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STUDIES IN MYCOLOGY 50: 401–407. 2004.
401
Hypocrea flaviconidia, a new species from Costa Rica with yellow conidia
Irina S. Druzhinina1, Priscila Chaverri2,4, Payam Fallah2,5, Christian P. Kubicek1 and Gary J. Samuels3*
1Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9-
1665, A-1060 Wien, Austria; 2Department of Plant Pathology, The Pennsylvania State University, 301 Buckhout Laboratory,
University Park, PA 16802, U.S.A.; 3United States Department of Agriculture-ARS, Systematic Botany and Mycology Labo-
ratory, Rm. 304, B-011A, Beltsville, MD 20705 U.S.A.; 4Current address:Department of Plant Pathology, Cornell Univer-
sity, 334 Plant Science Building, Ithaca, New York 14853, U.S.A.; 5Current address:Environmental Microbiology Labora-
tory, Inc., 10636 Scripps Summit Court Suite 103, San Diego, California 92131, U.S.A.
*Correspondence: Gary J. Samuels, gary@nt.ars-grin.gov
Abstract: The new species Hypocrea flaviconidia and its Pachybasium-like Trichoderma anamorph are described based on
collections from the Southern region of Costa Rica. This species is distinguished by its yellow conidia, a character that is
rare and unusual in Hypocrea/Trichoderma. The phylogenetic relationship of H. flaviconidia to other species of Hy-
pocrea/Trichoderma is explored based on ITS1 and 2 regions of rDNA, tef1 and ech42 gene genealogies. Phylogenetic
analyses show that H. flaviconidia belongs in Trichoderma sect. Trichoderma, where it forms a sister group to a clade that
includes T. hamatum and T. pubescens. A key to species of Hypocrea with known anamorphs with yellow conidia is pre-
sented.
Taxonomic novelty: Hypocrea flaviconidia Chaverri, Druzhinina & Samuels sp. nov.
Key words: Hypocreales, Hypocreaceae,Trichoderma, systematics, trees.
INTRODUCTION
Three specimens of a species of Hypocrea that pro-
duced an anamorph with yellow conidia were col-
lected by Chaverri and Samuels in the southern region
of Costa Rica (Puntarenas Prov.). This specimen was
encountered during a National Biodiversity Institute
(INBio) inventory of the fungi of Costa Rica. Only
two species of Trichoderma are described as having
yellow conidia, viz. T. flavofuscum (J. Miller et al.)
Bissett and T. croceum Bissett. Trichoderma flavo-
fuscum is a synonym of T. virens (J. Miller et al.) Arx,
the anamorph of H. virens Chaverri et al. (Chaverri &
Samuels 2003), and T. croceum is a synonym of T.
polysporum (Link : Fr.) Rifai, the anamorph of H.
pachybasioides Yoshim. Doi (Lu et al. 2004). These
species are not closely related to each other nor to the
new species. In the present paper we describe a new
species of Hypocrea and its Trichoderma anamorph,
show its phylogenetic relationships to other species of
Hypocrea/Trichoderma based on multiple gene gene-
alogies and provide a key to species of Hy-
pocrea/Trichoderma with yellow conidia.
MATERIALS AND METHODS
Isolates
Single ascospores were isolated from fresh collections
of Hypocrea with the use of a micromanipulator and
placed on cornmeal-dextrose agar (CMD), consisting
of Difco cornmeal agar (Difco Laboratories, Detroit,
MI, U.S.A.), 2 % dextrose; 1 % antibiotic solution
(0.2 % Sigma [Sigma-Aldrich Corp., St. Louis, MO,
U.S.A.] Streptomycin Sulfate + 0.2 % Sigma Neomy-
cin Sulfate + distilled water) was added after autoclav-
ing. The cultures obtained are maintained at the U.S.
National Fungus Collection (BPI) on Difco cornmeal
agar (CMA) slants at 8 ºC and in liquid nitrogen in
cryovials with 10 % glycerol. Representative isolates
have been deposited Centraalbureau voor Schimmel-
cultures, Utrecht, The Netherlands (CBS). Herbarium
specimens have been deposited in BPI and in the
Department of Botany Herbarium of INBio, the Na-
tional Biodiversity Institute of Costa Rica (INB).
