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Hypocrea voglmayrii sp. nov. from the Austrian Alps represents a new phylogenetic
clade in Hypocrea/Trichoderma
Walter M. Jaklitsch
1
Monika Komon
Christian P. Kubicek
Irina S. Druzhinina
Institute of Chemical Engineering, Research Area Gene
Technology and Applied Biochemistry, Vienna
University of Technology, Getreidemarkt 9-166.5,
A-1060 Wien, Austria
Abstract: The holomorph of the new species
Hypo-
crea voglmayrii
(Hypocreales, Ascomycota, Fungi) is
described by a combined approach, using morphol-
ogy of the teleomorph, morphology of the anamorph,
culture studies and phylogenetic analyses of ITS1 and
2,
ech42
and
rpb2
gene sequences. Its anamorph
Trichoderma voglmayrii
is described as a new ana-
morph species. Unlike most other species of
Hypocrea
the teleomorph of
H. voglmayrii
occurs on dry
standing trunks and exhibits well defined black
ostioles. Although exclusively collected at higher
altitudes, this species grows at 35 C in culture.
Hypocrea voglmayrii
develops pale yellowish to green-
ish conidia, a yellowish pigment and a coconut-like
odor on CMD. Phylogenetically,
H. voglmayrii
forms
a distinct, isolated branch between the section
Trichoderma
and the
H. pachybasioides
clade but does
not associate with any of these clades in different gene
trees.
Key words: Ascomycetes,
ech42
,
Hypocrea
,
Hypo-
creales
, ITS, phylogenetic analysis,
rpb2
, systematics,
tef1
,
Trichoderma voglmayrii
INTRODUCTION
Hypocrea
is a genus of saprotrophic and fungicolous
species in which more than 400 names of species and
varieties have been published (cf. http://www.
indexfungorum.org), mostly on the basis of morpho-
logical characters only. However teleomorph mor-
phology is highly conserved in
Hypocrea
and reveals
only subtle variations in macro- and micromorpho-
logical characteristics, which are mostly of little value
in the differentiation and delimitation of species
(Lieckfeldt et al 1998; Dodd et al 2002, 2003;
Druzhinina et al 2004; Lu and Samuels 2003; Lu et
al 2004). For example, many
Hypocrea
spp. collected
worldwide show reddish to brown stromata in varying
tints and contain asci with hyaline ascospores. Most
mycologists identify such teleomorphs as
Hypocrea
rufa
Pers. : Fr., the type species of
Hypocrea
. However
true
H. rufa
is actually a rare species and most isolates
with this teleomorph morphology likely represent
other potentially new species. This underlines the
necessity of combining phenetic examinations of the
holomorph with molecular and physiological analyses
for the description and characterization of new
species of
Hypocrea
(cf. Kraus et al 2004). Most of
the
Hypocrea
holomorphs (i.e. combined anamorph
and teleomorph) known today have been described
from Japan, South East Asia, Australasia, North and
Latin America by Y. Doi and G. J. Samuels and co-
workers (Doi 1966, 1968, 1972, 1973, 1975a, b, 1976,
1978; Samuels et al 1990; Chaverri et al 2001a, b;
Dodd et al 2002, 2003; Druzhinina et al 2004; Lu et al
2004; Samuels et al 1998; Samuels and Lodge 1996;
Seifert and Samuels 1997). In contrast, little is known
about
Hypocrea
spp. from continental Europe.
In a project designed to assess the biodiversity of
Hypocrea
in Central Europe, an apparently unde-
scribed species with peculiar reddish stromata was
collected. This species exhibits remarkable ecological,
phenotypic and genotypic characteristics, which are
described below.
MATERIALS AND METHODS
Isolates and specimens.—Isolates including accession
numbers of gene sequences investigated in this study
are provided (T
ABLE
I). Isolates listed as C.P.K. are those
maintained in the collection of the institute of the
corresponding author. Representative isolates have
been deposited at the Centraalbureau voor Schimmel-
cultures, Utrecht, The Netherlands (CBS). Specimens
of Hypocrea voglmayrii including the type specimen were
deposited in the Herbarium of the Institute of Botany,
University of Vienna (WU).
Single-ascospore isolates were prepared in the following
way from fresh specimens of
Hypocrea
stromata: parts of
stromata were crushed in sterile distilled water. The
resulting suspension was transferred to cornmeal agar
plates (Sigma, St Louis, Missouri) supplemented with 2%
(w/v) D(+)-glucose-monohydrate (CMD), and 1%(v/v) of
an aqueous solution of 0.2%(w/v) streptomycin sulfate
(Sigma) and 0.2%(w/v) neomycin sulfate (Sigma). Plates
were incubated overnight at 25 C. Few germinated asco-
spores or a part of an ascus were transferred to fresh plates
of the same medium, using a thin platinum wire. The plates
Accepted for publication 17 Jul 2005.
1
Corresponding author. E-mail: jaklitsc@mail.zserv.tuwien.ac.at
Mycologia, 97(6), 2005, pp. 1365–1378.
#2005 by The Mycological Society of America, Lawrence, KS 66044-8897
1365
TABLE I. Isolates and sequences used
Species Strain
GenBank Accession Numbers
ITS1 and 2
rpb2 ech42
H. voglmayrii
sp. nov. CBS 117710 DQ086145 DQ086151 DQ086149
CBS 117711 DQ086142 DQ086150 DQ086148
C.P.K. 951 DQ086144
C.P.K. 941 DQ086143
Hypocrea sp.
/
T. viride
Pers. Vd CBS 245.63 Z48819
G.J.S. 89-127 AF545521
ATCC 28020 AF118928
T. pubescens
Bissett DAOM 166162 AF011978/
AF398496
DQ087235
CBS 345.93 AF545552
T. hamatum
(Bon.) Bain. DAOM 167057 Z48816 DQ087237
CBS 102160 AF545548
T. strigosum
Bissett CBS 348.93 AF545556
H. minutispora
B.S. Lu et al/
T. minutisporum
Bissett
DAOM 167069 AY965634 AF399273
CBS 901.72 AY481588
H. pachybasioides
Yoshim. Doi/
T. polysporum
(Link : Fr.) Rifai CBS 820.68 Z48815 DQ087238 AF486001
H. pilulifera
J. Webster & Rifai/
T. piluliferum
J.
