Mycol Res 106 (7) 757-767 (July 2002) © The British Mycological 757

Society DOI 10 1017/S0953756202006172 Printed in the United Kingdom

Phylogeny and evolution of the genus Trichoderma: a

multigene approach

Cornelia M. KULLNIG-GRADINGER1, George SZAKACS2 and Christian P. KUBICEK1*

1
Arbeitsgruppe fur Mihobielle Bwchemie und Gentechmk, Institut fur Veifahrenstechmk, Umnelttechnik und techmsche
Bwwissenschaften, Techmsche Umversitat Wien, Getreidemarkt 9, A-1060 Wien, Austria
2
Department of Agricultural Chemical Technology, Technical University of Budapest, 1111 Budapest, Gellert ter 4, Hungary
E-mail ckubicek@mail.zserv.tuwien.ac.at
Received 3 August 2001 accepted 16 June 2002

The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony
analysis and distance analysis of DNA sequences from multiple genetic loci 18S rDNA sequence analysis suggests that the
genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago 28S rDNA
sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae, which also
includes Arachnocrea and Aphysiostroma m basal positions Gene trees inferred from a combined analysis of the nuclear
ribosomal internal transcribed spacer (ITS1 and 2), the Dl and D2 region of the 28S rDNA, the small subunit of the
mitochondrial rDNA (mitSSU), the fifth and part of the sixth exon of translation elongation factor 2 (tef1), and a
fragment of ech42 provide strong statistical support for a phylogeny consistent with the existence of four clades clade A
comprises species of Bissett's (1991) sect Trichoderma but also T. hamatum, T. pubescens, T. asperellum, and T.
strigosum, clade C comprises all the species contained m section Longibrachiatum as revised by Samuels et al (1998), and
clade D contains only T. aureoviride which is genetically most distant to all other species Clade B, on the other hand,
contains a large and taxonomically heterogeneous mixture of species, among which several subclades could be identified
subclade B1 containing H. lactea, H. citrina, H. citrina var americana, H. lutea, and an unnamed T. sp 1, subclade B2
containing T. stromaticum, and an unnamed T. sp PPRI3559, subclade B3 containing T. fertile, T. oblongisporum, and
H. hunua, subclade B5 containing T. polysporum, T. croceum, Hypocrea pilulifera, and T. minutisporum, and a larger
subclade (B4), in which three strain clusters could be distinguished one comprising T. harzianum, T. inhamatum, H.
vinosa, and T. aggressivum, another one containing T. fasciculatum, T. longtpile, and T. strictipile, and a third
containing T. virens, T. flavofuscum, and T. crassum The position of the remaining species of subclade B4 (T. spirale,
T. cfr aureoviride, H. tawa, and T. tomentosum) was not resolved A comparison of the topologies of the individual gene
trees was concordant with the topology of the combined tree in most cases, but also revealed incongruent positions for a
few species (T. oblongisporum, T. longipide, T. fasciculatum) which was most pronounced in the ITS1 and 2 tree The
results confirm the recent concept for sects Longibrachiatum and Trichoderma, indicate that the sects Hypocreanum and
Pachybasium cannot be distinguished phylogenetically, and provide a first phylogenetic basis for dissection of the latter
two sections

INTRODUCTION level has proved difficult, due to the degree of

morphological similarities between them. As a solution,
Species of the deuteromyceteous genus Trichoderma
Rifai (1969) adopted the concept of 'aggregate' species,
are cosmopolitan and typically soil-borne or
and distinguished nine aggregates, some of which
wood-decaying fungi (Samuels 1996, Esposito & Da
comprised two or more morphologically indistinguish-
Silva 1998). Some of them are economically important
able species. In contrast, Bissett (1984, 1991a-c, 1992,
because of their production of industrial enzymes
Gams & Bissett 1998) perceived non-continuous mor-
(cellu-lases and hemicellulases), antibiotics, and their
phological characteristics of biological species. On this
action as biocontrol agents (Kubicek & Penttila 1998,
basis, he elevated Rifai's aggregate species to the
Sivasitham-param & Ghisalberti 1998, Hjeljord &
sectional level, and expanded the morphological criteria
Tronsmo 1998). Identification of the respective
to accommodate the wider range of morphological
isolates at the species
variation expressed by some anamorphs of Hypocrea
and also to include some forms previously included in
* Corresponding author. Gliocladium.
Present address: Arbeitsgruppe fuer Mykologie, Institut fuer Molecular methods have recently been introduced
Verfahrenstechnik, Umwelttechnik und technische
Biowissenschaf-ten, TU Wien, Getreidemarkt 9, A-1060 Wien,
Austria.
