Copyright © Copyright 2006 Centraalbureau voor Schimmelcultures, P.O.
Box 85167, 3508 AD Utrecht, The Netherlands. Taxonomy and phylogenetic relationships of nine species of
Hypocrea with anamorphs assignable to Trichoderma section
Hypocreanum *Correspondence: Barrie E. Overton,
boverton/at/lhup.edu You are free to share–to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights. | ||||
Abstract Morphological studies and phylogenetic analyses of DNA sequences from the
internal transcribed spacer (ITS) regions of the nuclear ribosomal gene
repeat, a partial sequence of RNA polymerase II subunit (rpb2), and a
partial sequence of the large exon of tef1 (LEtef1) were
used to investigate the taxonomy and systematics of nine Hypocrea
species with anamorphs assignable to Trichoderma sect.
Hypocreanum. Hypocrea corticioides and H. sulphurea are
reevaluated. Their Trichoderma anamorphs are described and the
phylogenetic positions of these species are determined. Hypocrea
sulphurea and H. subcitrina are distinct species based on
studies of the type specimens. Hypocrea egmontensis is a facultative
synonym of the older name H. subcitrina. Hypocrea with anamorphs
assignable to Trichoderma sect. Hypocreanum formed a
well-supported clade. Five species with anamorphs morphologically similar to
sect. Hypocreanum, H. avellanea, H. parmastoi, H. megalocitrina, H.
alcalifuscescens, and H. pezizoides, are not located in this
clade. Protocrea farinosa belongs to Hypocrea s.s. Keywords: Ascomycetes, Hypocreales, Hypocreanum, Hypocrea corticioides, H. egmontensis, H. parmastoi, H. alcalifuscescens, H. subsulphurea, H. farinosa, H. subcitrina, H. sulphurea, H. victoriensis, ITS rDNA, Lentinula edodes, systematics, rpb2 gene sequences, tef1 gene sequences, Trichoderma | ||||
INTRODUCTION Nine species of Hypocrea Fr. (Ascomycetes, Hypocreales, Hypocreaceae) with effused stromata from Japan, Australia, New Zealand, North America, Europe, and Central America, are newly described or redescribed. Anamorphs of these species are morphologically similar, having acremonium- or verticillium-like conidiophores with hyaline conidia, and are assignable to Trichoderma sect. Hypocreanum Bissett. Hypocrea sulphurea (Schw.) Sacc. is a common, yellow, effused fungicolous species recorded from North America and Europe that occurs on Exidia spp. Dingley (1956) considered H. subcitrina Kalchbr. & Cooke, recorded from Africa, as a synonym of the older H. sulphurea, but this synonymy has never been critically examined. Dingley (1956) published the new name H. egmontensis from New Zealand based on a fungus with yellow effused stromata. The relationship between H. egmontensis and H. sulphurea has not been established. Doi (1972) described Hypocrea sulphurea f. macrospora Yoshim. Doi. We compared morphologically type material (NY) of this forma with collections of H. sulphurea from North America and Europe. A specimen identified as Hypocrea subsulphurea Syd. in De Wild. was recently collected and cultured in Japan and redescribed. Hypocrea corticioides Speg. is similar in appearance to H. sulphurea, but H. corticioides occurs on decorticated wood and has a tropical distribution. Hypocrea corticioides Speg. is a later homonym of H. corticioides Berk. & Broome. The type material of H. corticioides Berk. & Broome is indistinguishable from and therefore synonymous with Stilbocrea macrostoma (Berk. & M.A. Curtis) Höhn., a member of the Bionectriaceae (Rossman et al. 1999). A new name is proposed for H. corticioides Speg. Two apparently new species of Hypocrea with hyphal stromata were studied. Their relationship to Hypocrea spp. with pseudoparenchymatous tissue was unclear. In addition, the relationship of these hyphal species to Protocrea farinosa (Berk. & Broome) Petch, which also has a hyphal stroma, had to be examined. Kullnig-Gradinger et al. (2002) showed that some Trichoderma species with anamorphs in Trichoderma sect. Hypocreanum form a highly supported subclade of sect. Pachybasium sensu lato and suggested that sections Hypocreanum and Pachybasium are phylogenetically indistinguishable. Their analysis included a limited number of taxa with acremonium- or verticillium-like anamorphs. More recently, Chaverri et al. (2003) used partial sequences of the RNA polymerase II subunit (rpb2) and the large exon of tef-1α (LEtef1) and found that anamorphs referable to sect. Hypocreanum do not form a monophyletic group, as H. pezizoides Berk. & Broome and H. avellanea S.T. Carey & Rogerson were situated in the H. rufa clade. Chaverri et al. (2003) showed that H. citrina (Pers.: Fr.) Fr. and H. pulvinata Fuckel form a highly supported clade, the limits of which were not established. Dodd et al. (2002) showed, using the ITS1-5.8S-ITS2 rDNA (ITS) region, that H. pulvinata and H. sulphurea form two distinct subclades of a strongly supported but phylogenetically unresolved clade. These authors did not conclude that sect. Hypocreanum and sect. Pachybasium were phylogenetically indistinguishable. The results of Dodd et al. (2002) and Chaverri et al. (2003) support the conclusion of Kullnig-Gradinger et al. (2002) that section Pachybasium is paraphyletic. The seven species included are compared to selected species treated by Overton et al. (2006) to establish the phylogenetic limits of Trichoderma sect. Hypocreanum. The objectives of this study are: (1) to determine whether H. sulphurea, H. subcitrina, and H. egmontensis are distinct species; (2) to verify the phylogenetic relationship between H. subsulphurea and H. sulphurea; (3) to verify the relationship between H. corticioides and H. sulphurea; (4) to determine the relationships of two new hyphal species to Protocrea farinosa; (5) to investigate the phylogenetic boundaries of Hypocrea with anamorphs in Trichoderma sect. Hypocreanum; and (6) to describe the phylogenetic species delineated in this study according to criteria developed by Taylor et al. (2000). | ||||
MATERIALS AND METHODS Collections and isolates Doi's illustrations and descriptions
(1971,
1972,
1975) were used in making
initial species determinations. Table
1 lists the accession numbers used in this study. Frequently cited
collectors are abbreviated: B.E. Overton (B.E.O.), G. J. Samuels (G.J.S.), and
K. Põldmaa (K.P.). All isolates with G.J.S. designations were obtained
by isolating single ascospores on CMD with the aid of a micromanipulator. All
isolates with B.E.O. designations were obtained from plating the entire
contents of individual perithecia. Unless otherwise noted, host and substratum
data are taken from herbarium labels. The presentation of measurements is the
same as in Overton et al.