Webster & Rifai CBS 814.68 Z48813 AF545519 Z48813
H. citrina
(Tode : Fr.) Fr. CBS 977.69 AY865633 AF486004
CBS 894.85 AF545561
H. pulvinata
Fuckel G.J.S. 98-104 AF545559
H. melanomagna
Chaverri & Samuels/
T.
melanomagnum
Chaverri & Samuels CBS 114236 AY391926
H. lutea
(Tode) Petch/
Gliocladium viride
Matr. G.J.S. 89-129 AF545517
T. oblongisporum
Bissett CBS 343.93 AF149854 AF400746
CBS 344.98 AF545551
T. fertile
Bissett DAOM 167161 AF400260 AF399262
CBS 339.93 AF545546
H. chlorospora
Berk. & M.A. Curtis/
T. chlorosporum
Chaverri & Samuels CBS 114231 AY391903
H. sinuosa
Chaverri & Samuels/
T. sinuosum
Chaverri & Samuels CBS 114247 AY391942
H. aureoviridis
Plowr. & Cooke/
T. aureoviride
Rifai CBS 245.63 AF545509
H. strictipilosa
Chaverri & Samuels/
T. strictipile
Bissett G.J.S 98-91 AF545538
T. longipile
Bissett CBS 340.93 AF545550
T. spirale
Bissett CBS 346.93 AF400262 AF545553 AF399274
T. aggressivum
Samuels & W. Gams CBS 100525 AF345950 AF545541
DAOM 222156 AF399282
H. tawa
Dingley/
T. tawa
CBS 246.63 AF400258 AF399258
Chaverri & Samuels CBS 114233 AY391956
H. lixi
Pat./
T. harzianum
Oudem. CBS 226.95 AY605713 AF545549 AF276646
H. catoptron
Berk. & Broome/
T. catoptron
Chaverri & Samuels CBS 114232 AY391900
T. tomentosum
Bissett DAOM 178713a AF545557
H. gelatinosa
(Tode : Fr.) Fr./
T. gelatinosum
Chaverri & Samuels CBS 114246 AY391924
T. helicum
Bissett et al DAOM 230021 DQ087239
T. rossicum
Bissett et al TUB F-718 AF149857 DQ087240 DQ087236
H. jecorina
Berk. & Broome/
T. reesei
E.G. Simmons TUB F-430 DQ087241
T. longibrachiatum
Rifai CBS 816.68 DQ087242
T. saturnisporum
Hammill CBS 330.70 DQ087243
H. schweinitzii
(Fr.) Sacc/
T. citrinoviride
Bissett G.J.S. 01-364 AF545565
1366 MYCOLOGIA
were sealed with Parafilm (Pechiney, Chicago, Illinois) and
incubated at 25 C. The cultures were maintained at 15 C. In
addition the mycelia and spores were scraped off the plates
and kept at 280 C.
Growth characterization.—Optimum temperature of
growth and colony characteristics were determined.
The strains were pregrown on CMD until they reached
55–65 mm diam. Agar plugs 0.5 cm diam were cut from
the margin of the colonies and transferred to fresh
medium, 1.0–1.5 mm from the edge of the 9 cm diam
Petri dish with the mycelium facing down on the surface
of the agar. CMD, PDA (potato-dextrose agar, Merck,
Darmstadt, Germany) and low nutrient agar (SNA,
Nirenberg 1976) with the pH adjusted to 5.5 were used.
The tests were performed at 15 C (with alternating 12 h
nUV light and 12 h darkness), 25 C (with alternating
12 h cool white fluorescent light and 12 h darkness)
and 30 C and 35 C (both in darkness). For growth at
25 C the Petri dishes were sealed with Parafilm to avoid
drying out of the agar caused by the ventilator of the
cooling incubator (MIR 153, Sanyo, Gunma, Japan).
The maximum colony radius was measured once daily
for at least 7 d or until the plates were entirely covered
with mycelium. The growth rate was calculated by linear
regression of log t versus log r(t 5time of incubation, r
5radius measured from the edge of the agar plug),
using only measurements from the phase where the
logarithmic increase of the colony radius was linear over
time. The data given are ranges obtained from six
experiments for all media and temperatures.
In addition the plates also were examined daily under the
compound microscope at low magnification and the time of
first appearance of conidia, autolytic behavior of marginal
hyphae, presence of chlamydospores, formation of pig-
ments and odor and colony appearance were noted.
Morphological observations.—Conidiation structures were
examined, measured and photographed on a com-
pound microscope from cultures grown 3–7 d on CMD
at 25 C (see above) on the plates under low magnifica-
tion and after mounting in tap water. These characters
were measured: length of conidia, width of conidia,
length of phialides, width of phialides at the base, width
of phialides at the widest point. The size of chlamydos-
pores was measured by examining colonies grown on
CMD or on SNA under the conditions described above
using the 403objective of a compound microscope.
Dry stromata of
Hypocrea
were rehydrated briefly in 3%
KOH, imbedded in Tissue-Tek O.C.T. Compound 4583
(Sakura Finetek Europe B.V., Zoeterwoude, The Nether-
lands) and sectioned at a thickness of 12–15 mm with
a freezing microtome. Permanent preparations of the
sections were made as described by Volkmann-Kohlmeyer
and Kohlmeyer (1996). These teleomorph characteristics
were evaluated: diameter, height, color and shape of the
stroma; texture of the surface of the stroma; perithecium
shape, length and width; color, width of perithecium wall;
length and diameter of ostioles; color and reaction of the
stroma surface to 3%KOH; shape, thickness of the surface
region, size and wall thickness of cells of the supraperithe-
cial (between the perithecia and the surface region),
subperithecial and basal regions of the stroma; ascus length
and width; distal and proximal part-ascospore length and
width. Measurements of asci, ascospores and anamorph
characters are reported as maxima and minima in paren-
theses and the mean plus and minus the standard deviation
of a number of measurements given in parentheses.
