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Phylogenetic studies on large subunit riboso-mal

DNA sequences of smut fungi and related taxa.
Can J Bot
ARTICLE in CANADIAN JOURNAL OF BOTANY FEBRUARY 2011
Impact Factor: 1.4 DOI: 10.1139/b97-916

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Phylogenetic studies on nuclear large subunit

ribosomal DNA sequences of smut fungi and
related taxal

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Dominik Begerow, Robert Bauer, and Franz Oberwinkler

Abstract: To show phylogenetic relationships among the smut fungi and their relatives, we sequenced a part of the
nuclear LSU rDNA from 43 different species of smut fungi and related taxa. Our data wcrc 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 septate 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, Ustilaginomycetes,
and Hyrnenon~yceteswas confirmed and two groups of smut fungi appeared. The smut genera Airrctrztiosporiurrz,
Microbotryurn, Ful\~isporinm,and Ustiler~tylorr1aare members of the Urediniomycetcs, whereas the other smut species
tested are members of the Ustilaginomycetes with Er~torrlzizaas a basal taxon. The second set of 4 6 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
Entorrlriza species. The Ustilaginomycetidae contain at least two groups. the Urocystales and Ustilaginales. The
Exobasidiomycetidae include five orders, i.e., Doassansiales, Entylomatales, Exobasidiales, Georgefischeriales, and
Tilletiales, and Grnphiola pl~oenicisand Microstrornn jrcglarldis. Our results support a classification mainly based on
ultrastructure. The description of the Glomosporiaceae is emended. The Doassansiopsaceae, Melanotaeniaceae. and
Urocystaceae are proposed as new taxa.

Key words: basidiomycete systematics, LSU rDNA, Microbotryales, n~olecularphylogeny, smut fungi, Ustilaginomycetes.

R6sum6 : Afin de dCmontrer les relations phylogCnCtiques parmi les champignons du charbon et espkces apparenties,
les auteurs ont sCquencC une partie de la grande sous-unit6 nuclCaire de I'ADN ribosomique (LSU rDNA), chez 43 espkces
diffkrentes de cha~npignonsdu charbon et taxons apparentCs. Les donnCes ont CtC combinCes avec les sequences dCji
connucs de sept champignons du charbon et 17 autres basidiomycktes. Ils ont analysC deux groupes dc sequences. Le
premier comporte un nombre representatif de basidiomycktes i septations sinlples, de basidiomycktes septations
con~plexeset de champignons du charbon, en utilisant la mCthode de jonction avec les voisins pour estimer la topologie
gCnCrale de la phylogCnie des basidioniycktes et les positions des champignons du charbon. La subclassification tripartite
des basidiomycktes en UrCdinomycktes, Ustilagoinonlycktes et HyrnCnomycktes est confirniCe et on dttecte deux groupes
de champignons du charbon. Les champignons du charbon des genres Aurantiosporiuttz, Microbotryrcrn, F~rlvisporiumet
Ustilet1tylort1osont membres des Uredinomycktes, alors que les autres espkces examinees sont membres des Ustilaginomyc6tes,
les Er~torrl~izn
Ctant le taxon de base. Le second groupe de 4 6 Ustilaginomycktes a CtC analysC en utilisant les mCthodes
de jonction avec les voisins et de parscimonie maximum pour illustrer la topologie interne des UstilaginomycCtes. Les
rCsultats montrent trois IignCes majeures parmi les Ustilaginomycktes, lesquelles correspondent aux Entorrhizomycetidae,
Exobasidiomycetidae et Ustilaginomycetidae. Les Entorrhizomycetidae sont reprCsentCs par les espkces d'Errtorrl~iza.
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 que le
Graphioln phoerlicis et le Microstrornn jrcglar~dis. Les resultats des auteurs supportent la classification basee
principalement sur les ultrastructures. La description des Glomosporiaceac est amendCe et les auteurs proposent les
Doassansiopsaceae, les Melanotaeniaceae et les Urocystaceae comme nouveaux taxons.

Mots elks : systematique des basidiomycktes, LSU rDNA, Microbotryales, phylogCnie niolCculaire, champignons du
charbon, Ustilaginomycktes.
[Traduit par la rCdaction]
Received February 3, 1997.

D. Begerow,' R. Bauer, and F. Oberwinkler. Universitat Tiibingen, Institut fur Biologie I, Lehrstuhl Spezielle Botanik und
Mykologie, Auf der Morgenstelle I, D-72076 Tiibingen, Germany.

I'

Part 141 of the series "Studies in Heterobasidiomycetes".

Author to whom all correspondence should be addressed. e-mail: doniinik.begerow@uni-tuebingen.de

Can. J. Bot. 75: 2045-2056 (1997)

1997 NRC Canada

Can. J. Bot. Vol. 75, 1997

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introduction
A new system of smut fungi and allied taxa based on ultrastructural studies was recently proposed by Bauer et al. (1997).
This system differs significantly from the traditional classification characterized by a basic dichoton~y separating the
phragmobasidiate Ustilaginaceae or Ustilaginales from the
holobasidiate Tilletiaceae or Tilletiales (e.g., Tulasne and
Tulasne 1847; Oberwinkler 1977, 1987). In the system of
Bauer et al. (1997) six genera of smut fungi are grouped in
the Microbotryales, whereas the other smut fungi are placed
together with the Exobasidiales, Graphiolales, and Microstromatales in the Ustilaginomycetes. Owing to the profound
differences between traditional concepts and the system proposed by Bauer et al. (1997), studies with independent
markers are required to test the classifications and to develop
phylogenetic hypotheses.
Analyses of molecular sequence data have made important contributions to the understanding of basidiomycete
phylogeny (e.g., Berbee and Taylor 1993; Swann and Taylor
1993, 1995; Berres et al. 1995; Boekhout et al. 1995).
Unfortunately, sequence data from smut fungi are available
only from a few species (Blanz and Gottschalk 1984: Swann
and Taylor 1993, 1995; Boekhout et al. 1995; Berres et al.
1995). However, the previous results indicated the existence
of two distantly related groups of smut fungi. Because the
existing molecular sequence data base is too small to identify
natural groups among the smut fungi, we sequenced the 5 '
end of the large subunit of riboson~alD N A (LSU rDNA) of
38 smut fungi and five presumptively related species as a
semiconserved region (Qu et al. 1988; Bruns et al. 1991) to
validate the system proposed by Bauer et al. (1997). Data
sets were completed with the sequences of seven further smut
fungi and 17 other basidiomycetes from literature.