Morphological characterization
Morphological observations of the anamorph were
based on cultures grown on 15–20 mL CMD in vented
plastic, 9 cm diam Petri dishes in an incubator at 20–
21 °C, with 12 h cool white fluorescent light and 12 h
darkness. Morphological characters of the teleomorph
and anamorph are as described in Chaverri & Samuels
(2004). Observations of the anamorph within ap-
proximately 1 wk or when the first mature conidia
were formed. The presence of chlamydospores was
recorded by examining the reverse of a colony grown
on CMD for ca. 1 wk with the 40 × objective of a
compound microscope.
DRUZHININA ET AL.
402
Table 1. Sequenced strains of Hypocrea (H) and Trichoderma (T) species, their provenance and GenBank numbers of their
DNA sequences
Species Strain GenBank no. Provenance
ITS1 & ITS2 tef1 ech42
H. flaviconidia G.J.S. 99-49 AY665696/AY665700 AY665710 AY665691 Costa Rica
H. flaviconidia G.J.S. 99-57 AY665697/AY665701 AY665711 AY665692 Costa Rica
T. asperellum CBS 433.97 AJ230668 AF456907 AY665688 U.S.A., Maryland
T. atroviride DAOM 165779 Z48817 AF348113 AF276650 U.S.A., North Carolina
T. hamatum DAOM 167057 Z48816 AY665702 AY665690 Canada, Ontario
T. koningii CBS 979.70 Z79628 AY665703 AF188918 Germany
T. pubescens CBS 345.93 AF011979/AF398496 AY665704 AY665712 U.S.A., North Carolina
T. strigosum CBS 348.93 AF011982/AF398497 AY665705 - U.S.A., North Carolina
T. viride BW.J. 2450* AY665593 AY665595 AY665591 Sweden
T. viride DC.P.K. 998 AY665698 AY665706 AY665693 Russia, South Taiga
T. viride DC.P.K. 999 AY665699 AY665707 AY665694 Russia, South Taiga
T. viride DCBS 111094 AJ507084 AY665708 AY665689 Austria
T. viride DCBS 111096 AJ507138 AY665709 AY665695 Austria
T. viride DW.J. 2374* AY665592 AY665594 AY665590 Austria
*Isolated from the teleomorph by Walter Jaklitsch.
Measurements of continuous characters were made
using the beta 4.0.2 version of Scion Image software
(Scion Corporation, Frederick, MD, U.S.A.). Confi-
dence intervals (α = 0.05), minimum and maximum
values for the anamorph and teleomorph morphologi-
cal characters measured were calculated using Systat
8.0 (SPSS, Inc., Chicago, IL, U.S.A.). Colony appear-
ance was described from CMD at 20 °C and potato-
dextrose-agar (PDA, Difco) at 25 °C, and included
observations on the formation, distribution and shape
of tufts or pustules. Colour terminology is from
Kornerup & Wanscher (1978).
DNA extraction, PCR amplification and sequenc-
ing
Mycelia were harvested after 2–4 d growth on MEA at
25 °C and genomic DNA was isolated using QIAGEN
DNeasy® Plant Maxi Kit (Qiagen, Inc., Valencia, CA,
U.S.A.) following the manufacturer’s protocol. Am-
plification of nuclear rDNA, containing the ITS1 and
2 and the 5.8S rRNA gene, and of a 0.4 kb fragment
of ech42 was done as described previously (Kullnig-
Gradinger et al. 2002). A 0.3 kb fragment of tef1,
containing the large intron, was amplified by the
primer pair EF1-728F (5’-CATCGAGAAGTTCGAG
AAGG-3’) and EF1-986R (5’-TACTTGAAG
GAACCCTTACC-3’) (Druzhinina et al. unpubl.
data). Amplicon purification and sequencing was also
done as described in detail previously (Kullnig-
Gradinger et al. 2002). All sequences obtained in this
study have been submitted to NCBI GenBank, their
accession numbers are indicated in Table 1. Previ-
ously published sequences used for phylogenetic
analyses in this study are given by accession numbers
as they were retrieved from GenBank.