Nomarski differential interference contrast (DIC) and
bright field microscopy were used for observations and
measurements. Colors were determined and cited accord-
ing to Kornerup and Wanscher (1981).
DNA extraction, PCR amplifications and sequencing.— My-
celium for DNA extraction was grown on PDA covered
by sterile cellophane. Genomic DNA was extracted with
the Plant DNeasy Minikit (QIAgen GmbH, Hilden,
Germany) according to the manufacturer’s instructions,
using approximately 150 650 mg fresh mycelium. A
region of nuclear DNA, containing the ITS1 and 2
region, was amplified by PCR with the primer combina-
tions SR6R and LR1 (White et al 1990), following the
protocol of Kullnig-Gradinger et al (2002). A 1.26 kb
fragment of the tef1 gene encoding translation elonga-
tion factor 1 alpha was amplified using the primer pair
EF1728F (Chaverri and Samuels 2003) and TEF1LLErev
(59-AAC TTG CAG GCA ATG TGG -39). This fragment
includes the fourth and the fifth intron and a significant
portion of the last large exon. A 0.7 kb fragment of RNA
Polymerase II subunit B (rpb2) was amplified using the
primer pair fRPB2-5f and fRPB2-7cr (Liu et al 1999). A
0.5 kb fragment of the endochitinase 42 gene was
amplified using the primer pair ech42-1a and ech42-2a
as described by Lieckfeldt et al (2000).
Template DNA (100 mL) was directly prepared from PCR
products by purification with the QIAquick PCR Purifica-
tion Kit (QIAgen) and sequenced with a capillary sequencer
ABI 3730 XL (Applied Biosystems, Foster City, California).
All sequences used in this study are listed (TABLE I) by their
GenBank accession numbers.
Molecular phylogenetic analysis.—DNA sequences were
aligned visually with Genedoc 2.6 (Nicholas and
Nicholas 1997). The interleaved NEXUS file was
formatted with PAUP*4.0b10 and manually formatted
for the MrBayes v 3.0B4 program. The Bayesian
approach to phylogenetic reconstructions (Rannala
and Yang 1996, Yang and Rannala 1997) was imple-
mented with MrBayes 3.0B4 (Huelsenbeck and Ron-
quist 2001). The model of evolution and prior settings
for individual loci were used as estimated by Druzhinina
et al (2004) for different taxa of Hypocrea/Trichoderma.
Metropolis-coupled Markov chain Monte Carlo
(MCMCMC) sampling was performed with four in-
crementally heated chains that were simultaneously run
for 1 000 000 and 3 000 000 generations. To check for
potentially poor mixing of MCMCMC, each analysis was
repeated three times. The convergence of MCMCMC
was monitored by examining the value of the marginal
likelihood through generations. Convergence of sub-
stitution rate and rate heterogeneity model parameters
also were checked. Bayesian posterior probabilities (PP)
JAKLITSCH ET AL:
HYPOCREA VOGLMAYRII
SP. NOV. 1367
were obtained from the 50%majority rule consensus of
trees sampled every 100 generations after removing the
500 first trees using the ‘‘burn-in’’ command. Accord-
ing to the protocol of Leache and Reeder (2002), PP
values lower than 0.95 were not considered significant
while values below 0.9 are not shown on phylograms
and radial trees.
The MSA file and phylogenetic trees have been deposited
in the TreeBASE database (http://www.treebase.org/
treebase/submit.html) under the submission code SN
2355. Genetic distance was computed in PAUP*4.0b10
under the GTR +G model.
RESULTS
Phylogeny.—Barcode identification of ITS1 and
two sequences from four unknown Hypocrea
isolates using TrichOKEY v. 1.0 (Druzhinina et al
2005, www.isth.info) indicated that they probably
represent a new species. All five markers specific
for the genus Hypocrea/Trichoderma were found
while nucleotide stretches in hallmark areas
characteristic for species and/or clades were
unique. Therefore it is unlikely that these isolates
represent a member of any of the sections or
clades known in the genus (Chaverri and Samuels
2003, Druzhinina et al 2004). To prove this
hypothesis and to find an exact position of the
potentially new species in the phylogenetic tree of
the genus we applied the genealogical concor-
dance phylogenetic species recognition (GCPSR)
concept (Taylor et al 2000). To do this we
amplified and sequenced fragments from three
additional phylogenetic markers (i.e. a fragment
of the RNA polymerase II subunit B (rpb2) gene,
a portion of the last exon of the endochitinase 42
(ech42) encoding gene and a 1260 nt long frag-
ment of the translation elongation factor 1-alpha
(tef1) gene which includes sequences of the forth
and fifth introns and a portion of the last large
exon). The corresponding sequences and vou-
chered sequences of Hypocrea/Trichoderma spp.
from currently established sections and clades were
subjected to phylogenetic analyses. Following Cha-
verri and Samuels (2003) we selected the rpb2
phylogenetic marker to find the exact position of
the potentially new species in the generic tree. As
shown (F
IG
.1)Hypocrea voglmayrii was placed with
significant statistical support (P.0.95) between
the species of section Trichoderma (Rufa Clade XII
and Pachybasioides ‘‘A’’ Clade XIII, Druzhinina
et al 2005) and Pachybasioides Clade IX (Lu et al
2004, Druzhinina et al 2005). This position was
confirmed fully by individual ITS1 and 2 and ech42
trees that include only species from neighboring
groups (F
IGS
. 2, 3). Thus our analyses show that the
isolates fulfill the criteria of the GCPSR concept,
and therefore they represent a new species.
