae basidia and hyphal anamorphs that produce ballistoconidia fiom the other species studied (Fig. 3). This correlates well (Bauer et al. 1997). Except for Erratoml'ces patelii, all with the ultrastructural data and is in agreement with the members occur on grasses and the sori appear predominantly family concept proposed by Bauer et al. (1997). In contrast in ovaries. The occurrence of Erratom-\ces pateLii on with the other species, Rhamphospora ny'mphaeae fbrms Leguminosae may be a result of parasite transfer from haustoria (Bauer et al. 1997). grasses to Leguminosae or vice versa. Two evolutionary trends within the Tilletiales are conceivable. Either the Graphiolales and Exobasidiales: The Graphiolales and species on grasses and those on Leguminosae constitute two Exobasidiales are represented by Graphbla phoeni<:is. Ero-

iar 1997 NRC Canada Begerow et al. 2055

basidium rhododendri, Exobasidium rostrupii, and Exo- genetic relationships in auriculariaceous basidiomycetes based basidium vaccinii, respectively. A bootstrap value of on 25S ribosomal DNA scquences. Mycologia, ST: 821-840. of 55 ribo- 100% supports the union of Graphiolales and Exobasidiales Blanz, P.A., and Gottschalk, M. 1984. A comparison sequences fiom smut fungi. System. (Fig. 3). This monophyly is congruent with ultrastructural somal RNA nucleotide Appl. Microbiol. 5: 518-526. analysis (Bauer et al. 1991) . The Exobasidiales and Graphio- Boekhout, T., Fell, J.W.. and O'Donnell, K. 1995. Molecular sys- lales are united in having complex interaction apparatus with tematics of some yeast-like anamorphs belonging to the Usti- interaction rings (Bauer et al. 1997). Interestingly, Gott- laginales and Tilletiales. Stud. Mycol.38: 175-183. (1985) sequence schalk and Blanz reported that the 55 rRNA Bruns, T.D., White, T.J., and Taylor, J.W. 1991. Fungal molecular of Graphiola phoenicis is identical to that of Erobasidium systematics. Annu. Rev. Ecol. Syst. 22: 525-564. vaccinii. Graphiola and Exobasidium lack teliospores, are Celerin, M., Day, A.W., Castle, A.J., and Laudenbach, D.E. holobasidiate, and the basidiospores become septate during 1995. A glycosylation pattern that is unique to fimbriae from the germination. Graphiola spp. have cup-shaped to cylindrical taxon Microbotryales. Can. J. Microbiol. 41 452-460. fruit bodies in which basidia are produced in chains between Chapela, 1.H., Rehner, S.A., Schultz, T.R., and Mueller, U.G. sterile hyphal strands (Oberwinkler et al. 1982), whereas in 1994. Evolutionary history of the symbiosis between fungus- Exobasidium, the basidia protrude from the intercellular growing ants and their fungi. Science (Washington, D .C .) ' 266: space directly to the surface of host organs (Oberwinkler t691 - t694. family (Ustilaginales). Riv. 1982). Significant morphological differences separate Cif'erri. R. 1963. The Glomosporiaceae Patol. Veg. 3: 141- 143. Graphiola and Exobasidium and may justify distinct orders. De Bary, A. 1874. Protoml'ces microsporus und seine Verwandten. Bot. Zeitung (Berlin), 32: 97 -108. Gonclusions Deml, G. 1987. of phragmobasidial smut fungi. Stud. Mycol. 30: 127 - 135. The results of our analysis correlate well with the system Deml, G., and Oberwinkler, F. 1981. Studies in Heterobasidiomy- proposed by Bauer et al. (1997). The traditional classifica- cetes. Part 4. Investigations on Entorrhiza casparyana by light tion mainly based on the morphology of basidia must be and electron microscopy. Mycologia, 83: 392-398. re-evaluated and requires a new interpretation of basidial Dietel, P. 1897. Hemibasidii (Ustilagineae und Tilletianae). 