Phylogenetic analysis
DNA sequences were visually aligned using Genedoc
2.6 (Nicholas & Nicholas 1997). The interleaved
NEXUS file was formatted using PAUP v. 4.0b10 and
was manually edited in order for it to be recognized by
MrBayes v. 3.0B4 programme. The Bayesian ap-
proach to phylogenetic reconstructions (Rannala &
Yang 1996, Yang & Rannala 1997) was implemented
using MrBayes 3.0B4 (Huelsenbeck & Ronquist
2001). The model of evolution and prior settings for
individual loci and the combined dataset were used as
has been estimated by Druzhinina et al. (unpublished)
for different taxa of Hypocrea/Trichoderma. Metropo-
lis-coupled Markov chain Monte Carlo (MCMCMC)
sampling was performed with four incrementally
heated chains that were simultaneously run for 106
generations. To check for potentially poor mixing of
MCMCMC, each analysis was repeated four to six
times. The convergence of MCMCMC was monitored
by examining the value of the marginal likelihood
through generations. Convergence of substitution rate
and rate heterogeneity model parameters was also
checked. Bayesian posterior probabilities (PP) were
obtained from the 50 % majority rule consensus of
80 000 trees sampled every 100 generations after
removing the 2 000 first trees as the "burn-in" stage.
According to the protocol of Leache & Reeder (2002),
PP values lower then 0.95 were not considered sig-
nificant while values below 0.9 were not shown on
phylograms.
The MSA file and phylogenetic trees have been
deposited in the Treebase http://www.treebase.org/tree
base/submit.html) database under the submission code
SN1926.
RESULTS
Phylogenetic analysis
Preliminary analysis of the ITS1 and 2 sequence of
two isolates of the unknown Hypocrea indicated that it
is likely a new taxon belonging to section Tricho-
HYPOCREA FLAVICONIDIA, A NEW SPECIES FROM COSTA RICA
403
derma. In order to prove this by the Genealogical
Concordance Phylogenetic Species Recognition
(GCPSR) approach (Taylor et al. 2000), we amplified
and sequenced fragments from three genomic loci
from the two isolates and other taxa of section Tricho-
derma, i.e. the internal transcribed spacers 1 and 2
(ITS1 and 2) of the ribosomal rDNA; the large intron
of the translation elongation factor 1-alpha gene (tef1);
and a portion of the last exon of the endochitinase 42-
encoding gene (ech42). The corresponding sequences
were subjected to Bayesian phylogenetic analysis,
using the General Time Reversible model (GTR,
Rodriguez et al. 1990). As shown in Fig. 1, when the
trees were rooted with T. asperellum as an outgroup,
the remaining taxa of section Trichoderma split into
two major clades: one containing the species of the
“H. rufa” species complex (H. rufa, T. viride, T.
atroviride, T. koningii); and a second clade containing
T. hamatum,T. pubescens and the two isolates of the
unknown Hypocrea species. As no ech42 sequence
was available for T. strigosum, it was analyzed only in
the ITS1 and 2, tef1 and combined trees. While it
clustered in the first with high posterior probability
basal to the species of the H. rufa clade, this position
received no PP support in the tef1 tree and its phy-
logenetic position remains therefore unclear. The
unknown Hypocrea consistently formed a sister clade
to T. hamatum and T. pubescens in all three single-
gene trees and the combined tree, which was charac-
terized by high posterior probabilities in the ITS1 and
2 and the tef1 tree. Although the clade of the unknown
Hypocrea received low PPs in the ech42 tree, this
does not reject its phylogenetic position. These analy-
ses therefore confirm that the two isolates of the
unknown Hypocrea fulfill the criteria of GCPSR to be
considered as a separate phylogenetic taxon.