In addition we compared genetic distances (GTR +
G) between
H. voglmayrii
and species from closest
clades and also representatives of the next neighbor
(
H. citrina
from Hypocreanum Clade X and
H. lixii
from Catoptron-Lixii Clade I). The distances to both
neighboring clades were approximately the same
while they were significantly lower than those to
genetically more diverse fungi (TABLE II), confirming
that the species under description is neither a mem-
ber of section
Trichoderma
nor the Pachybasioides
Clade IX.
DESCRIPTION OF THE SPECIES
Hypocrea voglmayrii W.M. Jaklitsch, sp.nov. FIGS.4,5
Anamorph: Trichoderma voglmayrii W.M. Jaklitsch,
sp.nov.
Etymology. In honor of Dr Hermann Voglmayr,
mycologist at the Institute of Botany, University of
Vienna, who, in collaboration with the correspond-
ing author, collected many Hypocrea teleomorphs
in Europe.
Stromata in ligno
Alni alnobetulae
et rarius
A. incanae
in
altitudine 1100–1400 m s.m., solitaria vel gregaria, erum-
pentia per corticem, discoidea, basi sterili presente an non,
(1–)1.3–3(–5.1) 3(0.7–)1–2.2(–3.2) 3(0.3–)0.3–0.7(–1)
mm, lateritia ad ianthino-purpurea, ostiolo fusco ad atro,
leviter prominente. Asci cylindrici, (60–)67–85(–94) 3
(3.3–)3.8–4.6(–5.4) mm. Ascosporae bicellulares, verruculo-
sae, hyalinae, ad septum disarticulatae, pars distalis sub-
globosa ad late ellipsoidea (3–)3.3–3.9(–4.3) 3(2.5–)2.8–
3.3(–3.5) mm, pars proxima oblonga ad quasi ellipsoidea,
saepe leviter attenuata ad basim (3.9–)4.1–4.8(–5.3) 3(2–)
2.3–2.9(–3.2) mm.
Anamorphosis
Trichoderma voglmayrii
. Incremen-
tum in agaro ‘‘CMD’’ ad 35 C post 7 d. Phialides
lageniformes vel rarius ampulliformes, (6.5–)7.1–
11.3(–16.3) 3(2.7–)3.1–4.1(–4.7) mm. Conidia hya-
lina, pallide viridia in acervis, ovoidea usque ad
subglobosa, glabra, (3.1–)3.4–4.5(–5.2) 3(2.2–)2.6–
3.4(–4.2) mm.
Stromata (FIG. 4.1) solitary or in small cespitose
groups, breaking through fissures in the bark with the
sterile and light-colored margin surrounded by the
epidermis of the host (FIG. 4.2), or on wood. Size:
length 3width 3height 5(1–)1.3–3(–5.1) 3(0.7–)
1–2.2(–3.2) 3(0.3–)0.3–0.7(–1) mm (n 530) in the
dry state. Stromata circular to elongate in outline,
discoid, with a nearly plane surface and free regular to
undulate sharp margins, and often a short sterile
constricted stipe (FIG. 4.4) or/and a radiating white
basal mycelium. Stromata sometimes annular with the
1368 MYCOLOGIA
center sunken appearing hollow. Fresh stromata from
brick red (7CD6-7) to pinkish or grayish red (9C5-6),
and of dry stromata, as usually collected, grayish red
or brownish red (9C5-6) to Cuba red (9E7-8) or violet
brown (10E7-8), with the margin concolorous or, like
the stipe, whitish to yellowish to pale orange. Slight
differences between fresh and dry stromata except for
wrinkles and fine fissures in dry stromata sometimes
forming stellate structures around the ostioles.
Ostioles densely arranged with openings visible as
conspicuous dark brown to black, well-defined,
slightly raised dots (FIGS. 4.3, 4.5) in dry stromata,
with an outer basal diameter of (26–)32–49(–55) mm
(n 530), projecting beyond the surface of the stroma
by (12–)18–36(–55) mm. Stroma surface discolored
dark reddish-brown to nearly black in 3%KOH,
consisting of thick-walled compressed angular cells of
indistinct outline ca. 3–7 mm diam, and in sections
appearing as a thin compact amorphous orange layer,
(6–)8–16(–22) mm(n520) thick. Cells between this
FIG. 1. Position of
H. voglmayrii
in the Bayesian phylogenetic tree as it was inferred from partial
rpb2
sequences. Black
circles indicate nodes supported by posterior probabilities higher than 0.95; gray circles show nodes with support between 0.89
and 0.95. Arrows and vertical bars indicate branches that lead to sections and clades as it has been established by Chaverri and
Samuels (2003) and Druzhinina et al (2004).
JAKLITSCH ET AL:
HYPOCREA VOGLMAYRII
SP. NOV. 1369
layer and the perithecia (FIG. 4.8) subglobose to
angular, (3–)5.4–10.7(–12.4) 3(2.5–)4.4–8.3(–9.9)
(n 530), hyaline, but orange to reddish directly
below the surface layer. Thickness of the entire tissue
above the perithecia (30–)41–67(–77) (n 520). The
subperithecial tissue (FIG.4.9)ofhyphaewith
strongly constricted septa and hyaline, refractive,
elongate to, directly below perithecia, subglobose
cells of (7–)12–38(–57) 3(6.5–)8.5–17.5(–24) mm(n
530) with walls approximately 1–2 mm thick. The
basal tissue (FIG. 4.10) a hyaline, loose textura
intricata of (2.3–)2.7–5.2(–7.5) mm wide hyphae (n
530). Perithecia (FIG. 1.6) entirely immersed in the
upper part of the stroma, ellipsoidal or broadly
cylindrical to flask-shaped, height 3diam 5(186 –)
214–258(–283) 3(81–)100–167(–228) (n 531),
laterally compressed and approximately by 40%
higher than wide on average. Peridia of adjacent
perithecia usually in close contact laterally, of re-
fractive, narrow and strongly compressed orange cells.
