Mycologia, 97(4), 2005, pp. 888–900.
q 2005 by The Mycological Society of America, Lawrence, KS 66044-8897
Phylogenetic analysis of Tilletia and allied genera in order Tilletiales
(Ustilaginomycetes; Exobasidiomycetidae) based on large subunit
nuclear rDNA sequences
Lisa A. Castlebury1
supported clades corresponding to host subfamily.
The results of this work suggest that morphological
characters used to segregate Neovossia, Conidiosporomyces and Ingoldiomyces from Tilletia are not useful
generic level characters and that all included species
can be accommodated in the genus Tilletia.
Key words: Conidiosporomyces, Erratomyces, germination, Ingoldiomyces, molecular systematics, Neovossia, smut and bunt fungi
USDA ARS Systematic Botany and Mycology
Laboratory, 10300 Baltimore Avenue, Beltsville,
Maryland 20705-2350
Lori M. Carris
Department of Plant Pathology, Washington State
University, Pullman, Washington 99164–6430
Kálmán Vánky
Herbarium Ustilaginales Vánky, Gabriel-Biel-Str. 5, D72076 Tübingen, Germany
INTRODUCTION
Abstract: The order Tilletiales (Ustilaginomycetes,
Basidiomycota) includes six genera (Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria and Tilletia) and approximately 150 species. All
members of Tilletiales infect hosts in the grass family
Poaceae with the exception of Erratomyces spp.,
which occur on hosts in the Fabaceae. Morphological
features including teliospore ornamentation, number and nuclear condition of primary basidiospores
and ability of primary basidiospores to conjugate and
form an infective dikaryon were studied in conjunction with sequence analysis of the large subunit nuclear rDNA gene (nLSU). Analysis based on nLSU
data shows that taxa infecting hosts in the grass subfamily Pooideae form one well supported lineage.
This lineage comprises most of the reticulate-spored
species that germinate to form a small number of
rapidly conjugating basidiospores and includes the
type species Tilletia tritici. Two tuberculate-spored
species with a large number of nonconjugating basidiospores, T. indica and T. walkeri, and Ingoldiomyces
hyalosporus are also included in this lineage. Most of
the species included in the analysis with echinulate,
verrucose or tuberculate teliospores that germinate
to form a large number (.30) of nonconjugating
basidiospores infect hosts in the subfamilies Panicoideae, Chloridoideae, Arundinoideae and Ehrhartoideae. This group of species is more diverse than the
pooid-infecting taxa and in general do not form well
The genus Tilletia Tul. & C. Tul. comprises ca. 140
species restricted to hosts in the grass family (Poaceae) and is the largest genus in order Tilletiales
(Basidiomycota, Ustilaginomycetes, Exobasidiomycetidae) (Vánky 2002). Tilletia is characterized by the
formation of pigmented teliospores intermingled
with hyaline sterile cells, and in most species the teliospores are formed in host ovaries. Teliospore ornamentation ranges from reticulate, echinulate, verrucose, tuberculate to smooth. In many species teliospore masses have a fetid, herring brine odor due to
the production of trimethylamine. Teliospores germinate to form an aseptate basidium, frequently with
multiple retraction septa, and a terminal whorl of aerial primary basidiospores (FIG. 1). The type species,
T. tritici, produces 8–12 filiform to narrowly falcate
monokaryotic basidiospores (Goates 1996). Most of
the basidiospores conjugate while attached to the basidium to form an ‘‘H-body,’’ giving rise to dikaryotic
mycelium that infects host plants at seedling stage,
resulting in a systemic infection (Vánky 1994). A second type of germination pattern, consisting of the
production of large numbers of nonconjugating primary basidiospores, is found in species of Neovossia
Körn., Conidiosporomyces Vánky (Vánky and Bauer
1992) and some species of Tilletia, such as T. indica,
T. horrida and T. walkeri (Castlebury and Carris 1999,
Durán 1987). Oberwinkleria Vánky & R. Bauer produces nonconjugating primary basidiospores (Vánky
and Bauer 1995) but their nuclear condition was not
reported.
Five of six genera in Tilletiales, Conidiosporomyces,
Ingoldiomyces Vánky, Neovossia, Oberwinkleria and Tilletia, are known to infect only grass hosts. Most species within these genera produce teliospores in host
Accepted for publication 23 March 2005.
Corresponding author. E-mail: castlebury@nt.ars-grin.gov
Mention of trade names or commercial products in this article is
solely for the purpose of providing specific information and does
not imply recommendation or endorsement by the U.S. Department of Agriculture.
888
CASTLEBURY
ET AL:
PHYLOGENETIC
FIG. 1. Tilletia tritici germinated teliospore with conjugating primary basidiospores. Bar 5 20 mm.
ovaries, with the exception of nine Tilletia species
that form teliospores in leaves and stems (Zogg
1972). The sixth genus, Erratomyces M. Piepenbr. &
R. Bauer, comprises five species that produce teliospores in leaves of Fabaceae and have a teliospore
germination pattern similar to Tilletia (Piepenbring
ANALYSIS OF
TILLETIALES
889
and Bauer 1997). Conidiosporomyces and Ingoldiomyces
are based on Tilletia ayresii and Tilletia hyalospora,
respectively. Conidiosporomyces is distinguished from
Tilletia by the formation of an apically open, sac-like
sorus and presence of Y-shaped conidia (FIG. 2G) in
the sorus (Vánky and Bauer 1992). Two additional
species have been transferred to the Conidiosporomyces from Tilletia and Ustilago (Pers.) Roussel (Vánky
1993, 2001).
The monotypic Ingoldiomyces is distinguished from
Tilletia by formation of ballistosporic primary basidiospores and a unique type of teliospore ornamentation (Vánky and Bauer 1996). Oberwinkleria, also
monotypic, was erected for a new species, O. anulata
K. & C. Vánky, and is characterized by greatly reduced basidia and primary basidiospores produced
on pedicels (Vánky and Bauer 1995). Neovossia was
erected based on Neovossia moliniae (Thüm.) Körn.,
a Tilletia-like species producing teliospores with a hyaline appendage, local infection, a large number
(.40) of nonconjugating primary basidiospores and
without sterile cells (Vánky 1994). Ten or more species have been placed in Neovossia, but the generic
boundary between Neovossia and Tilletia is not clear
FIG. 2. Spore types from various species in the Tilletiales. A. Blastospores from T. ixophori. B. Denticulate sporogenous
cells from T. kimberleyensis. C. Formation of ballistospores and proliferation of ballistospore. D. Formation of blastospores.