Fig. 1. Phylogenetic position of H. flaviconidia by Bayesian analysis of ITS1 and 2, tef1 large intron, ech42 large exon and a
combined dataset of all three loci. Posterior probabilities are given in italic numbers over the branches, and values below 0.9
are not shown. Arrows indicate position of taxa which were incongruent between different trees.
DRUZHININA ET AL.
404
Phenotype analysis
The teleomorph of the unknown Hypocrea is almost
nondescript. The stroma is small, pulvinate, light
brown; the ostiolar openings are barely visible as paler
colored dots. The surface of the stroma is plane and
cells of the surface are almost angular. The stroma
surface region is 60–70 µm wide and composed of
angular cells ca. 10 µm diam. Ascospores are hyaline.
In fresh isolates on CMD conidia formed in
sharply delimited yellow pustules. After six years
storage at 5–9 °C on cornmeal agar (no dextrose) in
culture tubes conidia no longer form on CMD. On
SNA yellow-green conidia form slowly in pustules,
after more than 1 wk under ambient laboratory condi-
tions; on oatmeal agar (Gams et al. 1998) large, flat,
pale yellow pustules form after 2 wk under ambient
laboratory conditions, although in one oatmeal plate
incubated at 25 °C with 12 h dark/12 h cool white
fluorescent a green pustule formed.
Conidiophores from SNA are variable in morphol-
ogy. In part they are somewhat Verticillium-like (Figs
11, 14), with a discernible main axis from which
single phialides arise at an angle < 90 ° with respect to
the main axis, or phialides terminate short lateral
branches and are then held in a verticil; these phialides
tend to be long, narrow and to taper uniformly from
base to tip. In part there is no discernible main axis;
conidiophores are irregularly and frequently branched
and the septa were conspicuous (Figs 12, 13); phi-
alides are Pachybasium-like, being relatively short
and broad and are often crowded at the tips of short
branches or along the length of branches. Intercalary
phialides are common.
Conidia are light yellow by transmitted light in
fresh cultures; green cultures conidia formed derived
from old stock cultures grown on SNA. Conidia in
G.J.S. 99-49 are ellipsoidal to oblong or subcylindri-
cal, while in G.J.S. 99-51 and G.J.S. 99-57 they are
ellipsoidal with at most few oblong conidia.
Chlamydospores were observed in one (G.J.S. 99-
49) of three cultures grown on CMD after 2 wk.
TAXONOMY
Hypocrea flaviconidia Chaverri, Druzhinina &
Samuels, sp. nov. MycoBank MB500100.
Anamorph:Trichoderma sp.
Stromata pulvinata, brunnea ad subbrunnea, 1–1.7(–2) mm
diam. Ascosporae bicellulares, verruculosae, ad septum
disarticulatae, hyalina, parte distali globosa ad subglobosa,
(4–)4.5–4.7(–45.5) × (3.7–)4.2–4.3(–5) µm, parte proximali
cuneiformi ad subcylindrica, (4.2–)5.2–5.5(–6.7) × (3.2–)
3.7–4(–4.5) µm. Anamorphosis Trichoderma sp. Phialides
(4–)6.2–7(–12.7) × (2.2–) 2.7–3(–4) µm, longitudo/latitudo
(1.4–)2.2–2.5(–5.6). Conidia oblonga ad ellipsoidea, flava,
glabra, (3–)3.7–4(–4.7) × (2–)2.2–2.5(–3) µm,
longitudo/latitudo (1.2–)1.5–1.6(–2) µm.
Holotypus: INB 3862698, isotypus BPI 746538.