Phylogeny of Trichoderma
into Trichoderma taxonomy with a revision of sects
Longibrachiatum and Trichoderma, respectively, and
related teleomorphs (Kuhls et al 1996, 1997, Samuels
et al 1998, 2000,2002, Samuels, Lieckfeldt & Nirenberg
1999, Lieckfeldt et al 1998, 1999, 2001) However, the
majority of species which Bissett (199la, b) included in
sect Pachybasium have not yet been treated in this
detail Kindermann et al (1998) attempted a first
phylogenetic analysis of the whole genus, using sequence
analysis of the ITS1 region of the rDNA However, the
use of phylogenies based on single gene sequences is
now generally discredited, especially as regards the use
of ITS1 and/or ITS2, as some fungi and plants have
been shown to contain paralogous copies (O'Donnell
1992, Buckler et al 1997, O'Donnell et al 1998,
Lieckfeldt & Seifert 2000) Taylor, Jacobson & Fisher
(1999) proposed basing phylogenetic species concepts
on the concordance between five or more gene trees
Consequently, combined sequence analysis of ITS1 and
ITS2 with single-copy genes such as β-tubulin (Schardl
et al 1994, O'Donnell et al 1998) or hydrophobin
(Geiser, Frisvad & Taylor 1998) has been used with
success Lieckfeldt et al (2000) used endochitinase
(ech42) gene sequence analysis for Trichoderma sect
Trichoderma, and showed that the results were con-
cordant with those obtained from ITS1 and ITS2
sequence analysis
The purpose of the present work was to establish a
species phylogeny of the genus Trichoderma, based on
the sequence analysis of multiple independent loci, by
investigating all the species described by Bissett (1984,
1991a-c, 1992), and additionally including new species
described since then (Samuels et al 1998, 1999, 2000,
2002) To this end, we have chosen the multicopy loci
ITS1, ITS2, 28S-rDNA, and the small mitochondrial
rDNA subunit, and fragments from two single-copy
gene loci, viz translation elongation factor 1 (Berney,
Pawlowski & Zamnetti 2000), and endochitinase 42
(Lieckfeldt et al 2000) In addition, 18S- and
28S-rDNA sequence analyses were used to delimit the
genus and to estimate the chronology of its evolution
MATERIALS AND METHODS
Material studied
All strains listed in Table 1 are stored at — 70 °C in
50 % glycerol in the first author's (CPK) laboratory
The ingroup taxa were chosen to represent the complete
range of taxa described as members of the genus
Trichoderma by Bissett (1984, 1991a-c, 1992) and
Samuels et al (1998, 1999, 2000)
DNA extraction, PCR amplifications and sequencing
DNA was isolated from fresh mycelium as described
previously (Turner et al 1997) A region of nuclear
rDNA, containing the ITS1 and 2 and the 5 8S rRNA
gene, was amplified by PCR using the primer combi-
758
nations SR6R and LR1 in 50 µl volumes (White et al.
1990), in an automated temperature-cycling device
(Biotron, Biometra, Goettingen), using the following
parameters: 1 min initial denaturation at 94 °, followed
by 30 cycles of 1 min denaturation at 94 °, 1 min primer
annealing at 50 °, 90 s extension at 74 °, and a final
extension period of 7 min at 74 °.
28S r-DNA was amplified by PCR using the primer
combinations LS1 and LSD (White et al. 1990) in
100 µl volumes using the following amplification para-
meters : 1 min initial denaturation at 94 °, followed by
30 cycles of 1 min denaturation at 94 °, 45 s primer
annealing at 56°, 90 s extension at 72 ° and a final
extension period of 7 min at 72°.
A 0.7kb fragment of the mitSSU rDNA was
amplified with the primer pair MS1 and MS2 (White et
al. 1990), using the following amplification protocol:
1 min initial denaturation (94 °), 35 cycles each of 30 s
at 94°, 30s at 52°, and 90s at 72°, and a final
extension period of 7 min at 72 °.
A 0.2 kb fragment of tef1 was amplified by the
primer pair tef1fw
(5'-GTGAGCGTGGTATCACCA-TCG-3') and teflrev
(5'-GCCATCCTTGGAGACCA-GC-3'), and the
following amplification protocol: 1 min initial
denaturation (94 °), 30 cycles each of 1 min at 94°, 1
min at 59°, and 50s at 74°, and a final extension period
of 7 min at 74°.
A 0.4 kb fragment of ech42 was amplified by the
primer pair Chit42-la
(5'-GCTYTCCATCGGTGGC-TGGAC-3') and
Chit42-2a (5'-GGAGTTGGGGTA-GCTCAGC-3'),
and the following amplification protocol : 1 min initial
denaturation (94°), 30 cycles each of 1 min at 94 °, 1
min at 62 °, and 1 min at 74 °, and a final extension
period of 7 min at 74°.
Five µl from each PCR reaction were electrophoresed
on 1.5% agarose minigels (containing 0.5 mg 1-1
ethi-dium bromide) for 1h in Tris-acetate buffer
(Sambrook et al. 1989). The PCR products were
revealed under uv-light. Template DNA (100 µl) was
directly prepared from PCR products by purifying it
with a commercial kit (Cleanmix; Talent, Trieste), and
sequenced with the aid of a LI-COR 4000L automatic
sequencing system (Middendorf et al. 1992), using
cycle-sequencing (Robo-cycler 40; Stratagene, La
Jolla) with the Thermo-Sequenase-kit (Amersham
Biosciences, Piscataway) as described previously
(Kindermann et al. 1998). The sequences of isolates
submitted to GenBank, as indicated in Table 1, are
the sequences used in all phylogenetic analyses.
Sequences not obtained during this work are given by
their respective GenBank accession numbers.