Thickness of the peridium at the base of the
perithecia (12–)13–18(–20) mm(n520) and at half
FIG. 2. Position of
H. voglmayrii
in a radial Bayesian phylogenetic tree inferred from partial exon sequences of the
ech42
gene. Black circles indicate nodes supported by posterior probabilities higher than 0.95; gray circles show nodes with support
between 0.89 and 0.95. * indicates
H. protopulvinata
CBS 739.83 which has ITS1 and 2 sequences identified as
H. pulvinata
by
Trich
OKey (Druzhinina et al 2005). GenBank accession numbers are not available for this isolate.
FIG. 3. Position of
H. voglmayrii
in a radial Bayesian phylogenetic tree inferred from complete ITS1 and 2 sequences. Black
circles indicate nodes supported by posterior probabilities higher than 0.95.
1370 MYCOLOGIA
of their height (5–)6–12(–16) mm(n520). Ostioles
appearing apically as a palisade of elongate, narrow,
strongly compressed orange to reddish cells (FIG. 4.7),
resembling those of the peridium; ostioles conical
with a length of (50–)60–89(–99) mm(n520) and an
internal basal diameter of (22–)26–44(–50) mm(n5
20). Asci cylindrical, (60–)67–85(–94) 3(3.3–)3.8–
4.6(–5.4) mm(n530), with a flat subapical ring
(FIG. 4.11). Part-ascospores hyaline, uniseriate, finely
spinulose, dimorphic. Distal part-ascospores subglo-
bose to ovoid or broadly ellipsoidal, (3–)3.3–3.9(–4.3)
3(2.5–)2.8–3.3(–3.5) mm(n530). Proximal part-
ascospores oblong to nearly ellipsoidal, often slightly
attenuated toward the base, (3.9–)4.1–4.8(–5.3) 3
(2–)2.3–2.9(–3.2) mm(n530) (FIG. 4.11).
Cultures and anamorph.—Some growth character-
istics recorded are provided (T
ABLE
III).
On CMD (25 C): colony thin, compact, regularly
circular in outline, fast growing and covering the Petri
dish in 5(–6) d, hyaline, hyphae loosely arranged,
with conspicuous difference in thickness between
hyphae originating directly from the agar plug and
hyphae derived from further branching, macroscop-
ically scarcely visible, no zonation, mainly and faster
growing inside the agar. Autolytic activity slight.
Conspicuous coilings in surface hyphae. Surface
smooth, except for the distant margin appearing
slightly hairy to finely floccose due to long branched
aerial hyphae. A coconut-like odor developing and
a yellow pigment diffusing through agar after 4 d
(FIG. 5.1). After 14 d the yellow pigment sometimes
occurring as long needle-shaped crystals on agar
surface. Conidiation beginning after 2–3 d as solitary
phialides in rows on surface hyphae or as fascicles of
3–5(–6) phialides on very short erect slightly
branched conidiophores, effuse between the proxi-
mal margin and 2/3 of the growth radius. Within 4–
9 d visible as inconspicuous and ill-defined powdery
whitish granules mainly in the distant third of the
plate, subsequently spreading back nearly across the
whole plate. Granules 0.1–0.5(–1.0) mm diam, made
up of single or few coalescing conidiophores, bearing
conidia in heads of up to 60 mm diam and later
sometimes in chains, appearing white to pale yellow.
Macroscopically, the color of the granules difficult to
determine due to the yellow discoloration of the agar.
At the same time conidiation also occurring within
the agar (FIG. 5.9). The structure of typical conidio-
phores determined at 5–7 d.
Conidiophores
consisting
of a main axis up to 200 mm long, simple, forked or
with up to 5(–7) nearly pyramidal or tree-like primary
branches (FIGS. 5.4, 5.5), later covered by conidial
heads, becoming more irregular. Primary branches
(FIG. 5.11–14) with short secondary branches, mostly
paired or substituted by paired or unpaired phialides
(FIG. 5.11, 5.14). Secondary branches short, up to
35 mm long, in the lowest positions at right angles,
higher branches including phialides curved upwards,
bearing phialides singly, in fascicles of 3–5(–7), or
rarely on extremely short tertiary branches, each
bearing fascicles of 3–5 phialides, resulting in dense
complex and nontransparent structures. Sparse co-
nidial development on long aerial hyphae. Metulae
generally absent. Phialides (FIG. 5.13–15) (5.5–)7.2–
12.2(–16.5) 3(2.7–)3.2–4.1(–4.7) mm, width at the
base (1.5–)2.0–3.0(–4.0) (n 560), terminal phialides
often longer than the flanking ones in the fascicle,
lageniform to narrowly subcylindrical and sometimes
sinuous, less commonly ampulliform or sometimes
ventricose, inaequilateral and with a long neck,
thickest point median, sub- or supramedian, often
multiguttulate.
Conidia
(FIG. 5.16) hyaline, yellowish
in mass, oval to oblong, often attenuated toward one
TABLE II. Genetic distances (GTR +G) between
H. voglmayrii
and species from neighbor clades
H. voglmayrii
sp. nov.
Rufa Clade (XII) +Section
Pachybasium ‘‘A’’ (XIII) Pachybasioides Clade (IX)
Hypo-
creanum
Clade (X)
Lixii-Catoptron
Clade (I)
T. hama-
tum
T. viride
VD
T. pubes-
cens
mean
H. minutis-
pora
H. pachy-
basioides
H. piluli-
fera
mean
H.
citrina
H.
lixii
rpb2
CBS 117710 0.129 0.159 0.124 0.14 0.127 0.142 0.157 0.14 0.205 0.186
CBS 117711 0.128 0.159 0.125 0.14 0.126 0.143 0.146 0.14 0.206 0.188
ech42
CBS 117710 0.189 0.168 0.197 0.18 0.192 0.197 0.185 0.19 0.337 0.321
CBS 117711 0.189 0.168 0.197 0.18 0.193 0.197 0.186 0.19 0.338 0.321
ITS1 and 2
CBS 117710 0.166 0.155 0.175 0.17 0.067 0.079 0.081 0.08 0.117 0.158
CBS 117711 0.158 0.147 0.167 0.16 0.061 0.073 0.075 0.07 0.109 0.149
JAKLITSCH ET AL:
HYPOCREA VOGLMAYRII
SP. NOV. 1371
1372 MYCOLOGIA
end, smooth, with varying numbers of guttules often
in a group at each end, (3.0–)3.5–6.5(–10.5) 3(2.2–)
2.6–3.3(–4.2), l/w5(1.1–)1.2–2.2(–3.4) (n 575).