Stromata solitary, pulvinate, circular in outline, 1–
1.7(–2) mm diam, (0.5–)0.7–0.9(–1) mm high (n =
10), somewhat constricted at the base, smooth,
opaque, with small perithecial protuberances, pale
brown to brown, becoming darker brown in KOH;
ostiolar openings visible. Cells of the stroma surface
nearly angular in outline, (3.5–)4.5–7(–10) µm, walls
at most slightly thickened. Surface region of stroma
60–70 µm thick, comprising thin-walled angular cells,
hyaline, (5.7–)7.5–8.5(–14.5) µm diam. Tissue be-
tween the perithecia and below the outermost layer
consisting of intertwined hyphae. Internal tissue below
the perithecia of textura angularis, cells hyaline, thin-
walled, (7–)12.5–14.5(–21) µm diam. Perithecia
completely immersed in the stroma, generally closely
aggregated, subglobose in section, (216–) 243–275(–
330) × (147–)161–184(–215) µm, wall composed of
compacted cells, KOH–, ostiolar canal (54–)66–85
(–109) µm long. Cells of the ostiolar region not
sharply differentiated from the surrounding cells of
the stroma surface. Asci cylindrical, (72–)86–91(–102)
× (4.7–)5.2–5.5(–6.2) µm (n = 50); apex slightly
thickened and with a pore. Part-ascospores hyaline,
warted, dimorphic, distal part globose to subglobose,
(4–)4.5–4.7(–45.5) × (3.7–)4.2–4.3(–5) µm, proximal
part wedge-shaped to subcylindrical, (4.2–)5.2–5.5
(–6.7) × (3.2–)3.7–4(–4.5) µm (n = 70).
Colonies on CMD at 20 ºC after ca. 2 wks 9 cm
diam, flat, with no aerial mycelium, no distinctive
odour; agar not pigmented; conidia produced in pus-
tules mainly at the margins of the colony; pustules
compact, dry, yellow (3A–B8), 1–3 mm diam. Co-
nidiophores Trichoderma-like, branching irregularly,
generally terminating in short branches, branches
arising singly or in pairs from the main axis; phialides
arising in whorls of 2–3, rarely singly, intercalary
phialides common. Sterile elongations of conidio-
phores and long protruding conidiophores lacking.
Phialides cylindrical to flask-shaped, tapering towards
the tip, sometimes slightly hooked, (4–)6.2–7(–12.7)
µm long, (2.2–)2.7–3(–4) µm wide at the widest point,
(1.5–)2.2–2.5(–3) µm at the base, L/W (1.4–)2.2–
2.5(–5.6) (n = 90), arising from a cell 2.5–3.5(–4.5)
µm wide. Conidia pale yellow, smooth, ellipsoidal to
oblong, (3–)3.7–4(–4.7) × (2–)2.2–2.5(–3) µm, L/W
(1.2–)1.5–1.6(–2) (n = 90). Chlamydospores some-
times observed after 1 wk; globose to subglobose, (7–)
9.5–11.5(–20.5) × (5.2–) 7.5–9.5(–15.2) µm (n = 30).
Habitat:On bark.
HYPOCREA FLAVICONIDIA, A NEW SPECIES FROM COSTA RICA
405
Figs 2–15. Hypocrea flaviconidia. 2–4. Stromata. 5. Cells at the stroma surface. 6. Section through a stroma showing perithe-
cia. 7. Median longitudinal section through a perithecium showing the surface and subsurface regions of the stroma. 8. Stroma
tissue below perithecia. 9. Two asci with ascospores. 10. Conidial pustules on CMD. 11–14. Conidiophores, from SNA. 15.
Conidia, from CMD. Figs 2–5, 8, 9, 15 from G.J.S. 99-51; 6, 9, 10, 12, 13 from G.J.S. 99-49; 7, 11, 14 from G.J.S. 99-57.
Scale bars: Figs 1–3 = 2 mm, 4, 10 = 1 mm, 5, 9, 11, 13, 15 = 10 µm, 6 = 500 µm, 7, 8, 12, 14 = 20µm.
DRUZHININA ET AL.
406
Known distribution:Costa Rica.
Specimens examined: Costa Rica, Puntarenas. Coto Brus,
Sabalito, Sitio Las Tablas, Sendero Siénega, elevation 1500
m, on bark, 29 Jun. 1999, G.J. Samuels (8477), P. Chaverri,
H.L. Chamberlain (INB 3862187, BPI 746540, culture
G.J.S. 99-49); Sendero Siénega, elevation 1500 m, on bark;
29 Jun. 1999, G.J. Samuels (8475), P. Chaverri, H.L.
Chamberlain (holotype INB 3862698, isotype BPI
746538); Sendero Siénega, elevation 1350 m, on bark, 1
Jul. 1999, G.J. Samuels (8498), P. Chaverri, H.L.