Sequence analysis
DNA sequences were aligned using MULTALIN
(http://prodes.toulouse.inra.fr/multalin/multalin. html)
based on the algorithm of Waterman (1986), and
corrected by visual inspection. Single gaps were
treated as a fifth nucleotide. All gap positions within
C. M. Kullnig-Gradinger, G. Szakacs and C. P. Kubicek 759

Table 1. Trichoderma strains used and gene sequence accession numbers obtained in this study*
GenBank accession nos
Collection
Species name and number ITS1 and ITS2 28S rDNA mitSSU tef1 ech42
Hypocrea andinensis CBS 345.97 X93957 AF400743 AF400748 AF401010 -
H. atrogelatinosa CBS 237.63 AF400751 - - - -
H. aureoviridis CBS 245.63 Z48819 AF399219 AF400750 AF401002 -
H. citrina CBS 977.69 AF400254 AF399220 AF399203 AF401028 AF486004
H. citrina var. americana CBS 976.69 AF400255 AF399221 AF399204 AF401026 -
H. hunua CBS 238.63 AF400257 AF399222 AF399208 AF401011 AF400745
H. jecorina QM 6a Z31016 AF400741 AF399176 AF401004 -
H. lactea CBS 853.70 AF400253 AF399223 AF399205 AF401027 AF399257
H. orientalis PPRI 3894 Z93930 AF400744 AF399211 AF401009 -
H. novaezelandiae CBS 639.92 X93968 AF399224 AF400749 AF401012 -
H. pilulifera CBS 814.68 Z48813 AF400739 AF399183 AF401014 AF486003
H. schweinitzii ICMP 1694 X93965 AF400738 AF399210 AF401008 -
H. tawa CBS 246.63 AF400258 AF399225 AF399191 AF401006 AF399258
H. vinosa CBS 960.68 AF191038 AF399226 AF399195 AF401007
Trichoderma asperellum CBS 433.97 AJ230668 AF127153 AF276100 AF401000 AF276652
T. atroviride DAOM 165779 Z48817 AF399227 AF276090 AF400997 AF276650
T. citrinoviride CBS 258.85 Z31017 AF399228 AF399210 AF401008 -
T. crassum CBS 336.93 AF011946/ AF399229 AF399189 AF401021 AF399259
T. croceum DAOM 167086 AF400259 AF399230 AF399185 AF400995 AF486002
T. fasciculatum CBS 118.72 AF011950/AF398491 AF399231 AF399199 - AF399260
T. flavofuscum CBS 248.59 AF011953/AF398492 AF399232 AF399190 AF401020 AF399261
T. fertile DAOM 167161 AF400260 AF399233 AF399207 AF401025 AF399262
T. ghanense CBS 259.85 Z31015 AF399234 AF399215 AF400987 -
T. hamatum DAOM 167057 Z48816 AF399235 AF399181 AF401019 -
T. harzianum CBS 226.95 AF222720 AF399236 AF276094 AF401016 AF276646
T. harzianum NR5555 AF 194009 - - - AF399263
T. harzianum NR6938 AF194013 - - - AF399264
T. harzianum gt3 TUB-776 AF149866 - - - AF399265
T. harzianum gt4 TUB-781 AF149870 - - - AF399266
T. harzianum gt3 TUB-768 AF 149862 - - - AF399267
T. harzianum gt2a TUB-762 AF 149866 - - - AF399268
T. harzianum gt2a TUB-477 AF 1498 52 - - - AF399269
T. harzianum gtl TUB-743 AF149855 - - - AF399270
T. inhamatum CBS 273.78 Z68187 AF399237 AF399194 AF401015 AF399271
T. konilangbra GJS 96-146 AF400261 AF399238 AF399216 AF400988 -
T. koningii CBS 979.70 Z79628 AF399239 AF399177 AF400990 AF188918
T. longibrachiatum CBS 816.68 Z31019 AF399240 AF399212 AF400991 -
T. longipilis CBS 340.93 AF011975/AF398493 AF399241 AF399188 AF401323 AF399272
T. minutisporum CBS 341.93 AF011932/AF398494 AF399242 AF399186 AF400996 AF399273
T. oblongisporum CBS 343.93 AF011976/AF398495 AF399243 AF399196 AF401022 AF400746
T. polysporum CBS 820.68 Z48815 AF399244 AF399184 AF400989 AF486001
T. pubescens DAOM 162.162 AF011979/AF398496 AF399245 AF399182 AF401001 -
T. pseudokoningii CBS 408.91 Z31014 AF400740 AF399214 AF400986 -
T. saturnisporum ATCC 18903 Z48726 AF399246 AF399213 AF401013 -
T. spirale CBS 346.93 AF011978 AF399247 AF399187 AF401018 AF399274
T. strictipile CBS 347.93 AF400263 AF399248 AF399200 AF401324 AF399275
T. strigosum CBS 348.93 AF011982/AF398497 AF399249 AF399180 AF401023 -
T. stromaticum CBS 101.875 AF098287 AF399250 AF399201 AF400994 AF399276
T. tomentosum CBS 349.93 AF011984/AF398498 AF399251 AF399198 AF401024 AF399277
T. virens CBS 249.59 AF222865 AF399252 AF276089 AF400998 AF276654
T. viride ATCC 28020 AJ230678 AF127150 AF399178 AF400992 AF188928
T. cfr aureoviride DAOM 175924 AF191039 AF399253 AF399197 AF401017 AF399278
T. aggressivum f. europaeum CBS 435.95 AF400264 AF399254 AF399192 AF400993 AF399279
T. aggressivum f. europaeum CBS 450.95 AF400265 - - - AF399280
T. aggressivum f. aggressivum CBS 100.527 AF400266 - AF399193 - AF399281
T. aggressivum f. aggressivum DAOM 222.156 AF400266 - - - AF399282
T. sp. 1 Cornell 9 AF400267 AF399255 AF399206 AF401005 -
T.sp. PPRI 3559 AF400267 AF399256 AF399202 AF401003 AF486005
* Only strains sequenced in this study are given.
I1, ITS1; I2, ITS2; 28 S, Dl and D2 region of the 28S rRNA; mtSSU, fragment of the small subunit of mtRNA; tef1, translation elongation
factor alpha, fragment including intron 1; ech42, fragment of endochitinase 42.
ATCC, American Type Culture Collection, Manassas; CBS, Centraalbureau voor Schimmelcultures, Utrecht; IMI, CABI Bioscience UK
Centre, Egham; PPRI, Plant Protection Research Institute, Pretoria. Origin of strains designated with NR and TUB have been described by
Fujimori & Okuda (1994) and Kullnig et al. (2000). gt1, gt2a, gt3, and gt4 indicates the genotypes of T. harzianum, as defined by Kullnig
et al. (2000).