Materials and methods

Species studied for molecular analysis arc listed in Table I. DNA
was isolatcd from cultu~.esor herbarium specin~ensfollowing a
modified version of the SDS method of Edwards et al. (1991) and
Henrion et al. (1992): samplcs were ground in liquid nitrogcn and
incubated in 500 pL extraction buffer. for I h at 65C. The extracts
werc centrifuged at 13.793 x g for 15 min and thc supernatant was
transferred to a new Eppendorf tube. RNA was digested with 10 U
RNAse at 37C for I h. DNA was precipitated by addition of
400 pL isopropanol and LO pL 3-M sodium acetate bcfore centrifugation. After a washing step with 70% ethanol. the pellet was dricd
at room tcmpcraturc and dissolvcd in sterile water.
The 5' region of the nuclear LSU rDNA was arnplificd using the
polymerase chain reaction (PCR) with NLI and NL4 as primcrs
(Bockhout ct al. 1995). Thc PCR product was purified using thc
QIAquickT" Kit (QIAGEN) according to thc manufacturer's protocol. This dsDNA was scquenccd dircctly using the ABI PRISM'"'
Dyc Tcrrninator Cycle Sequencing Kit (Perkin Elmer) and an automated scqucnccr (ABI 373, Perkin Elmer).
An alignment was produccd using thc ClustalW program (Thompson ct al. 1994). Thc sequence data were analyzed with thc PI-IYLIP
packagc version 3 . 5 (Felsenstein
~
1993). We used neighbor-joining
and inaximum parsimony methods to reconstruct phylogcnctic trces
froill LSU rDNA scqucnces.
Neighbor-joining analysis (Saitou and Nei 1987) of a distancc
matrix produccd by DNAdist (Kin~ura2-paran~ctcrmodel, transition to transversion ratc: 2.0) was applicd with thc default parainctcrs of the program. Bootstrapping (Felsenstcin 1985) was made

of 1000 replicates using ScqBoot and Consens of thc P H Y L I P package with default paramctcrs (Fclsenstcin 1993). Maximum parsimony analysis was performed by DNApass (jumble option: 100)
and Consens. The alignments are available upon request.

Results
Sequence alignments
Two sets of sequences were analyzed for phylogenetic discussion of smut fungi and allied taxa. The first set with
sequences of 3 0 basidiomycetes was analyzed to determine
the general topology of the basidiomycetes, the positions o f
smut fungi within this basidiomycetous topology and the root
of the ~ ~ t i l a g i n o m ~ c e t The
e s . second set with sequences o f
4 6 species was used to show the phylogenetic relationships
within the Ustilaginomycetes. The total consensus length o f
the first set was 518 bp, whereas the second set was 527 b p
long. Although two small regions, positions 15-28 and
465 -473 of the alignment in the first set, 3 3 -45 and 462 47 1 in the second set, respectively, included several ambiguous positions owing to gap position, any manipulations were
avoided to achieve highest reproducibility.
Phylogenetic analysis of the first set
W e analyzed a representative number of simple septate
basidiomycetes, complex septate basidiomycetes. and smut
fungi with allied taxa. Results of neighbor-joining analysis
are shown in Fig 1. The three previously recognized classes
of basidiomycetes were confirmed. It was not the aim of this
paper to study the origin of basidiomycetes. Analyses with
several ascomycetes and zygomycetes as outgroups led to
different arrangements of the three groups and Ei1tori.lli:~
was not always included in the Ustilaginomycetes (data not
shown). Thus, the origin of basidiomycetes 111ust be placed
somewhere close to the basal trifi~rcation.T h e first group,
representing the Hymenomycetes. contains the heterobasidiomycetes T r e i n ~ l l ineserlterica.
~
Ccrlocercr viscosrr, and
Auric~llaria c ~ ~ ~ r i c ~ ~ 1and
~ ~ the
j ~ r homobasidioinycetes
lr~e~
Gculodeuiln rnio-ospor~orl,R~l.ssulr~
irlaii.ei, Boletus i.ubiilc~lllw,
C o r t i i l ~ r i ~ l~st ~ i t l t z i iM
, ~ ~ C I S I I Id~eI lI eSc t ~ ~ and
t ~ ~ ,Agaricus
urvetlsis. The second lineage representing the Urediniomy cetes is coinposed of the rusts (Melarrz~~.sora
liili, P~lccirlia
g'niniizi.~,and P~lccirliarccoi~rlita)together with some related
fungi (Helicol>asirli~~m
irlorr1pcr, Eouoncrrti~lin ~ ~ ~ ~ [ . s c i e o l e ~ ,
and Septoba.sidilltil care.stianlliiz), and Kriegeriu wiophori at
the base of the smut fungi (Microbot~lutilviolacc~~o~i,
Fulvisl~orilimi.est(facierz.s, A u r c r r ~ t i o s l ~ o r subr1iteils,
i~~i~~
and Ustilrrltylornci jl~litarls). The third lineage, representing the
Ustilaginomycetes, contains Esobasirli~lrn~~uccirlii
and the
smut fungi Eiltorrhizcr crsche~sorliaila,Erltori.lliza caspai:varlrr,
Urocystis mn~olculi,Tl~ecnplzomseinirlis-coilvolvll(i, Ustilago
hot-dei, Eiltylonla i11icro.s1>or~rrtl, Gcorgefisclzeria riverrc,
Tilletia caries, and Doasselrl.sicl c)pilobii. Whereas the {irst
lineage contains only complex septate basidiomycetes. the
other two lineages both contain smut fungi. Bootstrap values
support the Hylnenomycetes and Urediniomycetes but not
the Ustilaginomycetes, thus in this analysis the monophyly of
the Ustilaginomycetes is indicated but remains uncertain.

Phylogenetic analyses of the second set

This set contained sequences of 4 6 species. The 18 most
parsin~onioustrees required 1189 steps and their strict con-

1997 NRC Canada

Begerow et al

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Table 1. List of species studied in ~nolecularanalysis.

GenBank
accession no.

Species

Host

Source"

Aurrrrztiosporirr,n srrbrziterzs (Schroter & Henn.)