1n Die evolution. The groups of Ustilaginomycetes reported by natürlichen Pflanzenfamilien I. l++. Edited bv A. Engler and Bauer et al. (1997) are also evident in the phylogenetic K. Prantl, Leipzig, Germany. pp.2-24. hypothesis resulting from our analysis of diverse smut fungi Eclwards, K., Johnstone, C., and Thompson, C. 1991. A simple genomic DNA fbr and allied taxa. Four additional families are recognized; the and rapid mcthod for the preparation of plant Nucleic Acids Res. 19: 1349. Doassansiopsaceae, the Glomosporiaceae, the Melanotaenia- PCR analysis. 1985. Confidence limits on phylogenies: an ceae, and the Urocystaceae. Felsenstein, J. approach using the bootstrap. Evolution, 39: 783-'791 . Felsenstein, J. 1993. ruvlre (Phylogenetic Int-erence Package) Acknowledgments version 3.5c. Distributed by the author. Dcpartment of Genetics, University of Washington, Seattle, Wash. We thank Dr. Martin Grube, Dr. Meike Piepenbring, and Fineran, J.M. 1982. Teliospore germination in Entorrhi:a cd'\Pu^'atto and help- Michael Weiß for critically reading the manuscript (Ustilaginales). Can. J. Bot. 60: 2903-2913. Paul Blanz and Dr. Bernd Wissinger ful discussions; Dr. Fuckel, L. 1870. Symbolae mycologicae. Beiträge zur Kenntniss for the use of automated sequencers; Dr. Günther Deml, der rheinischcn Pilze. Jahrb. Nassauischen Vereins Naturk. Dr. Meike Piepenbring, and Dr. Kälmän Vänky for loan of 23-24: I -459. specimens; Michael Weiß for Latin diagnoses and loan of Gottschalk. M.. and Blanz, P.A. 1985. Untersuchungen an 55 sequences; Jacqueline Götze for technical assistance; and the ribosomalen Ribonukleinsäuren als Beitrag zur Klärung von Deutsche Forschungsgemeinschaft for financial support. Systcmatik und Phylogenic der Basidiomyceten. Z. Mykol. 51: 205 -243. Hennings, P. 1900. Exobasidiineae . In Die natürlichen Pflanzen- References familien 7, l**. Edited D1' A. Engler and K. Prantl. Leipzig, Cermany. pp. 103-105. Bandoni, R.J., and Johri, B.N. 1972. Tilletiarirz: a new genus in the Martin, 1992. Rapid identifica- Ustilaginales. Can. J. Bot. 50: 39-43. Henrion, 8.. Lc Tacon. F., and F. genetic variation of ectomycorrhizal fungi by amplifica- Bauer, R., and Oberwinkler, F. 1994. Meiosis, septal pore archi- tion of genes. New Phytol. 122: 289-298. tecture, and systematic position of the heterobasidiomycetous tion of ribosomal RNA 1987. Acrial sporidia of Ustilago hypodlres and fern parasite Herpobasidium Jilicinum. Can. J. Bot. 72: 1229 - Ingold, C.T. 1242. Sorosporium saponariae. Trans. Br. Mycol. Soc. 89:411-475. Bauer, R., Mendgen, K., and Oberwinkler, F. 1995a. Septal pore Kochman, J. 1939. Beitrag zur Kenntnis der Brandpilzflora Polens apparatus of the smut Ustacystis waldsteiniae. Mycologia, 87: II. Acta Soc. Bot. Pol. 16: 53-67. 18-24. Moncalvo, J.M., Wang, H.H., and Hscu, R.S. 1995. Phylogenetic Bauer, R., Mendgen, K., and Oberwinkler, F. 1995b. Cellular relationships in Ganociermtt inferred from the internal tran- interaction of the smut fungus Ustcr-l'stis waldsteiniae. Can. J. scribcd spacers and 25S ribosomal DNA sequences. Mycologia, Bot.73:867-883. 87:223-238. Bauer, R., Oberwinkler, F., and Vänky, K. 1997. Ultrastructural Müller, B. 1989. Chemotaxonomische Untersuchungen an Basidi- markers in smut fungi and allied taxa. Can. J. Bot.75: 1213- omycetenhefen. Ph.D. thesis, Lchrstuhl Spezielle Botanik und 1314. Mykologie, Universität Tübingen, Tübingen, Germany. Berbee, M.L., and Taylor, J.W. 1993. Dating the evolutionary Naglcr, A. 1986. Untersuchungen zur Gattungsabgrenzung von radiations of the true fungi. Can. J. Bot. 71: lll4-1127. Ginanniella Cit'erri und Urocl-stis Rabenhorst sowie zur Onto- Berres, M.8., Szabo, L.J.. and Mclaughlin, D.J. 1995. Phylo- gcnie von Thecaphora seminis-convoh,a/i (Desm.) Ito. Ph.D.