the alignment exceeding a single base in length were
considered as missing information and therefore re-
placed by question marks. Sequence insertions without
homology to any of the other sequences were deleted in
the alignment and a single base was left over causing a
minimal gap. In this way, gaps of all sizes were
weighted equal corresponding to a hypothetical single
event in evolution. Data were analyzed by the programs
contained in the PHYLIP version 3.5.c package
(http://evolution.genetics.washington.edu/phylip). To
Phylogeny of Trichoderma
find the most likely tree topology, phylogenetic trees
were generated by parsimony and distance methods.
The DNAPARS program was used to carry out
unrooted parsimony analysis for investigating the tree
topology, invoking the jumble option (n = 25). Support
for the branches based on parsimony criteria were
estimated by bootstrap analysis of 500 replicates
generated with SEQBOOT. Each bootstrap replicate
was analyzed by DNAPARS and a majority-rule
consensus tree was generated by CONSENSE. The
consensus of trees arising from phylogenetic analysis of
a large number of bootstrap resampled data sets
provides an indication of support for specific branches
in the trees; this support is proportional to the fraction
of resampled data sets supporting trees having the
specific branch (Felsenstein 1985). Distance matrices
were computed using DNADIST with the same
alignment used for parsimony analysis. Distance (vari-
ability) is defined as probability of nucleotide substitu-
tions per site, based on the two-parameter model of
Kimura (1981). Topology and branch lengths of
phylogenetic trees were also evaluated by the least
square method of Fitch & Margoliash (1967) from the
distance matrices, using FITCH programme.
R ES U LT S
Evolution on the basis of 18S and 28S rDNA sequence
analysis
Data from 18S and 28S rDNA analysis are useful in
establishing the coarse-scale phylogeny of ascomycetes
(Berbee & Taylor 1992, Berbee et al. 1995), and for
sorting them into statistically supported family-level
groupings. In addition, 18S rDNA sequence data have
been used to calibrate the time of evolution of fungi
(Berbee & Taylor 1993). We therefore first used 18S and
28S sequence analysis to establish the origin of
Trichoderma within the ascomycetes. Preliminary se-
quence analysis of 18S rDNA of several Trichoderma
spp. has shown that there is very little variation within
the genus, and that these variations do not lead to
statistically supported subclades in parsimony analysis
(data not shown). We have therefore used only the 18S
rDNA sequence of Hypocrea schweinitzii to test the
evolution of the genus Trichoderma within the ascomy-
cetes. To this end, we performed a BLAST search with
the H. schweinitzii I8S-rDNA gene to find most similar
sequences in GenBank. These were then used for
parsimony analysis using Emericella nidulans
(Euro-tiales) as an outgroup. As can be seen from Fig.
1, H. schweinitzii was contained in a well-supported
(74%) subclade together with H. lutea and
Aphysiostroma stercorarium, accompanied by
Hypomyces aurantius and Sphaerostilbella aureonitens
as a sister subclade. Together with two other clades
containing members of the Nectriaceae and the
Clavicipitaceae, this clade emerged from a basal
branch containing Neurospora crassa. Distance
analysis (DNADIST) showed that the
760
three subclades had approximately similar genetic
distances from Neurospora. Thus, in view of published
molecular clock estimates for Neurospora crassa and
Sporophagomyces chrysospermus (180 and 110 Myr,
respectively; Berbee & Taylor 1993) and Gibberella
fujikuroi (Nectriaceae; 110 Myr; O'Donnell et al. 1998),
and assuming a similar rate of nucleotide changes in the
three fungi and H. schweinitzii, it is reasonable to
assume that the genus Trichoderma also evolved about
100-110 Myr.
To analyse whether the genus Trichoderma is mono-
phyletic, a parsimony analysis of 28S rDNA-sequences
of selected species of Trichoderma, covering the whole
genetic variation observed in this study (see below) and
of other species belonging to possibly closely related
genera of the family Hypocreaceae (Aphysiostroma,
Arachnocrea, Hypomyces, Sphaerostilbella; Rossman et
al. 1999, Poldmaa 2000) was performed (Fig. 1). This
showed that the four species chosen for this study
(Hypocrea aureoviridis, T. harzianum, T. viride, T. longi-
brachiatum) formed a well (92%) supported cluster
with H. citrina and H. lutea, and to which Aphysiostroma
stercorarium, Arachnocrea stipata and Sporophago-
myces chrysostomus also clustered in strongly supported
basal positions. The genera Sphaerostilbella and Hypo-
myces formed corresponding, statistically well sup-
ported sister clades. These data show that the genus
Trichoderma is indeed monophyletic, but in itself only
arising at the merging front of a larger complex
containing several genera of the family Hypocreaceae.
Nevertheless, the high bootstrap support for its bran-
ching from its closest neighbor (A. stercorarium) justifies
treating Trichoderma, as currently defined, as mono-
phyletic.
Phylogeny based on analysis of multiple genetic loci
To establish a phylogeny of Trichoderma, we amplified
the region spanning the nuclear ribosomal internal
transcribed spacer (ITS1 and 2), the Dl and D2 region
of the 28S rDNA, the small subunit of the mitochondrial
rDNA (mitSSU), the fifth and part of the sixth exon of
translation elongation factor 2 (tef1), and a 610-bp
fragment of the endochitinase gene ech42, from 47
different species of Trichoderma and Hypocrea, which
include all the species described by Bissett (1991a-c,
1992) and Samuels et al. (1998, 1999, 2000, 2002).
Where available, the ex-type strain was used (Table 1).
Otherwise, strains whose identity has been confirmed
by morphological, physiological and genetic characters
were used. In addition, three still unnamed new species
(T. sp. 1, T. cfr aureoviride DAOM 175924, and T. sp.