M. Piepenbr., K. Vinky & Oberw
Citztmctin tzxicola (Berk.) Cornu
Corzitlios~~ororrzyces
nyresii (Berk.) K. Vinky
Dorrssnrzsin epilobii Farlow
Donssrrrzsin hygroplziltre Thirun~.
Docrssnr~siopsisrleforrnnrzs (Setch.) D~etel
Donssnrz~iopsislirr~rzochcrrir/is(Cif.) K. Vinky
Erztorrlzizcr nscllersorlinrzn (Magnus) Lagerh.
Erztorrllizcr ccrspnrynrla (Magnus) Lagerh
Erz~yIorrz~
crrllitricllis Liro
Erzrylorrzn tlrrctylidis (Pass.) Cif.
E~zplo~rzn
hohinyi H. & P. Sydow
Entylo~rzcr~rzicrosporrr~rz
(Unger) Schroter
E~ztylo~rztr
pol~~spor~trtz
(Peck) Farlow
Errrrto~nycespntelii (Pavgi & Thirum.)
M. Piepenbr. & R. Bauer
Exobnsirli~r~rz
rlzorlodenclri (Fuck.) Cram.
Exobnsidirr~rzrostrrrpii Nannf.
Esobasirlirr~nvrrccinii (Fuck.) Woronin
Fn~ysiacl~nrrlonin~zn
Zundel
Frrlvisporirr~nrestifrciens (D.E. Shaw) K . Vinky
Georgefisclzeria riveae Thirum. & Naras.
Grnphiolrr phoenicis (Moug.) Poiteau
Melnnopsichirr~nperr~zsylvnrrrcrrr~~
Hirschh.
Me/crrzotne~zirrr~z
brnclzinrine Viegas
Melnrzotnerzi~r~rr
e~rphorbine(Lenz) M.D.
Whitehead & Thirum.
Microbotryuttz violnce~r~n
(Pers.:Pers.) Deml &

Sclerin ~nelnle~lcn
Reichb. ex Schlecht
& Cham.
Firnbristylis tetrrrgorzn R. Br.
Prrnicrrtrl rr~r~virrz~rrrz
Jacq.
El~ilobirrrrzrrzorzttrrzrrm L.
Hygroplziltr spirzosn T. Anders
Sngittnritr Irrrrceolrrttr L. ssp. Itrtzcifolitr
Lirnrzoc1znri.s flcrvn (L.) Buchenau
Jrrrlcrrs brrforzirrs L.
Jrrrzcrrs crrticrrlntrrs L.
Ctrllitriclze stcrgrzcr1i.s Scop.
Agrostis stolorzifercr L.
Cosrrzo~cerrr(1rtrrs H. B.K.
Rarz~rrzcrrlrrsrepens L.
Arrzbro~innrterr~isi~fi~lin
L.
Plznseolrrs \lrrlgaris L.

H.U.V. 17460
H.U.V. 15197
FO 38252
H.U.V. 15474
MP 2066
H.U.V. 15198
H.U.V. 15899
H.U.V. 17623
RB 1079
RB 915
MP 1769
FO 37329
H.U.V. 2960
MP 1991

Rhorlorle~zrlro~z
jkrrrrgbzerrmr L.
Vrrccinirr~rrozrycoccos L.
Vrrccinirr~rlvitis-irkren L.
Cares polystnchyn Swartz ex Wahlenb.
Stipn strrposn Hughes
Riven h~pocrnterifor~nis
Chois
Phoenix cnnnriensis Chaub.
Polygonrr~r~
Iris~~irlrr~n
H.B.K.
Brtrcl~inrinrlistnclzytr (L.) Stapf
Euplzorbin genicrrlnrer (KI . & Garke)

RB 2050
RB 949
RB 945
MP 2062
H.U.V. 17637
H.U.V. 15614
FO 29350
MP 801
H.U.V. 17510
H.U.V. 17733

Ortega
Gypsoplliln repens L.

Oberw.
Microst~~o~rrn
jrrglan(1i.s (Bereng.) Sacc.
Moeszio~rzyccsb~r1lrrtu.s(Schroter) K. Vinky
M~rr~tlkurella
knlopn~zncisK. Vinky
Rhn~nplzosporany~~zphaene
D. D. Cunn.
Sclzizo~~elln
~ ~ z e l n ~ ~ o g r n(DC.)
~ n ~ nSchroter
n
Heterotolyposporirrr~zpilrrlifornze (Berk.)

Jrrg1er1z.s regin L.
Pns~~al~orr
clisticlz~r~n
L.
Kn/oi~ntzc~v
pictus (Thumb.) Nakai
Ny~r~plrnea
nlba L.
Crlres pilr~liferaL.
Ju~rcuspln~zifoli~rsR. Br.

AF
AF
AF
AF
AF
AF

009867
009868
009869
007526
009870
00987 1

F 0 392 1 1
H.U.V. 15514
H.U.V. 16732
RB 862
FO 37 174
H.U.V. 15732

Sorglzum bicolor (L.) Moench

A~nnrtrnttzllsIlybrid~rsL.
C o r z v o l v ~ ~anvnsis
l~~s
L.
A~zrlropogo~z
sacd~crroiclesSwartz
J ~ r n c ~brdotzilr.~
~s
L.

AF
AF
AF
AF
AF

009872
009873
009874
009875
009876

MP 2036a
H.U.V. 15882
GD 1391
H.U.V. 17816
H.U.V. 17169

K. Vinky
Sporisoriumz sorghi Ehrenb. ex Link
Tlzecaphorr~nnznrcrtztki (Hirsch.) K . Vinky
Tlzecnplzom .serr~irlis-corzvolv~tli(Desm.) Ito
Tol),posporelln brlrrzkii (Ell. & Gall.) Clinton
Tol),posporilrnz jurzci (Schroter) Woronin ex

Schroter
Triclzocintrnctin rrtric~~licola
(Henn.)

Rh)~nclzosporr~
cor~3111bosn
(L.) Britton

M . Piepenbr.
Urocystis colcl~ici(Schlecht.) Rabenh.
Urocystis rnnurzc~~li
(Lib.) Moesz.
Ustncystis ~r~nlclstei~zine
(C.H. Beck) Zundel
Ustilago cy~zoclo~ztis
(Henn.) Henn.
U.stile~ztylo~nn
fluitnns (Liro) K. Vinky

Colclzic~rtnnutu~rznnleL.
Ranrrnc~rl~rs
repens L.
Wrdrlsteinin geoirles Wild.
CJ~IIOC~OIZ
~Irrcty1011
(L.) Pers.
Glycerin plicntn (Fr.) Fr.

Species sequenced from other authors

Agaricus nrvensis (authors not cited)
A~rric~~lcrricr
nrrricrrln7jrrrlc~e(Bull.:Fr.) Schroter
B o l e t ~ ~rlrbinellris
s
Peck
Ct~locernviscosn (Pers. ex Fr.) Fr.

U11910
L 20278
L 20279
AF 01 1569

Chapelaetal.1994
Berres et al. 1995
Berres et al. 1995
M. Wcill (unpublished data)
O 1997 NRC Canada

2048

Can. J. Bot. Vol. 75. 1997

Table 1 (cot~clitderl)
.
Spccics

Cortit~nrirrsst~itlt~ii
(authors not cited)
Entylottrrr caler~clrrlne(Oudem.) Dc Bary
Et~tj~lotrra
,ficrrriae (Cornu & Rose) Fischer v.

GcnBank
accession no.