E. Proliferating blastospores. F. Uninucleate and multinucleate primary basidiospores. G. Y-shaped conidia formed in culture
of C. verruculosus.
890
MYCOLOGIA
and Vánky (2002) now considers Neovossia a monotypic genus.
Members of Tilletiales have been poorly represented in previous phylogenetic analyses of the smut
fungi (Begerow et al 1997, Begerow et al 2000). The
analysis of Begerow et al (1997) included only four
type species, Ingoldiomyces hyalosporus, Conidiosporomyces ayresii, Tilletia tritici and Erratomyces patelii. In
that analysis T. tritici and I. hyalosporus were related
most closely and formed a sister group of C. ayresii,
with E. patelii basal to these species. The present
study, using nLSU sequence data, was initiated to determine phylogenetic relationships among species of
Tilletia and segregate genera. Data on teliospore
morphology, teliospore germination, primary and
secondary basidiospore morphology, and nuclear
condition, when available, are presented.
MATERIALS AND METHODS
Isolation, maintenance and deposition of cultures and voucher
specimens.—Species used in this study are listed (TABLE I).
All available taxa for which teliospores could be germinated
were included. Teliospores were germinated after soaking
in water for 2 d and surface sterilization in 0.26% NaClO
(5% v/v commercial bleach) on 2% water agar at room
temperature (20–25 C), 15 C or 5 C depending on the
species. Teliospores of T. controversa were germinated at 5
C under a 8/16 h daylight/dark regimin. Primary basidiospores were fixed and stained with Giemsa-HCl following
Durán (1980) to determine nuclear condition or were
transferred to potato-sucrose agar (PSA) or M-19 agar (Trione 1964) to establish colonies for nucleic acid extraction.
Nucleic acid extraction and PCR amplification.—Mycelium
for DNA extraction was grown in shaker flasks at 125 rpm
in 100 mL liquid potato-dextrose broth at room temperature or 15 C under ambient light. Mycelium was harvested
by centrifugation. Alternatively, DNA was extracted directly
from actively growing surface mycelium scraped from PSA
or M-19 plates. DNA was extracted with the PureGene DNA
extraction kit (Gentra Systems, Madison, Wisconsin) according to the manufacturer’s instructions using approximately 15 mg dried tissue or 50 mg fresh mycelium.
The nLSU genes were amplified in 50 mL reactions on a
GeneAmp 9700 thermal cycler (Applied Biosystems, Foster
City, California) under these reaction conditions: 10–15 ng
of genomic DNA, 200 mM each dNTP, 2.5 units Amplitaq
Gold (Applied Biosystems, Foster City, California), 25 pmoles each of primers LR0R and LR7 (Vilgalys and Hester
1990, Rehner and Samuels 1994) and the supplied 103
PCR buffer with 15 mM MgCl2. The thermal cycler program
was: 10 min at 95 C followed by 35 cycles of 30 s at 94 C,
30 s at 55 C, 1 min at 72 C, with a final extension for 10
min at 72 C. After amplification, the PCR products were
purified with QIAquick columns (QIAGEN Inc., Chatsworth, California) according to the manufacturer’s instructions. Amplified products were sequenced with the BigDye
terminator kit (Applied Biosystems, Foster City, California)
on an automated DNA sequencer with these primers: LR0R,
LR3R, LR5R, LR7, LR5, LR3 (Vilgalys and Hester 1990,
Rehner and Samuels 1994, 1995).
Sequence analysis.—Raw sequences were edited with Sequencher version 4.1.4 for Windows (Gene Codes Corp.,
Ann Arbor, Michigan). Alignments were adjusted manually
with GeneDoc 2.6.001 (http://www.psc.edu/biomed/genedoc/). The alignment included sequences from 57 isolates,
with three species of Entyloma de Bary and one species of
Graphiola Poit. (WSP 71169) as outgroup taxa and consisted
of 1345 positions. Entyloma and Graphiola also are contained within the Exobasidiomycetidae in different orders
and have been placed close to the Tilletiales in previous
analyses (Begerow et al 1997). The sequence alignment was
deposited in TreeBase.
Trees were inferred by the neighbor joining (NJ) method
(Kimura 2-parameter distance calculation) and by maximum parsimony (MP) using the heuristic search option
with the random addition sequence (1000 replications,
maximum of 100 trees saved per replicate) and the branch
swapping (tree bisection-reconnection) option of PAUP*
4.0b10 (Swofford 2002). All aligned positions were included
in the analyses. All characters were unordered and given
equal weight. Gaps were treated as missing data in the parsimony analysis and the neighbor joining analysis; missing
or ambiguous sites were ignored for affected pairwise comparisons. Relative support for branches was estimated with
1000 bootstrap replications (Felsenstein 1985) with multrees and TBR off and 10 random sequence additions for
the MP bootstraps.
Phylogenetic trees also were inferred with Bayesian inference as implemented in MrBayes (http://morphbank.ebc.
uu.se/mrbayes/) with these commands: number of generations 5 500 000, sample frequency 5 100, number of
chains 5 4, temperature 5 0.2, save branch lengths 5 yes,
starting tree 5 random. Likelihood model assumptions
were as determined with Modeltest version 3.06 (Posada
and Crandall 1998) with the Akaike Information Criterion
(AIC) under the GTR1I1G model: base frequencies A 5
0.2664, C 5 0.1971, G 5 0.2907, T 5 0.2458; number substitution types 5 6; proportion of invariable sites 5 0.6672;
gamma shape parameter 5 0.5947; rate matrix 5 0.6253,
2.4765, 0.8936, 0.2228, 5.4558, 1.000. The first 100 000 generations were discarded as the chains were converging
(burn-in). Three independent analyses, each starting from
a random tree, were run under the same conditions.