Chlamydospores (FIG. 5.10, measured after 14 d) first
noted after 6–8 d, scanty, mainly in the center around
the point of inoculation, smooth, terminal on
hyphae, oval to subclavate, often truncated at one
end, and intercalary in hyphal cells, oval to ellipsoi-
dal, (6.9–)7.5–10.8(–12.9) 3(5–)5.6–7.4(–8.2) with
l/w 5(1.2–)1.2–1.6(–1.7) (n 512).
Similar but slower culture development was ob-
served at 15 C. At 30 C slightly faster development
and more abundant yellowish conidiation structures
inside the agar, morphologically indistinguishable
from granules on the surface of the agar, were noted.
The coconut-like odor also formed at all other
temperatures. More abundant chlamydospores and
yellow crystals (FIG. 5.8) formed at 30 and 35 C. At
35 C growth occurred for more than a week, but only
few hyphae were noted on the surface and sometimes
the agar dried out before the colony covered the
plate. Scanty effuse simple sporulation without any
granulation occurred from 4–5 d.
On PDA (25 C, FIG. 5.2) growth slower than on
CMD, with hyphae more thickly and densely arranged
than on CMD. Colony thick, dense, surface whitish,
finely granular, downy to floccose, with aerial hyphae
a net of thick strands and numerous fine branches
without any noticeable orientation; 2–3 mm high
between the thin, yellowish, finely granular area of
inoculation of extremely densely interwoven to
substromatically condensed hyphae and the ill-de-
fined, diffuse margin with surface hyphae forming
strands. Autolytic activity and coilings (FIG.5.7)
conspicuous at 25 and 30 C. Only inconspicuous
zonation noted. Conidiation finely granular, colorless
to white, on numerous single phialides or short
verticillioid structures seated on surface and aerial
hyphae, effuse, spreading across the whole colony.
Slight yellowish discoloration of the center and the
reverse from 4 d, spreading from the area of in-
oculation over the whole plate, mainly 3A3 to 3B5-6,
finally turning yellowish-brown (4B4-5). Odor in-
distinct to slightly mushroomy. Slow growth at 35 C,
forming small sterile, white, hairy colonies.
On SNA (25 C, FIG. 5.3) colonies thin, hyaline,
growth predominantly inside the agar, hyphae loosely
arranged and sometimes forming several separated
strands rather than a continuous colony, smooth,
aerial hyphae scanty, longer and denser at the distant
margin, which becomes whitish and downy. Autolytic
activity and coilings conspicuous, also at 30 C. Surface
mycelium soon degenerating. Conidiation slightly
more abundant and denser than on CMD, starting
within 3d with phialides sessile or on short conidio-
phores, mainly at the proximal margin, later also on
long aerial hyphae on the distant part of the colony.
From 5–7 d conidiation appearing as fine granules
containing heads up to 60 mm diam, spreading from
the distant margin back nearly across the entire plate,
or concentrated in two to three concentric zones, with
a light (yellowish-) green color (28CD5-6 to 30CD5-6).
Granules more regular than on CMD, remaining
small (up to 0.6 mm diam) and appearing waxy to
glassy in the stereomicroscope (FIG. 5.6). No pigment
formed, no odor detected. At 30 C conidiation
denser, granules more regular in 3 concentric zones,
with conidial heads up to 100 mm diam. At 35 C
colonies irregular, dense, hairy to floccose, sporula-
tion more abundant than on CMD.
Chlamydospores
on SNA (measured after 6 d at
35 C): spreading from the point of inoculation, more
r
FIG. 4. Teleomorph of
Hypocrea voglmayrii
. 1. Fresh stromata. 2. Dry stroma rupturing the bark. 3. Dry stroma showing
black ostioles. 4. Dry stipitate stroma. 5. Ostioles on stroma surface. 6. Perithecia. 7. Ostiole. 8. Supraperithecial layer of
stroma. 9. Subperithecial layer. 10. Basal layer of stroma. 11. Asci with ascospores. Bars: 1, 2 5700 mm, 3, 5 5300 mm, 4 5
500 mm, 6 5100 mm, 7, 8, 9 530 mm, 10 540 mm, 11 515 mm.
TABLE III. Some growth characteristics of
H. voglmayrii
. Data are based on six independent growth experiments using the
strains CBS 117710 and 117711
T
Colony radius after
72 h [mm]
Onset of
sporulation [d]
Chlamydospores
noted from [d]
Plate totally grown
(r 570 mm) [d]
CMD PDA SNA CMD PDA SNA CMD SNA CMD PDA SNA
15 12–15 8–10 8–13 4–6 4–6 4–5 9–17 (14–16)* 13–17
25 35–44 27–29 31–41 2 2 2 6 5–8 4–5 7–8 5–6
30 47–61 25–31 37–47 2 2 2 3–5 4–7 4–5 6–9 4–6
35 11–20 2–6 5–14 3–5 3–6 3–6 4–6 9–11
* values in parentheses are calculated by linear regression.
JAKLITSCH ET AL:
HYPOCREA VOGLMAYRII
SP. NOV. 1373
1374 MYCOLOGIA
abundant than on CMD, particularly at higher
temperatures (30–35 C). Terminal chlamydospores as
on CMD, smooth, oval to subclavate and often
truncated at one end, (4.5–)5.6–8.8(–9.4) 3(4.2–)
4.5–6.6(–7.7) with l/w 5(1–)1–1.6(–1.7) (n 511);
intercalary chlamydospores ellipsoidal to irregularly
elongate to sinuous, (7.7–)10.8–17.5(–19.8) 3(4–)
4.3–5.7(–6.9) with l/w 5(1.6–)2.1–3.7(–4.4) (n 511).