Host

Crrlet~il~ilrc
oficitlnlis L.
Ficrrriri \ 8 e r ~Huds.
~n

Sourcc"

U 11917
not submitted
not submitted

Chapcla ct al. 1994

Boekhout ct al. 1995
Bockhout ct al. 1995

L 20280
X 78779
L 2028 1
L 20288
U I1922
L 20283
not submitted
L 20284
L 08728
L 08729
U 11926
L 20289
not subniittcd
not subrnittcd
AF 01 1570
L 20286
L 20287

Bcrrcs ct al. 199.5

Moncalvo ct al. 1995
Berrcs et al. 1995
Berrcs et al. 1995
Chapela ct al. 1994
Bcrrcs et al. 1995
Bockhout ct al. 1995
Bcrrcs ct al. 1995
Bcrrcs ct al. 1995
Zambino and Szabo 1993
Chapcla et al. 1994
Bcrrcs ct al. 1995
Bockhout ct al. 1995
Boekhout ct al. 1995
M. Wcili (unpublishcd data)
Berrcs et al. 1995
Berrcs ct al. 1995

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For personal use only.

Waldh.

Eoont~r~rtirrtti
ttr~rscicola(Fr.) Fitz.
Grit~odennntrricrosporutn Hscu
Helicobnsidirrtt~ttlotnprr Tanaka
Kt-irgerin erioptlori Brcs.
Mrrrrrstnirrs rlelectrrt~s(authors not cited)
Mrlnrt~psomlitli (Ehrenb.) Dcsm.
Melnt~otnet~irrtn
et~dogetr~rnr
(Unger) Dc Bary
Prrdrt~oc~be
ferrrrgit~err (Sow.: Fr.) Berk.
P~rccit~ia
grrrtninis Pcrs. :Pcrs.
Prrccit~inremndita f.sp. tritici Huerta-Espino
Rrissuln tnnirei (authors not cited)
Septobnsirli~rtncnrr.stiat~rrtt~Brcs.
Tilletia caries (DC.) Tul.
Tilletinrin nt~otnrrlrrBandoni & Johri
Tretnella tt~esetitet-icnRctz. in Hook.
Ustilr~gohordei (Pers.) Lagcrh.
Ustilrrgo tnnyrlis (DC.) Corda

(host not cited)

(host not cited)

(host not cited)

Grrlirrtn tnoll~rgoL.
(host not cited)
Triti~.~rtt~
spp.

Triticrrtt~nesti17~rttiL.

(host not citcd)

(host not cited)

"H.U.V., Herbarium Ustilaginales Vrinky; collection numbers: FO. F. Obcrwinklcr; GD. G. Deml: MP. Mcikc Picpenbring; RB. R. Baucr.

sensus tree is shown in Fig 2. The topology resulting from

neighbor-joining analysis is illustrated in Fig. 3. The results
of the two analyses were similar and differed mainly in the
position of the order Georgefischeriales. However, the position of Georgefischeriales is not supported with an appropriate bootstrap value in the neighbor-joining tree. In the
following, the principal topology of the two analyses is
described in more details.
Based on the topology in Fig. 1, the trees were rooted
with Etztorrhiza; therefore ~ t z t o 6 h i z aforms a separate lineage. Two lineages were found within the inner group. The
first lineage representing the Ustilaginomycetidae is well
supported by a bootstrap value of 90% (Fig. 3) and includes
four groups of smut fungi. The first group in this lineage is
composed of 12 species of the Ustilaginales. The second
etzdogetuit~~and Melanogroup includes Melanotc~eniu~~z
taet~iurneuphorbiae. The third group is composed of Thecaphora arnaranthi and Thecaphorn seminis-convolvuli. The
fourth group includes species of the Urocystales. The second
lineage representing the Exobasidiomycetidae contains species
of Exobasidium, Graphiola phoetzicis, Microstroma juglattdis, the presumptive smut fungus Tilletiaria atzotnala and
an assortment of smut fungi. The groups that appear in the
maximum parsimony analysis and that are well supported by
bootstrap values in neighbor-joining analysis correspond to
the orders of the Exobasidiomycetidae recognized by Bauer
et al. (1997). These are the Georgefischeriales, Entylomatales, Tilletiales, Microstromatales, Doassansiales, Graphiolales, and Exobasidiales. Except for the Graphiolales and
the Microstromatales, they are represented by three to five
species. The differences in relationship of the orders between
the two analyses and the bootstrap values for the neighbor-

joining analysis lower than 50% leave the phylogeny between

the orders of Exobasidiomycetidae unresolved. The grouping
of Melanotaetziunz e ~ z d o g e t z ~and
~ ~ nMelatzotaeniutt~ euphorbiae, and possible arrangements within the Georgefischeriales that are different between the two analyses will be
discussed below.

Discussion
The three lineages shown in Fig. 1 are consistent with previous molecular sequence analyses of basidiomycetes (Berbee
and Taylor 1993; Swann and Taylor 1993, 1995; Berres
et al. 1995; Suh and Nakase 1995). The topology within the
Ustilaginomycetes illustrated in Fig. 1 differs in some details
from the results illustrated in Figs. 2 and 3. These differences may be founded in the different numbers of taxa used
in the respective analyses. Thus, in the analysis illustrated in
Fig. 1, the number of taxa is too small, or the amount of
signal in the data set is too low, to interpret natural relationships among the Ustilaginomycetes. Within the Hymenomycetes, the three representatives of the heterobasidiomycetous
orders Tremellales, Dacrymycetales, and Auriculariales are
basal to the representatives of homobasidiomycetes. Except
for the common branch of Trenzella mesetzterica and
Calocerzl viscosa, the results illustrated for the Hymenomycetes in Fig. 1 are in agreement with the morphological
and ultrastructural data (Oberwinkler 1985). However, the
differences between the Tremellales and Dacrymycetales,
e.g., in the septa1 pore apparatus (Oberwinkler 1985), as
well as a bootstrap value lower than 5076, do not support a
common origin of these two orders. Therefore, further
studies with more taxa and longer sequences are necessary to
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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 srnaller than 50% have not been taken into consideration.

.
.

.
.

-.

... ..
.-..

Urediniomycetes
interpret the phylogeny of the Hymenomycetes. Within the
Urediniomycetes, the previously studied taxa show the same
topology as presented in Berres et al. (1995).
Depending on sampling and outgroup, any of the three
lineages may be regarded as basal group of basidiomycetes.
This situation parallels results of other sequencing projects,
since all three possible topologies have been published (Swann
and Taylor 1993, 1995; Berres et al. 1995). Thus, it is possible that the relationships among the three lineages cannot be
resolved by this kind of sequence analysis. Certainly, ultrastructural data of septa1 pores and spindle pole body morphology and ontogeny suggest that the Urediniomycetes are
the basal group of the basidiomycetes and that the Ustilaginomycetes represent the sister group of the Hymenomycetes
(e.g., see Bauer and Oberwinkler 1994; Wells 1994, and
references therein). Because of the unclear molecular situation, however, we have not used a simple septate or complex
septate fungus as outgroup for the analysis of the Ustilaginomycetes. Erztorrlziza is very distant to other basidiomycetes
in sequence analysis, but in Fig. 1, Erzrorrhiza represents the
sister group of the rest of smut fungi. Therefore, this taxon
was used as a root for the analysis of the Ustilaginomycetes.
In the following sections, the results obtained with LSU

rDNA are discussed predominantly with respect to the system proposed by Bauer et al. (1997).