.cl 1aA? NPa (-qnedc 2056 can. J. Bot. Vol. 75,1997

thesis, Lehrstuhl Spezielle Botanik und Mykologie, Universität Suh, S.-O., and Nakase, T. 1995. Phylogenetic analysis ofthe bal- Tübingen, Tübingen, Germany. listosporous anamorphic genera Udeniomyces and Bullera, and Narasimhan, M.J., Thirumalachar, M.J., Srinivasan, M.C., and related basidiomycetous yeasts, based on 18S rDNA sequence. Govinda, H.C. 1963. GeorgefischerirT, a new genus of Microbiology (U.K.), 141: 901 -906. Ustilaginales. Mycologia, 55: 30-34. Swann, E.C., and Taylor, J.W. 1993. Higher taxa of basidiomy- Oberwinkler, F. 1977 . Das neue System der Basidiomyceten. 1n cetes: an 18S rRNA gene perspective. Mycologia, 85: 923- Beiträge zur Biologie der niederen Pflanzen. Edited by W. Frey, 936. H. Hurka, and F. Oberwinkler. G. Fischer Verlag, Stuttgart, Swann, E.C., and Taylor, J.W. 1995. Phylogenetic diversity of Germany. pp. 59- 105. yeast-producing basidiomycetes. Mycol. Res. 99: l2O5 -1212. Oberwinkler, F. 1982. The significance of the morphology of the Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. ClustalW: basidium in the phylogeny of basidiomycetes. 1n Basidium and improving the sensitivity of progressive multiple sequence basidiocarp. Edited by K. Wells and E.K. Wells. Springer- alignment through sequence weighting, position-specific gap Verlag, New York. pp. 10-35. penalties and weight matrix choice. Nucleic Acids Res. 22: Oberwinkler. F. 1985. Anmerkungen zur Evolution und Systematik 4673-4680. der Basidiomyceten. Bot. Jahrb. Syst. 107: 541-580. Tulasne, L., and Tulasne, C. 184'7. M6moire sur les Ustilagin6es Oberwinkler, F. 1987. Heterobasidiomycetes with ontogenetic compar6es Uredindes. Ann. Sci. Nat. Bot. 7: 12-121 . yeast-stages: systematic and phylogenetic aspects. Stud. Mycol. Vrinky, K. 1987. Illustrated genera of smut fungi. Cryptogam. 30 6l-'74. stud. l: 1-159. Oberwinkler, F., Bandoni, R.J., Blanz, P., Deml, G., and Vänky, K. 1990. The genus Mundkurella (Ustilaginales). Mycol. Kisimova-Horovitz, L. 1982. Graphiolales: basidiomycetes Res. 94: 269-273. parasitic on palms. Plant Syst. Evol. 140: 251 -2'7'7. Vänky, K. 1994. European smut fungi. Gustav Fischer Verlag, Piepenbring, M., and Bauer, R. 1995. Noteworthy germinations of Stuttgart, Germany. some Costa Rican Ustilaginales. Mycol. Res. 99: 853-858. Vänky, K., and Bauer, R. 1995. Oberwinkleria, a ncw genus of Piepenbring, M., and Bauer, R. 1997. Erratomyces, a new genus Ustilaginales. Mycotaxon, 53: 361 -368. of Tilletiales with species on Leguminosae. Mycologia. 89: Vänky, K., Bauer, R., and Begerow, D. 1991 . Fulvisporium. anew 924-936. genus of Ustilaginales. Mycotaxon, 64: 57 *66. Prillinger, H., Oberwinkter, F., Umile, C., Tlachac, K., Wells, K. 1994. Jelly fungi, then and now! Mycologia, 86: l8-48. Bauer, R., Dörfler, C., and Taufratzhofer, E. 1993. Analysis of Wolters, J. 1987. Phylogenie der Organismen und Zellorganellen: cell wall carbohydrates (neutral sugars) from ascomycetous and kladistische Strukturanalysen ribosomaler RNAs. Ph.D. thesis, basidiomycetous yeasts with and without derivatization. J. Gen. Fachbereich Chemie, Freie Universität Berlin, Berlin, Germany. Appl. Microbiol. 39: 1-34. Zambino, P.J., and Szabo, L.J. 1993. Phylogenetic relationships of Qu, L.-H., Nicoloso, M., and Bachellerie, J.-P. 1988. Phylo- selected cereal and grass rusts based on rDNA sequences analy- genetic calibration of the 5' terminal domain of the large rRNA sis. Mycologia, 85: 401-414. achieved by determining twenty eucaryotic sequences. J. Mol. Zundel, G.L. 1945. A change in generic name. Mycologia, 37 Evol. 28: ll3-124. 795 -796. Saitou, N., and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.

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