Chamberlain (BPI 746561, INB 3862702; culture: G.J.S.
99-57, CBS 116238).
Notes: We know this species from three collections,
all made in the same area of Costa Rica. Ascospores
isolated from all three gave identical cultures; only
G.J.S. 99-49 and G.J.S. 99-57 are now viable, and
these were sequenced. Unfortunately the only speci-
men suitable to be the type specimen is BPI 746538
from which the no longer viable culture G.J.S. 99-51
was derived. There is no doubt in our minds of the
identity of this collection with the other two paratype
specimens, and their cultures, cited above.
Because of its morphology, the anamorph would
be assigned to Trichoderma sect. Pachybasium (Sacc.)
Bissett sensu Bissett (1991), a section that is now
known to be paraphyletic (Kullnig-Gradinger et al.
2002). The combined phenotype and genotype data
lead us to conclude that this unknown Hypocrea is not
one of the species for which the whole life cycle is
known and, accordingly, we describe it as a new
species.
Using cultures stored for 6 years as described
above, the optimum temperature for growth in dark-
ness on PDA and SNA was 25 °C. After 72 h the
radius of cultures grown on PDA after 72 h was 27–30
mm; on SNA < 5 mm; there was no growth at 15 or 30
°C. On SNA the colony was transparent and the
margin deeply dissected or lobed; on PDA the myce-
lium was more or less cottony and formed in concen-
tric rings.
Although yellow conidia sometimes form in some
cultures of some species, very few isolates have
permanently yellow isolates. As was said in the intro-
duction, T. flavofuscum and T. croceum were de-
scribed on the basis of yellow conidia, but these have
been shown to be synonyms of T. virens and T. poly-
sporum, respectively. Conidia of T. virens are typi-
cally green while conidia of T. polysporum are typi-
cally white to cream-colored. Even were it not for the
peculiar conidium pigmentation, this new species is
morphologically different from other described mem-
bers of the morphological T. sect. Pachybasium.
Conidia of H. flaviconidia were unmistakably yellow
on CMD when first isolated. Conidium pigmentation
was equivocal after storage, yellow-green on SNA but
white on OA.
Key to species of Hypocrea/Trichoderma with sometimes yellow conidia
1. Ascospores green; anamorph Gliocladium-like, producing mostly green,
rarely yellow-brown conidia ................................................................................................. H. virens/T. virens
1. Ascospores hyaline; anamorph not Gliocladium-l ike ......................................................................................... 2
2. Distal part-ascospores 3.2–4.5 × 3–4 µm, proximal part-ascospores 3.7–5.2 ×
2.5–3.5 µm; anamorph Pachybasium-like; conidia hyaline (white in mass)
in most isolates, rarely yellow, 2.5–3.5 × 1.5–2.5 µm; known only from
temperate regions ...........................................................................................H. pachybasioides/T. polysporum
2’. Distal part-ascospores 4.5–4.7 × 4.2–4.3 µm, proximal part-ascospores
5.2–5.5 × 3.7–4 µm; anamorph Trichoderma- to Pachybasium-like; conidia
light yellow, 3.7–4 × 2.2–2.5 µm; known only from
Costa Rica . ................................................................................................................................. H. flaviconidia
ACKNOWLEDGMENTS
P.C., P.F. and G.J.S. were supported part by INBio and by
National Science Foundation Grant 9712308, ‘Monographic
studies of the Hypocrealean fungi: Hypocrea and Hypomy-
ces,’ to the Department of Plant Pathology, The Pennsyl-
vania State University. C.P.K. and I.D. were supported in
part by the Austrian Science Fund grants P-12748-MOB
and FWF P-16601 to C.P.K. C.P.K. and I.D. thank Monika
Komon (first author’s laboratory) for her help in PCR
amplifications. G.J.S. and P.C. thank Lutorri Ashley for
technical assistance.
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