Microbotryales and Uredinioniycetes

This order is represented here by Aurarztiosporium subrzitens,
F~ilvisporizrmrestifnciens (VBnky et al. 1997), Microbotryurn
violaceurn, and Usrilerz~lomajluitarzs. The Microbotryales
are phytoparasitic, teliosporic, and dimorphic. Hosts are
monocots as well as dicots. They lack the interaction characters of the Ustilaginomycetes (Bauer et al. 1997). In contrast
to the phragmobasidiate members of the Ustilaginomycetes,
they do not produce intracellular hyphae or haustoria (Bauer
et al. 1997). Blanz and Gottschalk (1984) and Deml (1987)
suggested that the phragmobasidiate smut fungi occurring
either on dicots or monocots constitute two phylogenetic
lines. Interestingly, the ultrastructural results of Bauer et al.
(1997) suggest a dichotomy of the Microbotryales separating
the members occurring on dicots from those on monocots.
Thus, the members occurring on monocots have simple septal pores, whereas those occurring on dicots have poreless
septa. To verify this dichotomy with molecular data, further
studies on additional taxa are required. However, the monophyly is supported by a bootstrap value of 100%.
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Fig. 2. ltrict consensus tree of 18 most parsimonious trees (1 189 steps) of LSU rDNA sequences of 46 Ustilaginomycetes. Topology
was rol :d with Gltorrlzizu spp.

Entorrhiza aschersoniana
Entorrhiza casparyana
Exobasidiurn rhododendri
Exobasidiurn rostrupii
Exobasidiurn vaccinii
Graphiola phoenicis
Doassansia hygrophilae
Doassansia epilobii
Entylorna callitrichis
Rharnphospora nyrnphaeae
Microstrorna juglandis
Tilletia caries
Conidiosporornyces ayresii
Erratornyces patelii
Entylorna rnicrosporurn
Entylorna calendulae
Entylorna polysporurn
Entylorna holwayi
Entylorna ficariae
Tolyposporella brunkii
Entylorna dactylidis
Georgefischeria riveae
Melanotaeniurn brachiariae
Tilletiaria anornala
Sporisoriurn sorghi
Ustilago rnaydis
Melanopsichiurn pennsylvanicurn
Ustilago cynodontis
Ustilago hordei
Moesziornyces bullatus
Schizonella rnelanograrnrna
Farysia chardoniana
Cintractia axicola
Trichocintractia utriculicola
Tolyposporiurn junci
Heterotolyposporiurn piluliforrne
Melanotaeniurn endogenurn
Melanotaeniurn euphorbiae
Mundkurella kalopanacis
Urocystis colchici
Urocystis ranunculi
Ustacystis waldsteiniae
Doassansiopsis lirnnocharidis
Doassansiopsis deforrnans

:I;:;:I:

Entorrhizales

T
I$

Exobasidiales

l~raphiolales

Doassansiales

9
a-

~~icrostromatales

g.a

Tilletiales

%
II
D,
(D

Entylomatales

Georgefischeriales

g.
Ustilaginales

D,

e.
3

=.
II
(D

D,
(D

Melanotaeniaceae

Urocystales

Glomosporiaceae

~~:~~l",onvolvuli

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2051

Fig. 3. Topology obtained by neighbor-joining analysis of LSU rDNA scquences of 46 Ustilaginomycetes. Topology was rooted with
Enrorrhiza spp. Percentage bootstrap values of 1000 replicates are given at each furcation. Values smaller than 50% have not been
taken into consideration.
Entorrhiza aschersoniana
Entorrhiza casparyana
Exobasidium rhododendri
Exobasidium rostrupii
Exobasidium vaccinii
Graphiola phoenicis
Doassansia hygrophilae
Doassansia epilobii
Entyloma callitrichis
Rhamphospora nymphaeae
Microstroma juglandis
Entyloma microsporum
Entyloma calendulae
78
- Entyloma holwayi
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100

99

100

99

88

Erratomyces patelii
Tolyposporella brunkii
Entyloma dactylidis
Georgefischeria riveae
Tilletiaria anomala
Melanotaenium brachiariae
Sporisorium sorghi
Ustilago maydis
Melanopsichium pennsylvanicum

--

100

Doassansiales

Entylornatales

Moesziomyces bullatus
Ustilago cynodontis
Ustilago hordei
Trichocintractia utriculicola

Tilletiales

Georgefischeriales

Ustilaginales

--&Cintractia

axicola
Tolyposporium junci
Heterotolyposporium piluliforme
Schizonella melanogramma
Farysia chardoniana
Melanotaenium endogenum

-I

80

loo

1-

Exobasidiales

Entyloma polysporum
Entyloma ficariae
Tilletia caries
Conidiosporomyces ayresii

Entorrhizales

Melanotaenium euphorbiae
Urocystis ranunculi
Urocystis colchici
Ustacystis waldsteiniae

971

loo

Mundkurella kalopanacis
Doassansiopsis limnocharidis
Doassansiopsis deformans
Thecaphora amaranthi
Thecaphora seminis-convolvuli

Melanotaeniaceae

Urocystales

Glornosporiaceae

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Can. J . Bot. Vol. 75, 1997

Separation of the Microbotryales from the Ustilaginomycetes and their position in the Urediniomycetes (Fig. 1)
are also supported by 5 s rRNA, 18s rDNA, ultrastructural
features, and biochemical data (Blanz and Gottschalk 1984;
Muller 1989; Prillinger et al. 1993; Swann and Taylor 1993,
1995; Celerin et al. 1995; Bauer et al. 1997). Moreover, the
Microbotryales share a specific cell wall carbohydrate composition, i.e., dominance of mannose and presence of fucose,
with other members of the Urediniomycetes (Prillinger et al.
1993). In contrast with the Ustilaginomycetes and Hymenomycetes, the 5 s rRNA secondary structure of the Urediniomycetes is of type A (Gottschalk and Blanz 1985; Muller
1989). As discussed by Bauer et al. (1997), within the Urediniomycetes, the Microbotryales are closely related to a group
of potential or real mycoparasites possessing colacosomes.
The members of this group are predominantly dimorphic and
produce rhodotorulic acid as siderochrome (Deml 1987).
Analyses of 5 s rRNA and 18s rDNA sequences support this
line (Gottschalk and Blanz 1985; Wolters 1987; Muller
1989; Swann and Taylor 1995). Our data indicate that the
unusual phytoparasite Kriegeria erioplzori also belongs to
this group (Fig. 1).