Phylogenetic trees constraining monophyletic groups of
taxa were constructed as follows based on four major characters: (i) two based on spore ornamentation types (reticulate and echinulate/tuberculate/verrucose); (ii) four
based on host subfamily; (iii) two based on type of germination (conjugating primary basidiospores or nonconjugating); and (iv) two based on local- or systemic-infecting. Maximum parsimony analyses were run for each of the 10 resulting constraints (TABLE II) using the heuristic search option (1000 random sequence additions, TBR and multrees
off). The trees with the best 2ln likelihood score resulting
from each constrained analysis and all three Bayesian trees
CASTLEBURY
TABLE I.
ET AL:
PHYLOGENETIC
ANALYSIS OF
TILLETIALES
891
List of taxa, specimen numbers, hosts and GenBank accession number for nLSU
Taxon
Conidiosporomyces ayresii (Berk.) Vánky
C. verruculosus (Wakef.) Vánky
Erratomyces patelli (Pavgi & Thirum.) M. Piepenbr. & R. Bauer
Ingoldiomyces hyalosporus (Massee) Vánky
Neovossia iowensis Hume & Hodson
Tilletia aegopogonis Durán
T. anthoxanthi A. Blytt
T. asperifolia Ell. & Everh.
T. asperifolia
T. barclayana (Bref.) Sacc. & Syd.
T. barclayana
T. barclayana
T. boutelouae Durán
T. bromi (Brockm.) Brockm.
T. bromi
T. bromi
T. cerebrina Ell. & Everh.
T. chionachnes K. & C. Vánky & R.G. Shivas
T. controversa Kühn
T. ehrhartae Talbot
T. eremopoae Vánky & H. Scholz
T. fusca Ell. & Everh.
T. fusca
T. goloskokovii Schwarzman
T. goloskokovii
T. holci (Wesend.) J. Schröter
T. horrida Tak.
T. horrida
T. indica Mitra
T. ixophori Durán
T. kimberleyensis Vánky & R.G. Shivas
T. laevis Kühn
T. laevis
T. lycuroides Durán
T. menieri Har. & Pat.
T. obscura-reticulata Durán
T. olida (Riess) J. Schröter
T. opaca Sydow
T. polypogonis Vánky & N.D. Sharma
T. rugispora Ell. & Everh.
T. rugispora
T. savilei R.V. Gandhe & Vánky
T. setariae L. Ling
T. sterilis E. Ule
T. sumatii (S.D. Patil & Gandhe) Vánky
T. sumatii
T. togwateei Guillemette
T. trachypogonis Durán
T. tritici (Bjerk.) Wint.
T. tritici
T. vittata (Berk.) Mund.
T. walkeri Castlebury & Carris
T. whiteochloae R.G. Shivas & Vánky
Collection No.a
Host and geographic origin
GenBank No.
HUV 19.314
WSP 70430 (V
1116)
HUV 18.697
Panicum maximum, Argentina
Setaria sphacelata, Zimbabwe
AY819017
AY818984
Vigna mungo, India
AY818966
V 930
BPI 863664
WSP 67743
V 761
LMC 90
LMC 47
WSP 68658
WSP 68466
WSP 68654
WSP 68661
LMC 171
V 763
LMC 99
LMC 125
V 1083
V 764
HUV 19.754
HUV 19.420
LMC 141
LMC 214
LMC 321
LMC 315
V 765
LMC 339
LMC 358
BPI 863665
WSP 71170
HUV 19.174
LMC 178
V 766
WSP 68731
WSP 69115
WSP 68357
WSP 71076
V 837
V 931
WSP 60775
HUV 19.147
V 859
V 932
LMC 363
V 838
V 933
LMC 153
V 1134
LMC 4
LMC 97-136
HUV 19.160
BPI 746091
V 1087
Nassella mexicana, Venezuela
Phragmites communis, China
Aegopogon tenellus, Mexico
Anthoxanthum odoratum, New Zealand
Muhlenbergia asperifolia, USA
Muhlenbergia asperifolia, USA
Paspalum distichum, Mexico
Paspalum distichum, Mexico
Panicum obtusum, Mexico
Bouteloua gracilis, Mexico
Bromus japonicus, USA
Nardurus subulatus, Iran
Bromus tectorum, USA
Deschampsia danthonoides, USA
Chionachne cyathopoda, Australia
Hordeum glaucum, Iran
Ehrharta calycina, Australia
Eremopoa persica, Turkey
Vulpia microstachys, USA
Vulpia octoflora, USA
Apera interrupta, USA
Apera interrupta, USA
Holcus mollis, New Zealand
Oryza sativa, USA
Oryza sativa, USA
Triticum aestivum, USA
Ixophorus unisetus, Nicaragua
Chionachne cyathopoda, Australia
Triticum aestivum, Australia
Triticum aestivum, Iran
Lycurus phleoides, Mexico
Phalaris arundinacea, Germany
Bouteloua rothrockii, Mexico
Brachypodium pinnatum, Germany
Spinifex littoreus, Indonesia
Polypogon monspeliensis, India
Paspalum convexum, Mexico
Paspalum plicatulum, Argentina
Tripogon jacquemontii, India
Setaria intermedia, India
Poa secunda, USA
Coix lacryma-jobi, India
Coix lacryma-jobi, India
Poa reflexa, USA
Trachypogon spicatus, Zambia
Triticum aestivum,
Triticum aestivum, Australia
Oplimenus burmannii, India
Lolium multiflorum, USA
Whiteochloa cymbiformis, Australia
AY818976
AY818988
AY818967
AY819009
AY818968
AY818969
AY818970
AY818971
AY818972
AY818973
AY819001
AY818992
AY818993
AY818994
AY818990
AY818995
AY819013
AY819016
AY818997
AY818996
AY818998
AY818999
AY819008
AY818974
AY818975
AY818977
AY819010
AY818979
AY819004
AY819005
AY818980
AY819002
AY819011
AY819000
AY818981
AY819015
AY818982
AY818983
AY819018
AY819014
AY819003
AY818986
AY818987
AY818991
AY819012
AY819006
AY819007
AY818985
AY818978
AY818989
a BPI 5 U.S. National Fungus Collections, Beltsville, MD; HUV 5 Herbarium Ustilaginales Vánky, Tübingen; LMC 5 personal
collection of L. M. Carris; V 5 Vánky Ustilaginales Exsiccati; WSP 5 Washington State Department of Plant Pathology.