PPRI 3559), were included because of their unique
position in the ITS1 phylogenetic tree (Kindermann et
al. 1998, Lieckfeldt et al. 2001, C.K.-G. & C.P.K.,
unpubl.). tef1 could not be amplified from one species
(T. fasciculatum) and this was therefore coded as
unknown. In addition, we failed to amplify ech42 from
some species of section Longibrachiatum, and therefore
C. M. Kullnig-Gradinger, G. Szakacs and C. P. Kubicek 761

Fig. 1. Phylogenetic tree showing the position of the genus Trichoderma as inferred by parsimony analysis of 18S (A) and
28S sequences (B), and invoking the jumble option (15 different orders of input sequences) on 500 bootstrapped data sets.
The tree shown is the percentage-consensus of a number of equally parsimonious trees. The numbers given above the
branches indicate the number of times the group consisting of the species which are to the right of that fork occurred
among the trees out of 100 trees (Felsenstein 1985), and are given only for values > 50. The numbers below the branches in
the 18S tree are the distance values (x 103) determined by DNADIST. The boxed species and the arrow in the 18S tree
indicate the phylogenetic position of the species which were analyzed in more detail in the 28S tree.
Sequences used for this comparison were obtained under the following GenBank accession numbers: (18S tree)
Cladobotryum obdavatum (AF049145), Hypomyces aurantius (AF055297), Sphaerostilbella aureonitens (U32416), Hypocrea
schweinitzii (AF164357), Aphysiostroma stercorarium (U32398), Hypocrea lutea (AB027338), Gibberella avenacea
(AF141946), Bionectria ochroleuca (AB003950), Gibberella pulicaris (AF081467), Fusarium oxysporum (AF141951),
Fusarium solani (E17084), Hypocrea schweinitzii (AF164357), Claviceps purpurea (U44040), Lecanicillium psalliotae
(AJ292390), Lecanicillium lecanii (AF 108490), Neurospora crassa (X04971), Emericella nidulans (U77377); (28S tree)
Hypomyces aurantius (U00742), Hypomyces australis (U00743), Hypocrea lutea (U00739), Aphysiostroma stercorarium
(U47820), Hypocrea pallida (U00741), Bionectria ochroleuca (U00750), Arachnocrea stipata (AF160227), Sporophagomyces
chrysostomus (AF160235), Sphaerostilbella aureonitens (U00755), Sphaerostilbella lutea (U00757). Sequences of Trichoderma
spp. were obtained during this work and are given in Table 1.

this gene was omitted from all species of section
Longibrachiatum. Further, the β-tubulin encoding gene
tub1, which has been used successfully with other fungi
(Schardl et al. 1994, O'Donnell et al. 1998), was used in
preliminary trials but later abandoned as we found that
some species contain two copies with different sequences
(C.K.-G. & C.P.K., unpubl.). During this analysis, the
5.8S rDNA sequence was found to be identical in all
species, and thus subsequently excluded from the
analysis. Twenty-eight out of 210 positions of ITS1,
and fourteen out of 188 positions of ITS2 were excluded
as their alignment was ambiguous. The sequence of the
intron between exon 5 and 6 of tef1 was also highly
variable and could only be safely aligned between
closely related species, and this region therefore was
also omitted from the alignment, leaving 156-bp
informative nucleotides in tef1. All other gene sequences
could be well aligned. The corresponding sequences
were fused to form a continuous 1973-bp nucleotide
sequence which was subjected to parsimony analysis,
using H. aureoviridis as an outgroup (clade D). The
combined gene tree inferred from these sequences (Fig.
2) showed a highly supported clade (C) comprising the
species belonging to sect. Longibrachiatum as recently
redefined (Samuels et al. 1998). The remaining species
split into two strongly supported clades, A and B.
Clade A contained three species assigned to section
Trichoderma (T. viride, T. atroviride, T. koningii) and
also T. asperellum, T. hamatum, T. strigosum, and T.
pubescens. The occurrence of the latter four Trichoderma
species in clade A is in accordance with previous data
based on ITS1 and 2 and ech42 sequence analysis, and
thus confirms earlier results based on single gene
analyses that section Pachybasium is paraphyletic
(Kindermann et al. 1998, Lieckfeldt et al. 1999, 2000).
Clade B contained most of the species investigated. It
formed five strongly supported subclades: subclade Bl
(Hypocrea lactea, H. citrina, H. citrina var. americana,
and Trichoderma sp. 1); subclade B2 (T. stromaticum,
T. sp. PPRI3559); subclade B3 (T. fertile, T. oblongi-
sporum and H. hunua); subclade B5 (T. polysporum, T.
croceum, H. pilulifera and T. minutisporum); and a
Phylogeny of Trichoderma 762

Fig. 2. Phylogenetic tree of 47 different species of Trichoderma as inferred by parsimony analysis of ITS1 and ITS2-, 28S-,
mitSSU DNA-, tef1- and ech42-sequences, and invoking the jumble option (15 different orders of input sequences) on 500
bootstrapped data sets Note that ech42 sequences for species of section Longibrachiatum were not included and thus coded
as unknown The tree shown is the percentage-consensus of a number of equally parsimonious trees Sequences obtained
during this study are given by their EMBL-numbers in Table 1
Sequences from species of section Trichoderma (clade A) were taken from Lieckfeldt et al (2000) See Fig 1 (legend)
re-numbers above the branches Branches supported by Bootstrap coefficients < 50 were collapsed Arrows indicate species
which were listed as species aggregates by Rifai (1969) Vertical grey bars indicate species considered by Bissett (199la) to
belong to sect Hypocreanum, black triangles indicate species considered by Rifai (1969) to belong to Gliocladium, black
diamonds indicate species considered by Bissett (1991b) to belong to sect Pachybasium Clades of strains, which were
considered by Bissett (1991a) to belong to sects Trichoderma and Hypocreanum, and to Longibrachiatum as revised by
Samuels et al (1998), are boxed The broad vertical bars indicate clades A-D and their putative name Despite of the
position of the type strain for sect Pachybasium (T. hamatum) in clade A, the name 'Pachybasium' is maintained in this
grouping until a more appropriate name has been found The thin vertical bars indicate subclades B1-B5

complex subclade B4, in which three further strain
clusters could be distinguished: one containing T.
harzianum, T. inhamatum, H. vinosa, and the two
formae of T. aggressivum, T. aggressivum Samuels &
W. Gams, sp. nov. forma aggressivum and T. aggres-
sivum Samuels & W. Gams, sp. nov. forma europaeum
(Samuels et al. 2002); a second one containing T.
fasciculatum, T. longipile, and T. strictipile; and a third
one containing T. virens, T. flavofuscum, and T. crassum.