Ustilaginornycetes
In contrast with the Urediniomycetes and Hymenomycetes,
almost all members of the Ustilaginomycetes are known as
phytoparasites. Because ~illetiariaanornalc~was discovered
in culture (Bandoni and Johri 1972), its life strategy in nature
is still unknown. The Ustilaginomycetes contain phragmobasidiate as weli as holobasidiate species. The members are
usually dimorphic. Teliospores are present or absent. They
share type B of 5 s rRNA secondary structure with the
Hymenomycetes (Gottschalk and Blanz 1985; Muller 1989).
In terms of ultrastructure, the Ustilaginomycetes are characterized by a unique interaction type designated by Bauer
et al. (1997) as interaction with primary interactive vesicles.
Glucose is the major cell wall carbohydrate component and
xylose is absent (Prillinger et al. 1993). The short basal
distances within the Ustilaginomycetes (Fig. 1) and the low
bootstrap values (Fig. 3) suggest radiation within a short
time. But further analyses with more taxa and more sequence
information are necessary before any final conclusions are
drawn. However, the maximum parsimony analysis supports
the subdivision of the Ustilaginomycetes into three lineages
(Fig. 2) corresponding to the Entorrhizomycetidae, Ustilaginomycetidae, and Exobasidiomycetidae (Bauer et al. 1997).
The topology and arrangement of these groups (Fig. 2)
matches the results obtained by ultrastructural analysis
(Bauer et al. 1997).

Ewtorrhizales: This order is represented by E~ztorrhiza

aschersorliana and Entorrhiza caspatyana. The basal position of the species of Entorrhizn in relation to the other
members of the Ustilaginomycetes is well supported by
genetic distances (Fig. l ) , ultrastructure, morphology, and
ecology (Bauer et al. 1997). Species of Entorrhiza cause
galls on roots of Juncaceae and Cyperaceae; the teliospores
are produced in living host cells (Deml and Oberwinkler
1981), and germinate internally by becoming four-celled

(Fineran 1982). The great genetic distance implicating a

separated evolution over a long period may reflect the isolated ecological niche of Entorrlziza species.

Ustilaginomycetidne
A bootstrap value of 90% strongly supports the Ustilaginomycetidae sensu Bauer et al. (1997). In contrast with the
Entorrhizomycetidae and Exobasidiomycetidae, the members of the Ustilaginomycetidae interact with their respective
hosts by enlarged interaction zones (Bauer et al. 1997).
Basidial morphology is variable within the Ustilaginomycetidae. Hosts are monocots and dicots. All species of Ustilaginomycetidae form teliospores. The results obtained from the
LSU rDNA with the two different analyses differ in the position of Melanotaeniaceae.
Urocy.stales: The Urocystales are represented by Donssnnsiopsis cleformans, Donssansiopsis limnochnridis, Mundk~~rella
kalopanacis, Urocystis colchici, Urocystis rcmuculi, and
Usracysris waldstei~liae.Mundkurella kalopanacis was not
studied by Bauer et al. (1997); however, all other species are
characterized by simple pores with membrane caps and nonmembranous bands (Bauer et al. 1995a) and haustoria. The
interaction of Ustacystis waldsteiniae was described in detail
by Bauer et al. (19956) and the same type was shown for
Doassansiopsis deforma~zs,Doassnnsiopsis limwocharidis,
Urocystis rc~nunculi,and Urocysris colchici (Bauer et al.
1997). Our analysis indicates a dichotomy within the Urocystales separating Doassansiopsis from the other members
(Figs. 2 and 3). Doassmzsiopsis spp. differ from the other
members of the Urocystales in the presence of complex spore
balls with a central mass of pseudoparenchymateous cells
surrounded by a layer of firmly adhered light colored teliospores and an external cortex of sterile cells, and their occurrence on paludal or aquatic plants (VBnky 1987). The sister
kalogroup is composed of diverse species. M~azdkurellc~
parzacis has several-celled spores (Vrinky 1990) and develops
phragmobasidia (R. Bauer, unpublished data). The species of
Urocysris have spore balls usually composed of several dark
colored teliospores surrounded by sterile cells (Vanky 1987).
Ustacysris ,\'aldsteiniae differs from the species of Urocystis
in the presence of phragmobasidia (Zundel 1945) and the
absence of sterile cells in the spore balls. Accordingly, LSU
rDNA analysis shows the close relationship of phragmobasidiate and holobasidiate taxa.
Doassansiopsaceae and Urocystaceae can be distinguished
as follows:

Doassansiopsaceae Begerow, Bauer et Oberwinkler.

fam .nov .
Fungi Urocystalium hypharum septis simpliciter perforatis,
poris fasciculis nonmembranaceis galeisque membranaceis
indutis, teliosporis incoloratis.
Members of the Urocystales having simple septa1 pores
with nonmembranous bands and membrane caps and colorless teliospores.
TY PUS F A M I L I A E : Doassansiopsis (Setchell) Dietel, in
Engler and Prantl 1897: 2 1.
Urocystaceae Begerow, Bauer et Oberwinkler, fam.nov.
Fungi Urocystalium hypharum septis simpliciter perforatis,
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poris fasciculis nonmembranaceis galeisque membranaceis

indutis, teliosporis pigmentosis.
Members of the Urocystales having simple septal pores
with nonmembranous bands and membrane caps and pigmented teliospores.
TYPUS FAMILIAE:
Urocystis Rabenhorst ex Fuckel, 1870:
4 1 (nomen conserv.).