892
TABLE II.
MYCOLOGIA
Shimodaira-Hasegawa likelihood test results for analyses constrained for host subfamily or morphological character
Topology
Treesa
Length
2ln Likelihood
P
Unconstrained MP
Bambusoid hosts
Chloridoid hosts
Panicoid hosts
Pooid hosts
Conjugating basidiospores
Non-conjugating basidiospores
Reticulate teliospores
Tuberculate teliospores
Local-infecting
Systemic-infecting
Bayesian
1509
6970
2820
5930
1073
2524
5000
1198
100
1853
1072
3
432
458
445
450
432
488
510
447
448
465
456
—
4213.892
4284.285
4255.662
4271.798
4214.326
4420.040
4501.826
4251.180
4253.268
4321.874
4280.336
4215.393
—
0.005*
0.167
0.046*
0.952
0.000*
0.000*
0.207
0.190
0.000*
0.011
0.948
a
P-values and 2ln likelihood scores only reported for the tree with best 2ln likelihood score.
* Indicates significant at P , 0.05 in a one-tailed test under the null hypothesis that all trees are equally good explanations
of the data.
were compared with the MP tree with the best 2ln likelihood score, using the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999). The range of 2ln likelihood
scores of trees from each constraint topology is shown (TABLE II). Likelihood settings were as determined by Modeltest as previously described.
RESULTS
Phylogenetic analyses.—Of 1345 characters, 144 were
parsimony informative, 1124 were invariable, 77 were
variable but not parsimony informative. For MP analyses with the multrees option on, heuristic searches
resulted in an excess of 5000 trees. By limiting the
number of trees saved per replicate to 100, 1509
equally parsimonious trees were generated. A strict
consensus of trees generated with multrees on (maxtrees 5 5000) was identical to the strict consensus of
trees generated from analyses with multrees limited
to 100 per replicate (trees not shown). Parsimony
tree scores were CI 5 0.637, RI 5 0.860, RC 5 0.547
and length 5 432. The MPT with the best 2ln likelihood score is shown (FIG. 3). MP bootstrap support
values are indicated (FIG. 3) above the respective
branches. NJ bootstrap support values did not differ
greatly from MP bootstrap support and are not
shown.
Three independent Bayesian analyses were run
with each starting from a random tree and probabilities and topologies were similar in all analyses. One
arbitrarily chosen Bayesian tree is shown (FIG. 4). Topologies differed only in the placement of the Conidiosporomyces/T. vittata branch as unresolved in relation to the pooid group in two runs but immediately basal to the pooid group in the third run, although this was not supported (trees not shown).
Minor differences in terminal branching also were
noted but also not supported. Posterior probabilities
were pooled and branches with pooled posterior
probabilities . 90% are indicated with thickened
lines (FIG. 4).
The analysis shows strong support (100% Bayesian,
88% MP) (FIGS. 3–4) for a monophyletic group that
contains species of Tilletia, Ingoldiomyces, Neovossiaand Conidiosporomyces. Within these taxa, four distinct lineages are apparent. Lineage I contains species infecting grasses in the Pooideae (100% Bayesian, 61% MP support), with three well supported subgroups of taxa consisting of T. tritici and related
species (100% in all analyses), I. hyalosporus and T.
polypogonis, and one for T. indica and T. walkeri
(.99% in all analyses). Each of these groups also is
characterized by different germination patterns and
teliospore ornamentation. Lineage II, recognized in
all analyses, contains 11 species that infect Panicoideae, Arundinoideae and Chloridoideae (PAC)
(100% Bayesian, 82% MP), including N. iowensis. All
species in this group have tuberculate/verrucose teliospores with the exception of N. iowensis, which has
foveolate teliospores and nonconjugating, uni- or
multinucleate basidiospores. Several species in this
group have been described or referred to as species
of Neovossia in the literature. Tilletia barclayana,
which falls in this group, appears to be a species complex with slight differences in sequence found
among all three isolates. However it is not clear
whether the differences found in the nLSU sequences warrant species level distinction. Variation in the
nLSU was not consistent across all species. Taxa in
the pooid-infecting clade (Lineage I) varied the least
with almost no differences among the reticulatespored taxa or between T. indica and T. walkeri. Larger numbers of differences were observed among taxa
CASTLEBURY
ET AL:
PHYLOGENETIC
ANALYSIS OF
TILLETIALES
893
FIG. 3. MP tree resulting from analysis of 1345 bp from the nLSU for the species in the Tilletiales. Numbers above the
branches indicate MP bootstrap support percentages (.50%) from 1000 pseudoreplicates with 10 random taxon addition
replicates per pseudoreplicate for major lineages only. Four major lineages are identified by Roman numerals I-IV and host
subfamily when limited to a single subfamily. PAC refers to Panicoidae, Arundinoideae and Chloridoideae. Representatives
from segregate genera are indicated in bold type as is the type species of Tilletia.
in the other three lineages. This could be due to
better sampling of taxa in Lineage I, a more recent
radiation of species in Lineage I or some combination of both.