Habitat. on dead corticated branches and small
trunks of Alnus alnobetula (Ehrh.) C. Koch (5A.
viridis (Chaix) DC.) and A. incana (L.) Moench,
standing or lying on the ground.
Known distribution. Austria, upper montane re-
gion (1100–1400 m s.m.) of the central Alps.
HOLOTYPE: AUSTRIA: SALZBURG:Bo¨ckstein, hik-
ing trail to Bo¨ ckstein close to the parking lot in front
of the Gasteiner Heilstollen, MTB 8944/1,
47u049580N13u069080E, 1280 m s.m., on dead partly
standing trunk of
Alnus alnobetula
, 5 Sep 2003,
W.
Jaklitsch WJ 2378
(WU 25711; ex-type culture CBS
117711 5C.P.K. 948). Holotype of
Trichoderma
voglmayrii
isolated from WU 25711 and deposited as
a dry culture together with the holotype of
H.
voglmayrii
as WU 25711a.
Paratype specimens
. AUSTRIA: STEIERMARK: Schladminger
Tauern, Kleinso¨lk, steep wood at the western side of Lake
Schwarzensee, MTB 8749/1, 47u179350N13u529150E,
1165 m s.m., on dead branch of
Alnus incana
on the
ground, 6 Aug 2003,
W. Jaklitsch & H. Voglmayr, WJ 2302
(WU 25712, isolate CBS 117710 5C.P.K. 1592); Schladmin-
ger Tauern, Kleinso¨lk, hiking trail between Schwarzensee
and Putzentalalm, MTB 8749/1, 47u169360N13u519440E,
1320 m s.m., on dead standing trunk of
Alnus alnobetula
,6
Aug, 2003,
H. Voglmayr & W. Jaklitsch, WJ 2304
,WU
25713; Schladminger Tauern, Kleinso¨lk, hiking trail be-
tween Schwarzensee and Putzentalalm, MTB 8749/1,
47u179000N13u529020E, 1190 m s.m., on dead standing
trunk of
Alnus alnobetula
, 6 Aug 2003,
H. Voglmayr & W.
Jaklitsch, WJ 2305
(WU 25714, isolate C.P.K. 941). KA
¨RNTEN:
Stappitz, from Gasthof Alpenrose up along the brook
parallel to the hiking trail 518, MTB 8945/3, 47u019070N
13u119140E, 1360 m s.m., on dead trunk of
Alnus alnobetula
on the ground, 5 Sep 2003,
W. Jaklitsch, WJ 2382
(WU
25715, isolate C.P.K. 951).
DISCUSSION
The new species
H. voglmayrii
is unique in the genus in
several respects. First, unlike most other
Hypocrea
spp.,
H. voglmayrii
occurs in the upper montane vegetation
belt of the Austrian central Alps. Only few other species
such as
Hypocrea psychrophila
E. Mu¨ ll. et al (Mu¨ ller et al
1972) and
H. subalpina
Petrak (Petrak 1940) have
been described from similar altitudes in the Alps.
While
H. psychrophila
develops yellow semiglobose
stromata on
Rhododendron ferrugineum
L. and
Vacci-
nium myrtillus
L. in the subalpine zone,
H. subalpina
is
likely to be encountered in the montane zone, because
it forms its yellow discoid stromata on dead branches of
various coniferous trees.
The occurrence at such altitudes, which are
characterized by average summer temperatures of
12–18 C (Central Institute for Meteorology and
Geodynamics Vienna personal communication), is
in contrast to the ability of this species to grow in
culture at 35 C. However this may be related to
another conspicuous habit of
H. voglmayrii
(i.e. the
ability to ascend trunks, thereby becoming exposed to
climatic influences, such as direct sunshine and
potential drought). The ability to grow at 35 C in
culture is rare in
Trichoderma/Hypocrea
.Notable
exceptions are members of
Trichoderma
sect.
Long-
ibrachiatum
, including
H. schweinitzii
/
T. citrinoviride
and
T. longibrachiatum
, which are able to grow and
sporulate at 40 C (Samuels et al 1998).
Third, only few species of
Hypocrea
, such as
H.
scutellaeformis
Berk. & Ravenel described from
Acer
in
North America (Gary Samuels pers comm), or the
green-spored
H. ceramica
Ellis & Everh., show reddish
stromata comparable to that of
H. voglmayrii
.In
addition
H. voglmayrii
is characterized by conspicu-
ous ostioles that are nearly black when dry. Such
conspicuous ostioles are not seen in other species
with reddish brown stromata, except
H. patella
Cooke
& Peck (Dodd et al 2002) and
H. lacuwombatensis
B.S.
Lu et al (Lu et al 2004). These species, unrelated to
each other or to
H. voglmayrii
, are not known from
Europe, have as yet not been collected at high
r
FIG. 5. Cultures and anamorph of
Hypocrea voglmayrii
(strains CBS 117711 and CBS 117710). 1, 2, 3: Cultures grown at
25 C for 14 d, 1. on CMD, 2. on PDA, 3. on SNA. 4. Simple conidiophore on agar plate (CMD, 25 C, 4 d). 5. Branched
conidiophore on agar plate (CMD, 25 C, 4 d). 6. Conidiophores with condensed, light green conidiation under
stereomicroscope (SNA, 25 C, 14 d). 7. Coilings and hyphal tip showing autolysis (PDA, 25 C, 5 d). 8. Crystals formed on
surface of CMD agar (35 C, 6 d). 9. Conidiation at the bottom of CMD agar (25 C, 14 d). 10. Chlamydospores (CMD, 25 C,
14 d). 11, 12, 13, 14: Conidiophores and phialides (CMD, 25 C, 5–7 d), 15. Fascicle of lageniform phialides (CMD, 25 C, 5 d).