The position of the remaining species (T. spirale, T. cfr
aureoviride, H. tawa, and T. tomentosum) was essentially
unresolved.
Separate analyses of the individual sequenced gene
fragments resulted in tree topologies that were con-
cordant with that of the combined sequence analysis at
the level of clades A-D. The topologies of clades A
(sect. Trichoderma) and C (sect. Longibrachiatum) were
essentially the same as in the combined tree, and, in
C. M. Kullnig-Gradinger, G. Szakacs and C. P. Kubicek
view of their previous detailed treatment (Kuhls et al.
1998, Lieckfeldt et al. 1999, 2000), therefore omitted
from the presentations. The best resolution of clade B
was obtained by ech42 and tef1, which were superior to
ITS1. ITS1 was still superior to mitSSU and ITS2. 28S
rDNA provided the poorest resolution.
763
Fig. 3. Resolution of clade B in individual gene
trees, i.e. ech42, tef1 and ITS1 +2. One of a
number of equally parsimonious trees is shown in
each case. Only two species of clade B1 (H.
citrina, H. lactea) were included in the ech42
alignment, as the species contained in clade B1
were consistent in all other gene trees; also sect.
Longibrachiatum, sect. Trichoderma and H.
aureviridis were omitted, and the tree rooted with
T. viride as an outgroup. The ITS1 and 2 sequence
of H. lutea (AF020799, AF048736, and
AB027384; all three sequences are identical) was
additionally included in the ITS1 and 2 tree. See
Fig. 1 (legend) re-numbers above the branches.
The dashed lines indicate branches that collapsed
in the strict consensus tree.
The individual gene trees for ech42, tef1 and ITS1
and 2 are shown in Fig. 3. Subclade B5 (T. polysporum,
T. croceum, T. minutisporum and H. pilulifera) con-
sistently clustered in a basal position of clade B.
Subclusters B1 (containing species attributed to sect.
Hypocreanum by Bissett 1991a), B2 (containing T.
Phylogeny of Trichoderma
stromaticum and T. sp. PPRI 3559) and B3 (T. fertile,
H. hunua) were also clearly apparent, although the
branches separating them from each other received less
clear bootstrap support in the ITS1 and 2 and in the
tef1 tree. It was thereby interesting to note that T.
oblongisporum clustered with subclade B4 in the ITS1
and 2 tree, whereas it received strong support as
member of subclade B3 in the tef1 and ech42 tree.
Subcluster B4 was poorly resolved in the ITS1 and 2
tree, although a clustering of T. harzianum with H.
vinosa and T. inhamatum, as well as one of T. virens
with T. crassum and T. flavofuscum was apparent, tef1
and ech42 further showed a cluster containing T.
fasciculatum, T. strictipile and T. longipile. T. longtpile
was thereby a further case of a phylogenetically
'migrating' species as it clustered with T. spirale in the
ITS1 and 2 tree.
When this manuscript was being prepared for
publication, the ITS1 and 2 sequences for Gliocladium
viride (AF048736), G. deliquescens (AF020799), and
Hypocrea lutea (AB027384) became available; all these
are considered to be the same species, H. lutea. Rehner
& Samuels (1994) already showed that this species,
although morphologically atypical in Trichoderma, is a
member of the genus. In order to investigate its
phylogenetic position within Trichoderma, we included
these sequences in the parsimony analysis of ITS1 and
ITS2. This analysis showed that it clustered in subclade
Bl with other species of sect. Hypocreanum (Fig. 3).
Dodd et al. (2000) reported that the presence of some
species of Trichoderma (e.g. T. inhamatum GJS 90-90)
which, according to the deposited sequence and
morphological analysis (G. J. Samuels, pers. comm.), is
T. minutisporum) in the alignment corrupts tree top-
ology. We therefore investigated whether the omission
of any of the five subclades described above, or of
individual species would result in a different degree of
resolution, particularly of subclade B4. However, while
the bootstrap coefficients at some of the nodes were
actually increased or decreased by 10-15%, this
procedure did not strongly change branch support, and
in no case was a new topology obtained (data not
shown). We thus conclude that the variability of the
gene sequences used is not sufficient to phylogenetically
resolve all the species in the large clade B.
Phylogenetic resolution of Trichoderma harzianum
genotypes
Trichoderma harzianum is so-far unique in Trichoderma,
as it exhibits a considerably higher intraspecific gene
sequence diversity than other species such as T. virens
or T. spirale, or species from sects Trichoderma or
Longibrachiatum (Gams & Meyer 1998, C.K.-G. &
C.P.K., unpubl.). We have previously defined five
different 'genotypes' (actually haplotypes) of T. harzi-
anum on the basis of hallmark sequences in ITS1 and 2
(Kullnig, Szakacs & Kubicek 2000). In order to find out
whether all of these genotypes would still be members
764
Fig. 4. Phylogenetic tree of Trichoderma spp. forming part of
clade B4 including several genotypes of T. harzianum (Kullnig
et al. 2000) inferred by parsimony analysis of combined ITS1
and 2 and ech42-sequences, and invoking the jumble option
(15 different orders of input sequences) on 500 bootstrapped
data sets. The tree shown is a single of 54 most parsimonious
trees. See Fig. 1 (legend) re-numbers over the branches. The
dashed lines indicate branches that collapsed in the strict
consensus tree.