Lineage III includes species infecting chloridoid
hosts ($85% in all analyses), including T. asperifolia,
T. lycuroides, T. aegopogonis and T. obscura-reticulata.
These are the only four taxa in the analysis with reticulate spores that infect hosts other than Pooideae.With the exception of T. asperifolia, which has
uninucleate, conjugating basidiospores, all form multinucleate, nonconjugating basidiospores. Lineage IV
contains three panicoid-infecting species and includes C. ayresii, C. verruculosus and T. vittata. Conidiosporomyces species have open sori and Y-shaped
conidia (either in sori or formed in culture). Tilletia
vittata causes hypertrophy of the infected ovary so
that it forms a conspicuous, spur-like outgrowth. Basidiospores of the three species in this lineage are
uninucleate, and conjugation was observed (but rare-
894
MYCOLOGIA
FIG. 4. Phylogenetic tree resulting from Bayesian analysis of 1345 bp of the nLSU of species in the Tilletiales. Thickened
branches indicate .90% pooled posterior probabilities obtained from three independent Bayesian analyses, each consisting
of 500 000 Markov chain Monte Carlo generations (GTR1G1I model), with a burn-in of 100 000 generations. Lineages
identified in FIG. 3 are indicated with host subfamily association. Morphological characters from TABLE III are labeled as
follows: Ret 5 reticulate spore, Tub 5 tuberculate/verrucose spores, Ridg 5 ridged spores, Fov 5 Foveolate, Conj 5 conjugating primary basidiospores, Nonconj 5 nonconjugating primary basidiospores, ,30 5 ,30 primary basidiospores, .30
5 .30 primary basidiospores, Local 5 local-infecting, and Syst 5 systemic-infecting. When a species in a group differs from
the labeled characters, the difference for that species is indicated in bold with underlining.
CASTLEBURY
ET AL:
PHYLOGENETIC
ly) only in C. ayersii. A few species do not fall into
any of the four lineages described above. The relationships of T. setariae (panicoid host), T. ehrhartae
(ehrhartoid host), T. rugispora (panicoid host) and
T. horrida (ehrhartoid host) to other species remain
unresolved. Morphological characters (TABLE III) for
lineages are labeled (FIG. 4). For taxa with differing
character states for a given character, differences are
indicated in bold underlined text inside the brackets
The MP tree had the best likelihood score (TABLE
II), although the Bayesian trees were not significantly
worse explanations of the data (P 5 0.05). Trees constraining pooid-infecting, chloridoid-infecting, reticulate-spored, and echinulate/verrucose/tuberculatespored taxa, respectively, also were not significantly
worse than the MP tree. Trees constraining local-infecting or systemic-infecting taxa, taxa with conjugating basidiospores or panicoid- or ehrhartoid-infecting taxa were significantly worse (P 5 0.05) than the
MP tree.
Teliospore germination and growth in culture.—Teliospore germination data for species in the analysis are
provided (TABLE III). Nuclear condition of primary
basidiospores could not be determined for seven species that had limited teliospore germination. The teliospore germination pattern in the type species T.
tritici involves rapid conjugation of adjacent primary
basidiospores. Teliospores germinate at 5–15 C, but
no germination occurs at room temperature. The
fungus infects the host at the seedling stage, forming
a systemic infection and growing to the developing
host ovaries, where the fungus proliferates and forms
teliospores. This pattern of dikaryon formation, systemic infection and low temperature requirement occurs in all species closely related to T. tritici, with the
exception of T. sterilis and T. cerebrina, which form
multinucleate, nonconjugating primar y basidiospores. Zogg (1967) reported conjugation in T. olida,
but it was was not observed in the T. olida specimen
germinated in this study. Infection by T. olida and T.
sterilis is systemic, but teliospores form in sori in host
leaves rather than in the ovaries. Ingoldiomyces hyalosporus, T. polypogonis, T. indica and T. walkeri infect hosts in subfamily Pooideae, but teliospores of
these species germinate at room temperature. Of
these species, only T. polypogonis has a germination
pattern similar to that of T. tritici.
Tilletia asperifolia, host Muhlenbergia asperifolia
(subfamily Chloridoideae, Lineage III) is the only
species outside the pooid-infecting clade (Lineage I)
that exhibits the same type of germination pattern,
systemic infection and temperature requirement as T.
tritici. Tilletia aegopogonis and T. lycuroides, which
form a well supported group with T. asperifolia, differ
ANALYSIS OF
TILLETIALES
895
in having teliospores that germinate at room temperature to form multinucleate, nonconjugating basidiospores. Erratomyces patelii, host Vigna mungo (Fabaceae), also germinates at room temperature and
produces conjugating basidiospores (Piepenbring
and Bauer 1997). The infection type was not reported for this species but is probably local based on the
isolated leaf spots that are formed.
In most of the taxa studied with hosts outside subfamily Pooideae, primary basidiospores germinated
directly through formation of hyphae or indirectly
through formation of ballistospores and did not conjugate under axenic conditions. Multinucleate and
uninucleate nonconjugating primary basidiospores
(FIG. 2F) germinate in a similar manner. Nonconjugating primary basidiospores were shown to be multinucleate in nine species, with hosts in Pooideae (I.
hyalosporus, T. cerebrina, T. sterilis), Chloridoideae (T.
aegopogonis, T. lycuroides, T. savilei) and Panicoideae
(T. opaca, T. trachypogonis) ranging across Lineages
I, II and III.
All species studied in culture produced allantoid
ballistospores (FIG. 2C) and filiform to fusiform blastospores (FIG. 2A, D, E), although the two spore types
were not produced in equal abundance in all isolates
studied. Isolates of some taxa grew in a mycelial manner with relatively few secondary basidiospores. Ballistospores formed from sterigma-like structures on
primary basidiospores, other ballistospores, or hyphae (FIG. 2C). Blastospores were aseptate, filiform,
curved to coiled, and resembled primary basidiospores and were more abundant than ballistospores
in cultures of most taxa in this study. Blastospores
formed from other blastospores (FIG. 2E), and from
hyphae, either singly on undifferentiated sporogenous cells (FIG. 2D), or from sporogenous cells with
multiple denticles (FIG. 2B). Blastospores were not
reported in Erratomyces (Piepenbring and Bauer
1997). In addition to the two types of secondary basidiospores just described, C. verruculosus also produced abundant Y-shaped blastospores in culture
(FIG. 2G), similar in shape to the conidia formed in
sori of C. ayresii. The Y-shaped spores germinated
readily. Y-shaped conidia were not present in the sori
of C. verruculosus, and this type of spore was not observed in cultures of C. ayresii or other species included in this study. All species included in this study,
except E. patelii, had sterile cells intermingled with
teliospores in the sorus.