16. Conidia (CMD, 25 C, 6 d). Bars: 1, 2, 3 520 mm, 4 530 mm, 5, 9 540 mm, 6 5150 mm, 7, 8 5100 mm, 10, 11, 12, 13 5
20 mm, 14, 15, 16 510 mm.
JAKLITSCH ET AL:
HYPOCREA VOGLMAYRII
SP. NOV. 1375
altitudes, do not develop yellow pigments or a co-
conut-like odor and do not grow at 35 C.
A conspicuous phenotypic character of cultures of
H. voglmayrii
is the consistent formation of a yellow
pigment on CMD, which gives even rise to formation
of crystals at 30 and 35 C (and less so at 25 C),
a feature also described for
H. aureoviridis
(Rifai and
Webster 1966). Although many strains of
Trichoderma
form yellow pigments of various shades in cultures on
various media (mostly PDA or malt-extract agar),
these pigments are often only the result of suboptimal
growth (unpublished observations).
H. voglmayrii
is unusual in that its conidiophores
form a continuous lawn and not in distinct pustules
and that the conidia are at most only pale green. Pale-
colored conidia occur in the Pachybasioides Clade.
The coconut-like odor formed by
H. voglmayrii
in
culture is regarded as typically found only for some
species of sect.
Trichoderma
(Gams and Bissett 1998,
Samuels pers comm). However, as shown in
FIGS. 1–3,
H. voglmayrii
cannot be assigned to any
major clade of
Trichoderma
.
An independent intercladal position of this new
species already has been shown in the large ITS1 and
2 tree (Druzhinina et al 2005 as ‘‘51 vogl’’
H
. sp.
WJ
2305
) where the largest number of known species
(88) was considered. However in the
rpb2
tree cited
above the location of this species was basal to clades
representing the section
Trichoderma
and this posi-
tion was not contradicted by other trees. Bayesian
trees obtained in the current study based on
sequences of the genes ITS1 and 2,
rpb2
and
ech42
(FIGS.1–3)consistentlyplace
H. voglmayrii
in
a position between clades of the section
Trichoderma
and the Pachybasioides clade (cf. Druzhinina et al
2005) with nearly identical genetic distances. There-
fore
H. voglmayrii
represents a well-supported iso-
lated lineage between the section
Trichoderma
and
the Pachybasioides Clade IX and cannot be assigned
to any of these clades.
In addition we also have analyzed
tef1
sequences of
H. voglmayrii
. However, while the exon did not yield
any useful phylogenetic resolution, sequences of the
large intron were poorly alignable with those from
species of neighboring clades (viz. FIGS. 1–3), and
thus
tef1
was not used for phylogenetic analysis (data
not shown). When the
tef1
large intron or both intron
sequences (GenBank DQ086146 and DQ086147)
were subjected separately to a similarity search by
TrichoBLAST (Kopchinskiy et al 2005, www.isth.info),
the most similar species were
H. rufa
CBS 111094 for
the forth large intron (92%of similarity),
T. rossicum
TUB F-752 for the fifth short intron (97%similarity
based on the significant alignment of only 44 nt from
100) and
H. sulphurea
G.J.S. 95–190 for the last large
exon (92%of similarity). Because all three matches
belong to different phylogenetic clades of
Hypocrea
/
Trichoderma
, this result clearly indicates that
H. voglmayrii
is different from all other species,
also based on the diagnostic parts of the
tef1
sequence.
Despite several attempts (Kullnig-Gradinger et al
2002, Chaverri and Samuels 2003), the phylogeny of
the genus
Hypocrea
/
Trichoderma
is not yet fully
understood. Only a part of clades strongly supported
in single gene trees coincide with the sections defined
by Bissett (1991). However so-called ‘‘lone lineages’’
represented by single species appear in many phylo-
genetic evaluations (cf. Kullnig-Gradinger et al 2002,
Chaverri and Samuels 2003).
Hypocrea voglmayrii
represents such a new lineage that now stabilizes
neighboring clades. The phylogenetic position of the
Pachybasioides Clade had been unclear in previous
studies, because it was placed from within the large
Pachybasium species cluster (Pachybasium B5; Kull-
nig Gradinger et al 2002), whereas it formed a sister
clade of sect.
Trichoderma
in the
rpb2
tree of Chaverri
and Samuels (2003).
Hypocrea voglmayrii
appears to
be a missing link because its inclusion in the
phylogenetic analysis now stabilizes the phylogeny of
the Pachybasioides Clade in the vicinity of the section
Trichoderma
(Rufa Clade XII and Pachybasioides ‘‘A’’
Clade XIII). Also the position of species of sect.
Hypocreanum
(Pachybasium B1; Kullnig-Gradinger et
al 2002) is now stabilized adjacent to the Pachyba-
sioides Clade. While an overall phylogeny of the
genus is still difficult to obtain because of an
insufficient number of phylogenetic markers that
provide sufficient resolution within the whole genus,
it appears that this part of the generic tree can
become better understood after inclusion of unde-
scribed species. Several new species collected during
the investigation of the diversity of
Hypocrea
in
Central Europe by the corresponding author eventu-
ally will reveal further aspects of the phylogeny of
Hypocrea
/
Trichoderma
.
ACKNOWLEDGMENTS
We thank Gary Samuels for most valuable information,
comments and suggestions, Walter Gams for the amend-
ment of the Latin description, Hermann Voglmayr for
his tireless and pertinacious search and detection of
Hypocrea
teleomorphs, Leopold Puchinger for the use
of the microtome, Christian Scheuer for organizing
the excursion to Schladminger Tauern and Kristina Bauch
for the permission to collect in the Nationalpark Hohe
Tauern. The financial support by the Austrian Science Fund
(FWF Project P16465-B03) to WMJ is gratefully acknowl-
edged.
1376 MYCOLOGIA
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