of the 'T. harzianum-cluster' of subclade B4, we
included members of each of these five genotypes in a
combined parsimony analysis of ITS1 and 2 and ech42
(Fig. 4). As can be seen, the ex-type strain (= genotype
5) clustered in a basal position with T. inhamatum and
two strains originally considered to be T. aureoviride
(Fujimori & Okuda, 1994) but recently reidentified as
T. harzianum (Lieckfeldt et al. 2001) and whose ITS1
and 2 sequence are different from any of the
ITS-genotypes identified by us earlier (Kullnig,
Szakacs & Kubicek 2000). Interestingly, the remaining
genotypes grouped with high support in the same way
(i.e. gt3 and gt4 vs. gt1 and 2a) as in RAPD analysis
(Kullnig, Szakacs & Kubicek 2000), thus supporting
the assumption that they are indeed clonal lines. They
all were contained in the ' T. harzianum cluster'.
DISCUSSION
Teleomorphs of the genus Trichoderma, where known,
are in the genera Hypocrea, Podostroma and Sarawakus
of the Hypocreaceae (Gams & Bissett 1998, Rossman et
al. 1999). This raises two possibilities, viz. either that
Trichoderma is paraphyletic or that these genera are
synonyms of Hypocrea. The bulk of evidence (Poldmaa
2000, G. J. Samuels, pers. comm.) strongly suggests the
latter, and that Trichoderma (anamorph) and Hypocrea
(teleomorph) are a single holomorph genus. Unfor-
tunately, the type species for Podostroma and Sarawa-
kus were unavailable for study, but the 28S gene tree
nevertheless shows that Trichoderma and Hypocrea
C. M. Kullnig-Gradinger, G. Szakacs and C. P. Kubicek
occurred in a well supported terminal subclade of a
larger clade that also contained the genera Aphysio-
stroma, and Arachnocrea; Sphaerostilbella was a sister
clade. Aphysiostroma stercorarium, which Barassa et al.
(1985) speculated to be related to the Hypocreaceae,
clustered in a strongly supported basal position to
Trichoderma. A close relationship of this fungus with
Hypocrea has also been found in 18S rDNA sequence
analysis, thereby confirming earlier data by Spatafora
& Blackwell (1993). Barassa et al. (1985) already noted
the morphological similarity of A. stercorarium with
certain Hypocrea spp. such as H. citrina and H.
pulvinata that have Verticillium-like anamorphs.
Morphologically based taxonomy has divided the
genus Trichoderma into five sections: Trichoderma,
Pachybasium, Longibrachiatum, Saturnisporum and
Hypocreanum (Doi et al. 1987, Bissett 1991a). Of these,
Longibrachiatum and Saturnisporum were merged by
Samuels et al. (1998). The use of molecular and
biochemical analysis has recently confirmed sect.
Longibrachiatum (including Saturnisporum) (Kuhls et
al. 1997). The sects Pachybasium and Trichoderma, on
the other hand, were found to be unnatural
(Kinder-mann et al. 1998, Lieckfeldt et al. 1998,1999).
However, most of these molecular approaches had
used rDNA sequence analyses only, and confirmation
by analyses of other loci was thus warranted (cfr Taylor
et al. 1999, Lieckfeldt & Seifert 2001). The present
paper shows that such an approach fully supports the
phylogenetic revisions based on the ITS1 and ITS2
sequence analyses, namely the genetically distant
position of T. aureoviride (Lieckfeldt et al. 2001), and
the clade of species belonging to sects
Longibrachiatum and Trichoderma. Although the latter
clade, because of the presence of T. hamatum, T.
pubescens and T. strigosum, is different from the sects
as defined by Bissett (1991a), the strong support for the
clades containing species of sections Longibrachiatum
and Trichoderma (clade A and C), suggests clade B is
interpreted as the phylogenetic equivalent of section
Pachybasium. Yet, this is problematic for two reasons:
first, the nomenclatural type of the section,
Trichoderma hamatum, does not occur in clade B, and
the name therefore is not consistent with the phylogeny
of this section. In addition, all the species attributed by
Bissett (1991a) to sect. Hypocreanum occured in a
strongly supported subclade (B1) of clade B,
suggesting that the two sections should be merged. We
agree with Lieckfeldt & Seifert (2001) in maintaining the
name 'Pachybasium' for this large heterogeneous
section, ' pending the eventual formal renaming of this
clade', as the majority of species of Bissett's (1991b)
sect. Pachybasium occur in clade B. However, as a
future perspective, the concept of this section should be
strongly revised or replaced by more appropriate terms
such as phylogenetic lineages or clusters.
The presence of species of section Hypocreanum as a
subclade (B1) in clade B, was unexpected. Samuels
(1996) hypothesized that the anamorphs of species of
Hypocrea with effused stromata having colourless
765
conidia on irregularly verticillate conidiophores, which
Bissett (1991a) placed in Trichoderma sect. Hypo-
creanum, may be synanamorphs or spermatial states,
and therefore inappropriately placed in Trichoderma.
Gams & Bisset (1998) therefore omitted sect. Hypo-
creanum from their treatment of Trichoderma. Our
work, however, leaves no doubt that phylogenetically
they are members of the holomorphic genus, despite the
striking contrast in the morphology of the anamorphs.
Seifert & Samuels (2001) discussed the speciation of
anamorphs, and stressed the existence of distinct
synanamorphs within one species which may play
different roles in the survival strategies of the organism.