DISCUSSION
A strict generic concept of Tilletia as characterized by
the reticulate teliospore ornamentation and pattern
of germination and infection exhibited by the type
896
TABLE III.
Morphological characters for each taxon listed in alphabetical order
Taxon
Host
subfamily
Teliospore
ornamentation
Panicoideae
Panicoideae
verrucose
echinulate
E. patelii
I. hyalosporus
Fabaceae
Pooideae
verrucose
ridged
N. iowensis
Arundinoideae
foveate
T. aegopogonis
Chloridoideae
reticulate
T. anthoxanthi
T. asperifolia
T. barclayana
Pooideae
Chloridoideae
Panicoideae
reticulate
reticulate
tuberculate
T. boutelouae
Chloridoideae
tuberculate
T. bromi
Pooideae
reticulate
T. cerebrina
Pooideae
T.
T.
T.
T.
T.
T.
T.
T.
Panicoideae
Pooideae
Ehrhartoideae
Pooideae
Pooideae
Pooideae
Pooideae
Ehrhartoideae
reticulate to
cerebriform
verrucose
reticulate
tuberculate
reticulate
reticulate
reticulate
reticulate
tuberculate
T. indica
Pooideae
tuberculate
T.
T.
T.
T.
T.
Panicoideae
Panicoideae
Pooideae
Pooideae
Chloridoideae
tuberculate
verrucose
smooth
reticulate
reticulate
chionachnes
controversa
ehrhartae
eremopoae
fusca
goloskokovii
holci
horrida
ixophori
kimberleyensis
laevis
menieri
lycuroides
conjugating (rare)
nonconjugating,
multinucleate
conjugating
nonconjugating,
multinucleate
nonconjugating,
uninucleate
nonconjugating,
multinucleate
conjugating
conjugating
nonconjugating,
uninucleate
nonconjugating,
uninucleate
conjugating
nonconjugating,
multinucleate
nonconjugating
conjugating
nonconjugating
conjugating
conjugating
conjugating
conjugating
nonconjugating,
uninucleate
nonconjugating,
uninucleate
nonconjugating
nonconjugating
conjugating
conjugating
nonconjugating,
multinucleate
# primary
basidiospores
Germination
temperature
Infection
type
Reference for germination
pattern
,20
,20
20–25
20–25
local
local
this study
this study
.30
2
20–25
20–25
local
systemic
Piepenbring & Bauer 1997
Vánky & Bauer 1996
10–15
20–25
local
this study
5–6
20–25
systemic
Durán 1987
,20
10–12
.60
5
9
20–25
systemic
systemic
local
this study
this study
Durán 1987, this study
30–50
20–25
local
Durán 1987
10–16
5–15
systemic
5
systemic
Boyd & Carris 1998, this
study
Siang 1954
3–8
,20
14–30
,20
60–100
10–16
,20
,20
.60
20–25
5
15
5–15
5–15
5–10
15
20–25
local
systemic
systemic
systemic
systemic
systemic
systemic
local
this study
Goates & Hoffmann 1987
this study
this study
this study
Boyd et al 1998
this study
this study
.60
20–25
local
Durán 1987
11–17
,20
4–16
,10
8–15
20–25
20–25
15
15
20–25
local
local
systemic
systemic
systemic
this study
this study
Goates & Hoffmann 1987
this study; Meiners 1957
Durán 1979, 1983
MYCOLOGIA
C. ayresii
C. verruculosus
Germination pattern
TABLE III.
Continued
Teliospore
ornamentation
T. olida
T. opaca
Pooideae
Panicoideae
reticulate
tuberculate
T. polypogonis
Pooideae
T. rugispora
T. savilei
Panicoideae
Chloridoideae
reticulate to
cerebriform
tuberculate
tuberculate
T. sterilis
Pooideae
reticulate
T. sumatii
Panicoideae
tuberculate
T. togwateei
T. trachypogonis
Pooideae
Panicoideae
reticulate
verrucose
T. tritici
T. walkeri
Pooideae
Pooideae
reticulate
tuberculate
T. vittata
T. whitechloae
Panicoideae
Panicoideae
verrucose
verrucose
nonconjugating,
multinucleate
conjugating?
nonconjugating,
multinucleate
conjugating
conjugating
nonconjugating,
multinucleate
nonconjugating,
multinucleate
nonconjugating,
uninucleate
conjugating
nonconjugating,
multinucleate
conjugating
nonconjugating,
uninucleate
nonconjugating
nonconjugating
Infection
type
Reference for germination
pattern
50–60
not reported
local
Durán 1987
3–5
30–50
15
20–25
systemic
local
,10
20–25
systemic
this study; Zogg 1967
Ingold 1997, Vánky 1993;
this study
this study
.30
,20
20–25
20–25
local
local
Durán 1987
this study
systemic
this study
2–4
5
20–50
20–25
local
Ingold 1997, this study
3–10
.30
5–10
20–25
systemic
local
Guillemette 1988, this study
Durán 1987
15
20–25
systemic
local
this study
Castlebury & Carris 1999
20–25
20–25
local
local
Durán 1987, this study
this study
4–16
60–150
20–30
.50
TILLETIALES
reticulate
Germination
temperature
ANALYSIS OF
Chloridoideae
# primary
basidiospores
PHYLOGENETIC
T. obscura-reticulata
Germination pattern
ET AL:
Host
subfamily
CASTLEBURY
Taxon
897
898
MYCOLOGIA
species, T. tritici, is not supported based on the results of the analyses of nLSU data. The T. tritici pattern of teliospore germination, with a relatively small
number of rapidly conjugating primary basidiospores
and systemic host infection resulting in most or all of
the host ovaries replaced by fungal sori, is restricted
mostly to species in the pooid-infecting clade. However some members of this clade produce nonconjugating primary basidiospores, including T. cerebrina
and T. sterilis. Several species that have been studied
extensively, including T. bromi, T. fusca and T. togwateei, form mostly uninucleate, conjugating basidiospores, but a small percent of spores may be multinucleate. Boyd and Carris (1998) showed evidence
that up to 12% of primary basidiospores produced
by T. fusca are dikaryotic based on the formation of
teliospores in cultures derived from single basidiospores. Multinucleate primary basidiospores may result either from migration of multiple nuclei from
the basidium into developing basidiospores or from
mitotic division in basidiospores as shown by Goates
and Hoffmann (1987). The T. tritici germination pattern also is found in T. asperifolia (Lineage III),
which has a chloridoid host and falls outside the
pooid-infecting clade. Erratomyces patelii, which is
strongly supported as a basal group to Tilletia and
infects dicotyledonous hosts, exhibits this germination pattern as well. Similarly, the reticulate teliospore ornamentation exhibited by T. tritici, is restricted mostly to species in the pooid-infecting clade (Lineage I) but also occurs in T. aegopogonis, T. asperifolia, T. obscura-reticulata and T. lycuroides in Lineage
III.