Melatzotaetzium euphorbiae was not studied by Bauer

is supet al. (1997), but the monophyly of Melc~notaeni~~rn
ported by a bootstrap value of 100% (Fig. 3). Melanotaetzi~ltnetzdogen~lmshares with the Urocystales the presence
of simple pores with membrane caps and haustoria (Bauer
et al. 1997). Therefore, this species was ascribed to the
Urocystales by Bauer et al. (1997). It differs from other
members of the Urocystales in the lack of inner nonmembranous bands in the pores. This type of pore apparatus was
interpreted by Bauer et al. (1997) as plesiomorphic for the
Ustilaginomycetidae. Melanotaerziurn endogetz~lmwas interpreted by Bauer et al. (1997) as the basal taxon of the Urocystales and appears in the maximum parsimony analysis in
that position (Fig. 2), but not in the neighbor-joining analysis
(Fig. 3). The position illustrated in Fig. 3 would mean that
the haustoria of Melanotaerziurn endogetzzlm and the Urocystales are a result of convergent evolution. The haustoria
of M. erldogenum are clearly of the Urocystales type (Bauer
et al. 1997). Therefore, additional studies are necessary to
clarify the discrepancy between the ultrastructural and
neighbor-joining analysis concerning Melanotaerziurn species
on dicots. Yet, both analyses reveal that Melarzotaetziutn is a
polyphyletic genus and that a higher taxon for Melatzotaenium spp. on dicots is justified. The family rank is therefore proposed here. This family is placed in the Urocystales.
Melanotaeniaceae Begerow, Bauer et Oberwinkler, fam.nov.
Fungi Ustilagino~ilycetidarum hypharum septis simpliciter
perforatis, poris galeis membranaceis neque fasciculis indutis.
Members of the Ustilaginomycetidae having simple septal
pores with membrane caps and without nonmembranous
bands.
TYPUS FAMILIAE: Melatzotaetzi~~trz
de Bary, 1874: 105.
Ustilaginales: In the emended classification (Bauer et al.
1997), the Ustilaginales are characterized by enlarged interaction zones and poreless septa. While this order is well
supported by a bootstrap value of 100% (Fig. 3), the relationships among the members of the Ustilaginales remain
unclear in the ultrastructural analysis (Bauer et al. 1997) as
well as in the LSU rDNA analysis (Fig. 3). Therefore, it is
obvious that the phylogenetic analysis of the Ustilaginales
requires large scale DNA investigations in the future.
Thecaphora was interpreted by Bauer et al. (1997) as one
of several basal taxa of the Ustilaginales. The position of
Thecaphora obtained from LSU rDNA analysis (Figs. 2 and
3) is different. The position of Tlzecaphora setninis-convolv~ili
and Thecaphora amaranthi at the base of Urocystaceae and
Doassansiopsaceae was stable in every analysis. This position suggests that intracellular hyphae and poreless septa of
Thecaphora and the Ustilaginaceae sensu Bauer et al. (1997)
are results of convergent evolution. According to VBnky

(1994), Thecaphora is related to Glomosporium and Sorosporiutn. Among the Ustilaginomycetes, these taxa are
characterized by light brown spore balls containing only
teliospores. Glomosporiutn leptideum (H. & P. Sydow)
Kochman produces holobasidia (Kochman 1939). Spore
germination among the species of Thecaphora is variable,
ranging from true holobasidia to aseptate or septate hyphae
that sometimes bear "conidia" (Nagler 1986; Piepenbring
and Bauer 1995). The spores of Sorosporiutn saponariae
Rudolphi usually germinate with long hyphae in which the
cytoplasm is located at the apex, where "conidia" develop
occasionally (Ingold 1987). We interpret the hyphal germination of these taxa as atypical germinations resulting from
nonoptimal environmental conditions. It is known that environmental conditions influence the germination pattern of
smut fungi. For example, for Oberwinkleria annulata K. & C .
VBnky (VBnky and Bauer 1995) and Thecaphora lzaumanii
Spegazzini (Piepenbring and Bauer 1995) both germination
types have been reported. Thus, 771ecaphora,Sorosporium,
and Glotnosporiz~tncan be interpreted as holobasidiate taxa.
Among the Ustilaginaceae sensu Bauer et al. (1997), exactly
these three genera are characterized by holobasidia, whereas
the other members are phragmobasidiate. As discussed above,
the LSU rDNA analysis indicates that Thecaphora stands
apart from the other members of the Ustilaginaceae. In interpreting the morphological, ultrastructural, and LSU rDNA
situation, we use the described Glomosporiaceae in an
emended form to separate Recaphora, Glotnosporiilm, and
Sorosporiutn from the Ustilaginaceae. The Glomosporiaceae
are tentatively placed in the Ustilaginales. More studies are
necessary to draw final conclusions about the affiliation.

Glornosporiaceae Ciferri 1963, emended

Members of the Ustilaginomycetidae having poreless septa,
intracellular hyphae, and light brown spore balls without
sterile elements. Spore germination results in holobasidia or
in septate or aseptate hyphae.
If Glotnosporiurn, Thecaplzora, and Sorosporiutn are
excluded from the Ustilaginaceae, the morphological and
ecological characters of the Ustilaginaceae as defined by
Bauer et al. (1997) are more uniform. Thus, the members of
the Glomosporiaceae parasitize dicots, whereas those of the
Ustilaginaceae predominantly occur on monocots. In addition, the Glomosporiaceae are holobasidiate, whereas the
Ustilaginaceae are phragmobasidiate.
Exobasicliotnycetidae
While the respective groups within the Exobasidiomycetidae
are well supported by high bootstrap values (88 - loo%), the
relationships among them are poorly resolved and inconsistent in some details (compare Figs. 2 and 3). The unclear situation obtained from LSU rDNA analyses reflects the results
of ultrastructural analysis (Bauer et al. 1997). The short
internal distances (Fig. 1) and the small bootstrap values
(Fig. 3) suggest that after their split from the Ustilaginomycetidae, the Exobasidiomycetidae separated rapidly into the
several groups. In contrast with maximum parsimony analysis (Fig. 2), the Exobasidiomycetidae are not supported by
neighbor-joining analysis (Fig. 3). However, there is no
statistical significance for the position of Georgefischeriales
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Can. J . Bot. Vol. 75, 1997

illustrated in Fig. 3. In addition, in most analyses with different assortments of taxa, the Exobasidiomycetidae was
consistent (not illustrated). In contrast with the Ustilaginomycetidae, the Exobasidiomycetidae are characterized by
local interaction zones (Bauer et al. 1997). The local interaction type was thought to be plesioinorphic for the Ustilaginomycetes by Bauer et al. (1997). Except for Tilletiaria
anomala (Bandoni and Johri 1972), all analyzed members of
the Exobasidiomycetidae for which basidial morphology has
been studied possess holobasidia. In addition, among the
species of the Exobasidiomycetidae, the hilar appendices of
forcibly discharged basidiospores are oriented adaxially.
Those of all other holobasidiate basidiomycetes forming ballistosporic basidiospores are oriented abaxially (Bauer et al.
1997). Teliospores are present or absent among the species
of the Exobasidiomycetidae.