In applying their concept to the species in Hypocrea
sect. Hypocreanum, we postulate that the ability to
form the Trichoderma-like anamorph of these species
may have been lost during evolution. A similar
explanation may account for the presence of H. lutea in
clade B1, whose phylogenetic affinity to H. gelatinosa
and T. virens has already been documented by Rehner
& Samuels (1994), but whose morphology makes an
integration into the species concept of Trichoderma
difficult (Samuels 1996). It should be noted that the 28S
gene tree implies that the genus Trichoderma has
evolved from genera having Verticillium-like anamor-
phs (Aphysiostroma, Arachnocrea), and we thus favour
the interpretation that the ability to form the Tricho-
derma-like anamorph morphology, which may have
supported the switch from fungicolous to saprophytic
habitats, was subsequently lost in some evolutionary
lines. It is further noted that another Hypocrea species,
H. hunua, which Bissett (1991a) included in sect.
Hypocreanum, was not contained in subclade B1 but
formed a strongly supported species pair with T. fertile
and T. oblongisporum (subclade B3). This indicates that
the loss of genes (or their expression) required to form
the Trichoderma-like anamorph occured more than one
time in evolution.
Both the combined gene analysis as well as the
individual gene trees provided convincing support for
close phylogenetic relationships among species of clade
B. In some cases, morphological relationships were
confirmed, e.g. subclade B1 contained T. polysporum
and T. croceum, which are closely similar in mor-
phology ; in the same way, the branches in subclade B4,
which lead to T. virens, T. crassum and T. flavofuscum,
to T. strictipile and T. fasciculatum, and to T. harzianum,
T. inhamatum and T. aggressivum, respectively, indicate
correlation between phylogeny and morphology. How-
ever, this correlation was contradicted in several other
cases: an example is the presence of T. minutisporum in
subclade B5, which does not show the apical
conidio-phore extensions of T. polysporum and T.
croceum, and has green instead of colourless or yellow,
respectively, conidia. Similarly, other species with
highly similar morphology (e.g. T. oblongisporum and T.
longipilis; T. spirale and T. fertile; T. minutisporum and
T. tomentosum) occurred at separated phylogenetic
positions. This indicates that many of the
morphological characters of
Phylogeny of Trichoderma
Trichoderma were independently gained or lost several
times during the evolution of the genus.
While the different gene trees, depending on their
resolution, showed consistent topologies for most
species, a few species were noted the exact position of
which was ambiguous (e.g. T. oblongisporum, T.
fasciculatum, T. longipile). These species shuttled be-
tween different subclades or branches in clade B,
strongest deviations thereby being observed in the ITS 1
and 2 tree. Similar results were also reported by Dodd
et al. (2000), who stressed that ITS1 and 2, while
successfully differentiating among morphologically dis-
tinct isolates, were not sufficient to resolve phylogenetic
relationships among species. One explanation for this
fact may therefore be that in these species ITS1 and 2
evolved at a much more different rate than tef1 and
ech42. Alternatively, these species may have arisen by
hybridization, with subsequent loss of one of the two
parental genes during anamorph speciation. Such a
mechanism has not yet been shown in Trichoderma, but
is known in Neotyphodium (Schardl et al. 1994).
However, with the exception of these examples,
phylogenies based on ITS1 and 2 sequence analysis
were consistent with those generated for other genes.
Thus, in contrast to Fusarium (O'Donnell et al. 1998),
paralogous copies of ITS1 and ITS2 appear to be
absent from Trichoderma, thus validating the use of ITS
sequence analysis for strain phylogeny and particular
strain diagnosis. In fact, with the exception of T. viride,
T. koningii and T. atroviride, which have very narrow
and overlapping nucleotide variations (Lieckfeldt &
Seifert 2001), and also of T. harzianum/T. inhamatum
(for reasons given below), all the described species
exhibited unique ITS1 sequences (C.K.-G. & C.P.K.,
unpubl.), which valididates their use as a diagnostic
marker for identification of strains at the species level.
We have already made use of this tool in biodiversity
studies of Trichoderma (Kullnig, Szakacs & Kubicek
2000, Kullnig-Gradinger et al. in prep.), which led to
the identification of at least ten new species, the formal
description of which is currently underway (J. Bissett,
G.S., C.K.-G. & C.P.K., unpubl.).
In contrast to the identity or high similarity of ITS1
and 2 sequences in most taxa, T. harzianum and T.
inhamatum exhibited a considerably higher variation in
the ITS1 (5-9 bp's), and also ITS2 (2-5 bp's) sequences
with respect to the neotype isolate. This prompted
Kullnig, Szakacs & Kubicek (2000) to define five
'ITS-genotypes' of T. harzianum, of which some pairs
(gtla and gt2, and gt3 and gt4, respectively) gave
identical RAPD profiles. Interestingly, these genotype
pairs also formed strongly supported subclades in
combined sequence analysis of ITS1 and 2 and
ech42. This supports the speculation that some of
these genotypes may be unrecognized species or
subspecies. However, in a study of a much larger
number of T. harzianum strains, neither nucleotide
nor morphological data supported taxonomic
subdivision of T. harzianum (P. Chaverri & G. J.
Samuels, unpubl.), while recognizing
766
the existence of phylogenetic lineages that may be in the
process of speciation. In addition, the present data
clearly show that phylogenetically, T. inhamatum and
T. harzianum are indistinguishable and their distinction
(Gams & Meyer 1998) is at the moment merely based
on minor morphological differences which may be
untenable (P. Chaverri & G. J. Samuels, pers. comm.).
ACKNOWLEDGEMENTS
This work was supported partly by OTKA grant No. T029387 and
FKFP 516/1999 grant to G.S., and an FWF-grant (P-12748-MOB)
to C.P.K. The authors are grateful to the two anonymous reviewers
for their comments on the manuscript, and to Gary J. Samuels for
critical suggestions and the communication of unpublished work. The
assistance of Robert L. Mach from the authors laboratory during an
early phase of this work is gratefully acknowledged.
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Corresponding Editor: D. S. Hibbett