The pathogens responsible for Karnal bunt of
wheat, T. indica, and kernel smut of rice, T. horrida,
were placed in Neovossia by some authors (Singh and
Pavgi 1972, Vánky 1994, Whitney 1989) and in Tilletia by others (Durán 1987, Levy et al 2001, Pimentel
et al 1998). Both species produce sterile cells in the
sorus and numerous nonconjugating primary basidiospores (Castlebury and Carris 1999, Durán 1987).
Durán and Fischer (1961) dismissed the value of
number of primary basidiospores to delimit genera
and our analysis supports their conclusion. Absence
of sterile cells and production of numerous basidiospores were two characters used to distinguish Neovossia from Tilletia (Vánky 2002). The type species
N. moliniae was shown by Brefeld (1895) to form 30–
50 nonconjugating primary basidiospores. However,
examination of specimens of N. moliniae, on Molinia
(WSP 34463) and N. iowensis on Phragmites (V 573)
revealed the presence of sterile cells in the sorus.
Two species of Neovossia infecting Phragmites communis have been described: N. iowensis from the USA
(Hodson 1900) and N. danubialis T. Săvulescu from
Europe (Săvulescu 1955). Neovossia danubialis and
N. iowensis were merged with N. molinae by Vánky
(1990) based on their similar teliospore morphology
and germination patterns. Săvulescu and Hulea
(1955) showed that N. danubialis germinated to produce 10–15 nonconjugating primary basidiospores,
similar to what was shown in this study for N. iowensis.
Based on the morphological similarity and occurrence on the same host species, N. danubialis and N.
iowensis are considered to be synonymous. Because
of the differences in numbers of primary basidiospores and host genus between N. moliniae and N.
iowensis, we are maintaining the two as distinct species. The results of this study suggest that there is no
basis for recognizing Neovossia as a genus distinct
from Tilletia and we consider N. iowensis to be a species of Tilletia. However we were not able to study
viable collections of N. moliniae and therefore the
status of Neovossia itself remains uncertain.
Our analyses place I. hyalosporus, with ridged teliospores and production of ballistosporic primary basidiospores, within the well supported clade of pooidinfecting species containing T. tritici and allied species, T. indica and T. walkeri (Lineage I). Two species
of Conidiosporomyces, C. ayresii and C. verruculosus,
were included in this analysis and were closely related
to T. vittata (Lineage IV). Conidiosporomyces is distinguished from Tilletia based on the formation of a saclike, apically open sorus and the presence of Yshaped conidia (Vánky and Bauer 1992). The unusual Y-shaped conidia are formed in the sorus in C.
ayresii and are formed in C. verruculosus in culture.
Based on the results of the nLSU analyses the characters that have been used to segregate Ingoldiomyces
or Conidiosporomyces from Tilletia cannot be considered generic level characters and at this point we consider both genera synonyms of Tilletia.
The phylogeny of Tilletiales appears to reflect that
of the hosts, with a well supported group of closely
related species evolving on hosts in the subfamily
Pooideae and a poorly resolved group of more diverse species infecting hosts in Chloridoideae, Ehrhartoideae, Arundinoideae and Panicoideae. The relationships elucidated by the phylogenetic analyses in
this study suggest a more rapid radiation of Tilletia
species on pooid hosts than on hosts in other subfamilies. Phylogenetic studies in the grass family (Poaceae) show two well supported clades comprising six
monophyletic subfamilies, the Bambusoideae plus
Ehrhartoideae and Pooideae (BEP) clade, and the
Panicoideae, Arundinoideae, Centothecoideae and
Chloridoideae (PACC) clade (Kellogg 2001). The relationships among subfamilies in the PACC clade are
not well resolved in existing phylogenies (Kellogg
2001). Host specificity for individual species of Tille-
CASTLEBURY
ET AL:
PHYLOGENETIC
tia remains problematic and species concepts vary
from author to author. Genetically distinct lineages
can be associated with specific hosts in nature (Boyd
and Carris 1997, Boyd et al 1998). However some
species of Tilletia, while apparently host specific in
nature, have retained the ability to infect other hosts
under artificial conditions (Royer and Rytter 1988).
More variable gene regions will be required to investigate issues of host specificity and morphological
species complexes in this group of fungi.
ACKNOWLEDGMENTS
The authors thank Aimee S. Hyten and Douglas Linn for
technical assistance and Amy Rossman for her comments
on the manuscript. We express gratitude to Mary Palm for
providing a viable collection of Neovossia iowensis.
LITERATURE CITED
Begerow D, Bauer R, Oberwinkler F. 1997. Phylogenetic
studies on nuclear large subunit ribosomal DNA sequences of smut fungi and related taxa. Can J Bot 75:
2045–2056.
,
, Boekhout T. 2000. Phylogenetic placements
of ustilaginomycetous anamorphs as deduced from nuclear LSU rDNA sequences. Mycol Res 104:53–60.
Boyd ML, Carris LM. 1997. Molecular relationships among
varieties of the Tilletia fusca (T. bromi) complex and
related species. Mycol. Res 101:269–277.
,
. 1998. Evidence supporting the separation
of the Vulpia- and Bromus-infecting isolates in the Tilletia fusca (T. bromi) complex. Mycologia 90:1031–
1039.