Georgejischeriales: This order is represented by Etltylotnrr

dactylidis, Georgejischeria riverre, Melanotaetziutn brachiariae, Tilletiaria anotnala, and Tolyposporella brutzkii and is
well supported by bootstrapping (Fig. 3). Among the
Exobasidiomycetidae, presence of poreless septa characterizes the Georgefischeriales (Bauer et al. 1997), while general
morphology and ecology are variable. Tilletiaria anotnala
forms phragmobasidia and was discovered in culture (Bandoni
and Johri 1972). The other species currently representing
dactylidis, Georgethis order possess holobasidia. Et~lylott~a
jischeria riveae, and Tilletiaria anotnala form ballistosporic
basidiospores, whereas the other taxa are gastroid. In addition, Melanotaetziutn brachiariae develops a true yeast phase
in the haploid state, whereas the other taxa form a
Tilletiopsis-like pseudohyphal anamorph with ballistoconidia
(see Bauer et al. 1997). Except for Geor-gejischeria riveae,
they parasitize grasses and the sori appear in leaves. The two
species of Georgejiscl~eria occur on Convolvulaceae and
cause hypertrophy and development of witches' brooins
(Narasimhan et al. 1963). The inner topology of the Georgefischeriales deduced from neighbor-joining analysis (Fig. 3)
correlates with the family concept proposed by Bauer et al.
(1997), while the topology from the maximum parsimony
analysis is not obviously related to morphological characters.
LSU rDNA analyses (Figs. 2 and 3) as well as ultrastructural
studies reveal that Entylotna and Melanotaeniutn are polyphyletic genera.
Tilletiales: This order is represented by Conidiosj>orotnyces
ayresii, Erratomyces patelii, and Tilletia caries. A bootstrap
value of 88% supports this order. Among the Exobasidiomycetidae, the Tilletiales are characterized by the presence
of dolipores (Bauer et al. 1997). The known species of the
Tilletiales are similar, morphologically and with the exception of Erratomyces patelii (Piepenbring and Bauer 1997)
also ecologically. All species of the Tilletiales form holobasidia and hyphal anamorphs that produce ballistoconidia
(Bauer et al. 1997). Except for Erratotnyces patelii, all
members occur on grasses and the sori appear predominantly
in ovaries. The occurrence of Erratotnyces patelii on
Leguminosae may be a result of parasite transfer from
grasses to Leguminosae or vice versa. Two evolutionary
trends within the Tilletiales are conceivable. Either the
species on grasses and those on Leguminosae constitute two

phylogenetic groups or the species with sori in leaves and

those with sori in ovaries constitute two phylogenetic groups.
Sequence analyses of additional species are required before
any conclusion concerning the subclassification of the
Tilletiales may be drawn.

Et~~lotnatales:
This order, represented by Etltylotna caletzdulae, E. jicariae, E. holvvrlyi, E. tnicrospor?irn, and E. polyspouitn, comprises species with light colored teliospores of
Et~tylon~a
on dicots. A bootstrap value of 100% strongly
supports the Entylomatales (Fig. 3). Among the Exobasidiomycetidae, presence of simple interaction apparatus characterizes the order (Bauer et al. 1997). The topology illustrated
in Fig. 3 with a long genetic distance of the group and short
distances between its species implies that a common ancestor
of the Entyloinatales separated recently into the several
species. In other words, the group seems to be relatively old,
whereas the several species seem to be relatively young. This
may be reflected in the morphological situation. The members of Entylomatales are holobasidiate, and light colored
sori appear generally in vegetative parts of the hosts. In fact,
the species are difficult to distinguish from each other
(Vdnky 1994).
Microstrotnatales: 'This order is represented by Microstrot~~a
juglandis. It differs from other studied species in most
characters (Oberwinkler 1982). Among the Exobasidiomycetidae, presence of simple pores and lack of interaction
apparatus characterize the order (Bauer et al. 1997). The
known species of Microstrortza are holobasidiate and gastroid.
They lack teliospores like the Exobasidiales (Oberwinkler
1982) and have traditionally been placed in the Exobasidiales
(Hennings 1900). Micmstmtna juglatzrlis, however, differs
totally from the Exobasidiales in the modc of parasitism
(Bauer et al. 1997). There is no statistical significance for
the positions of Microstroma juglrrtzrlis illustrated in Figs. 2
and 3.
Doassutzsiales: This order is represented by Etztylotna callitrichis, Doassatzsia epilobii, Doassatlsin hygrophilrre, and
Rhatnphospora tzytnphaeae and is well supportcd by bootstrapping (Fig. 3). A conlplex interaction apparatus including cytoplasmic compartments characterizes this order
(Bauer et al. 1997). The known species of thc Doassansiales
differ morphologically but have similar ecology. Rhrrtnphospora t~ytnphaeaeand Etztylotna callitrichis produce single
teliospores, whereas Doassansia species form complex
structured spore balls (VBnky 1994). Hosts are of different
systematic positions. Ecologically, however, the species of
the Doassansiales share occurrence on paludal or aquatic
plants. They apparently evolved in the ecological niche of
aquatic plants. The inner topology of the Doassansiales
shows a dichotonly separating Rhatnphosporn t~ytnphaeae
from the other species studied (Fig. 3). This correlates well
with the ultrastructural data and is in agreement with the
family concept proposed by Bauer et al. (1997). In contrast
with the other species, Rhatnj7hosj70rcr nytnphaeae forms
haustoria (Bauer et al. 1997).
Graphiolales atzrl Exobasirliales: The Graphiolales and
Exobasidiales are represented by Graphiol~rphoenicis, Exo@ 1997 NRC Canada

Begerow et al.

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basidium rhododendri, Exobasidiunz rostr-upii, and Exobasidiurn vaccirzii, respectively. A bootstrap value of
100% supports the union of Graphiolales and Exobasidiales
(Fig. 3). This monophyly is congruent with ultrastructural
analysis (Bauer et al. 1997). The Exobasidiales and Graphiolales are united in having complex interaction apparatus with
interaction rings (Bauer et al. 1997). Interestingly, Gottschalk and Blanz (1985) reported that the 5s rRNA sequence
of Graphiola phoenicis is identical to that of Exobasidiunz
vaccinii. Graplziola and Exobasidium lack teliospores, are
holobasidiate, and the basidiospores become septate during
germination. Graphiola spp. have cup-shaped to cylindrical
fruit bodies in which basidia are produced in chains between
sterile hyphal strands (Oberwinkler et al. 1982), whereas in
Exobasidiunz, the basidia protrude from the intercellular
space directly to the surface of host organs (Oberwinkler
1982). Significant morphological differences separate
Graplziola and Exobasidiurn and may justify distinct orders.

Conclusions
The results of our analysis correlate well with the system
proposed by Bauer et al. (1997). The traditional classification mainly based on the morphology of basidia must be
re-evaluated and requires a new interpretation of basidial
evolution. The groups of Ustilaginomycetes reported by
Bauer et al. (1997) are also evident in the phylogenetic
hypothesis resulting from our analysis of diverse smut fungi
and allied taxa. Four additional families are recognized; the
Doassansiopsaceae, the Glomosporiaceae, the Melanotaeniaceae, and the Urocy staceae.

Acknowledgments
W e thank Dr. Martin Grube, Dr. Meike Piepenbring, and
Michael WeiO for critically reading the manuscript and helpful discussions; Dr. Paul Blanz and D r . Bernd Wissinger
for the use of automated sequencers; Dr. Giinther Deml,
Dr. Meike Piepenbring, and Dr. Kilman Vinky for loan of
specimens; Michael WeiO for Latin diagnoses and loan of
sequences; Jacqueline Gotze for technical assistance; and the
Deutsche Forschungsgemeinschaft for financial support.

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