,
, Gray PM. 1998. Characterization of Tilletia
goloskokovii and allied species. Mycologia 90:310–322.
Brefeld O. 1895. Untersuchungen aus dem Gesammtgebiete der Mykologie. XI. Die Brandpilze II. Die Brandkrankheiten des Getreides. Münster i. W., Commissions
Verlag v. H. Schöningh, Münster.
Castlebury LA, Carris LM. 1999. Tilletia walkeri, a new species on Lolium multiflorum and L. perenne. Mycologia
91:121–131.
Durán R. 1979. Tilletia lycuroides: biological implications of
nuclear behavior in the basidium. Mycologia 71:
449–455.
. 1980. Tilletia aegopogonis, a homo-heterothallic
bunt fungus. Phytopathology 70:528–533.
. 1983. Tilletia lycuroides, another homo-heterothallic
bunt fungus. Mycologia 75:974–976.
. 1987. Ustilaginales of Mexico. Pullman, Washington: Washington State University Press. 331 p.
, Fischer GW. 1961. The genus Tilletia. Washington
State University at Pullman. 138 p.
Felsenstein J. 1985. Confidence limits on phylogenies: an
approach using the boostrap. Evolution 6:227–242.
Goates B. 1996. Common and dwarf bunt. In: Wilcoxon RD,
Saari EE, eds. Bunt and Smut Diseases of Wheat: con-
ANALYSIS OF
TILLETIALES
899
cepts and methods of disease resistance. Mexico City:
CIMMYT. p 12–25.
, Hoffmann JA. 1987. Nuclear behavior during teliospore germination and sporidial development in Tilletia caries, T. foetida and T. controversa. Can J Bot 65:
512–517.
Guillemette MK. 1988. Tilletia togwatii, new bunt species
from Poa reflexa. Mycologia 80:273–285.
Hodson ER. 1900. A new species of Neovossia. Bot Gaz 30:
273–274.
Ingold CT. 1996. Two kinds of ballistoconidia in the anamorph of Tilletia setariae. Mycol Res 100:173–174.
. 1997. Teliospore germination in Tilletia opaca and
T. sumatii and the nature of the tilletiaceous basidium.
Mycol Res 101:281–284.
Kellogg EA. 2001. Evolutionary history of the grasses. Plant
Physiology 125:1198–1205.
Levy L, Castlebury LA, Carris LM, Meyer RJ, Pimentel G.
2001. Internal transcribed spacer sequence-based phylogeny and polymerase chain reaction-restriction fragment length polymorphism differentiation of Tilletia
walkeri and T. indica. Phytopathology 91:935–940.
Meiners JP. 1957. Spore germination and cytology of Tilletia
scrobiculata. Phytopathology 47:528.
Piepenbring M, Bauer R. 1997. Erratomyces, a new genus of
Tilletiales with species on Leguminosae. Mycologia 89:
924–936.
Pimentel G, Carris LM, Levy L, Meyer R. 1998. Genetic variation among isolates of Tilletia barclayana, T. indica,
and allied species. Mycologia 90:1017–1027.
Posada D, Crandall KA. 1998. Modeltest: testing the model
of DNA substitution. Bioinformatics 49:817–818.
Rehner S, Samuels GJ. 1994. Taxonomy and phylogeny of
Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycol Res 98:625–634.
,
. 1995. Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of
their anamorphs. Can J Bot 73 (Suppl. 1):S816–S823.
Royer MH, Rytter J. 1988. Comparison of the host ranges
of Tilletia indica and T. barclayana. Pl Dis 72:133–136.
Săvulescu T. 1955. Noi specii de Ustilaginee. Comun Acad
Republ Populare Române 5:63–76.
, Hulea A. 1955. Schimbările morfo-citologice ale
clamidosporilor, basidiosporilor şi sporediilor de Neovossia danubialis Săvul Bul Şti, Sect Biol, Şti Agricol,
Geol, Geogr 7:501–516.
Shimodaira H, Hasegawa M. 1999. Multiple comparisons of
log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16:1114–1116.
Siang WN. 1954. Observations on Tilletia cerebrina. Mycologia 46:238–244.
Singh RA, Pavgi MS. 1972. Cytology of teliospore germination and development of Neovossia horrida. Riso 21:
259–268.
Swofford DL. 2002. PAUP* Phylogenetic Analysis Using Parsimony (*and other methods) Version 4.0b10. Sunderland, Massachusetts: Sinauer and Associates.
Trione EJ. 1964. Isolation and in vitro culture of the wheat
bunt fungi Tilletia caries and T. controversa. Phytopathology 54:592–596.
900
MYCOLOGIA
Vánky K. 1990. Taxonomical studies on Ustilaginales. V. Mycotaxon 36:473–482.
. 1993. Taxonomical studies on Ustilaginales. X. Mycotaxon 48:27–44.
. 1994. European Smut Fungi. New York: Gustav Fischer. 570 p.
. 2001. Taxonomical studies on Ustilaginales. XXI.
Mycotaxon 78:265–326.
. 2002. Illustrated genera of smut fungi, 2nd ed. St
Paul, Minnesota: A PS Press. 238 p.
, Bauer R. 1992. Conidiosporomyces, a new genus of
Ustilaginales. Mycotaxon 43:427–436.
,
. 1995. Oberwinkleria, a new genus of Ustilaginales. Mycotaxon 53:361–368.
,
. 1996. Ingoldiomyces, a new genus of Ustilaginales. Mycotaxon 49:277–287.
Vilgalys R, Hester M. 1990. Rapid genetic identification and
mapping of enzymatically amplified ribosomal DNA
from several Cryptococcus species. J Bacteriol 172:4238–
4246.
Whitney NG. 1989. Taxonomy of the fungus causing kernel
smut of rice. Mycologia 81:468–471.
Zogg H. 1967. Uber die Sporenkeimung von Tilletia olida
(Riess. ap. Rab.) Schröter und Tilletia brachypodii-ramosi n. sp. Ber Schweiz Bot Ges 77:49–56.
Zogg H. 1972. Die Tilletia-Streifenbrandkrankheiten der
Gräser. Phytopath. Z. 74:218–229.