(2006).Molecular phylogenetic analyses DNA sequence analysis was conducted using three gene sequences: ITS
1-5.8S-ITS2 (ITS), a partial sequence of the large exon of translation
elongation factor (LEtef1), and a partial sequence of the RNA
polymerase II subunit (rpb2). ITS and rpb2 sequences were
generated following the protocol and primers described in Overton et
al. (2006). The following
primers were employed for amplifying the LEtef1 regions which differs
from the tef1region amplified in Overton et al.
(2006): for LEtef1,
EF1-983F (5'-GC(C/T)CC(C/T)GG(A/C/T)CA(C/T)GGTGA(C/T)TT(C/T)AT-3') (Carbone
& Kohn 1999), EF1-2218R (5'-ATGAC(A/G)TG(A/G)GC(A/G)AC(A/G)GT(C/T)TG-3')
(S.A. Rehner, pers. comm.). Two percent dimethyl sulfoxide (DMSO) from
AMRESCO® was added to each 50 μL PCR reaction. PCR products were
purified and sequenced following the protocol in Overton et al.
(2006). Sequences were
assembled using SeqMan® II option and aligned using Clustal W in DNA Star
(DNA Star Inc., Madison, Wisconsin), and a phylogenetic analysis was performed
using PAUP* v. 4.0 b4 (Swofford
1999). Alignments were manually adjusted in PAUP*. Outgroup taxa
varied depending on the phylogenetic analysis to meet two different objectives
in this study. For the first objective, ITS, rpb2, and
LEtef1 were evaluated in single and combined analyses to establish
phylogenetic species limits. These analyses excluded the taxa H.
avellanea, H. parmastoi, H. cinereoflava Samuels & Seifert, and
H. alcalifuscescens, with isolates of T. cf.
citrinoviride, H. megalocitrina, H. pezizoides, and H. cf.
ochroleuca used as outgroup taxa. The second objective was to place
Hypocrea isolates with Trichoderma sect.
Hypocreanum anamorphs in phylogenetic context with other
Hypocrea/Trichoderma species. For the second objective,
Sphaerostilbella cf. aureonitens, Arachnocrea scabrida
Yoshim. Doi., and Hypomyces stephanomatis Rogerson & Samuels were
used as outgroup taxa for the combined LEtef1 and rpb2
analysis with representative isolates from the different sections of
Trichoderma included in the analysis. Maximum parsimony (MP) analyses
were done using the heuristic search option under the following conditions:
TBR branch swapping, 10 random addition sequences, and gaps
(insertions/deletions) treated as missing. Bootstrap analysis was performed in
500 replicates with random sequence addition (10 replicates). For the combined
LEtef1 and rpb2 analysis, sequences were trimmed to the same
starting position because some GenBank sequences not generated in this study
were significantly shorter. All sequences and alignments were deposited in
GenBank (Table 1).Alternate phylogenetic hypotheses reflecting different species relationships were compared by the Kishino-Hasegawa (K-H) test (Table 2) in PAUP* for the combined LEtef1 and rpb2 data set. The most parsimonious trees recovered with and without constraints were compared by likelihood scores (Table 2). The likelihood model implemented in the K-H test assumed equal rates of substitution and empirical base frequencies. Models of sequence evolution were tested and model parameters obtained for the LEtef1, rpb2, and combined alignments using MODELTEST 3.06 (Posada & Crandall 1998) as implemented in PAUP*. For the LEtef1 data, the likelihood ratio test (LRT) implemented in MODELTEST, selected the TIM+I+G model with unequal base frequencies; nucleotide frequencies were set to A: 0.2133, C: 0.3337, G: 0.2211, T: 0.2320; a gamma-shape parameter of 0.5234; and substitution rates set to 1.0000 (A–C), 3.1252 (A–G), 1.6847 (A–T), 1.6847 (C–G), 10.5209 (C–T), and 1.0000 (G–T). For the rpb2 data, the LRT implemented in MODELTEST, selected the TrN+I+G model with unequal base frequencies; nucleotide frequencies were set to A: 0.2413, C: 0.2787, G: 0.2551, T: 0.2248; a gamma-shape parameter of 1.1736; and substitution rates set to 1.0000 (A–C), 6.5499 (A–G), 1.0000 (A–T), 1.0000 (C–G); 9.0762 (C–T), and 1.0000 (G–T). For the combined LEtef1 and rpb2 data set, the LRT implemented in MODELTEST, selected GTR G+I model with unequal base frequencies; nucleotide frequencies were set to A: 0.22590, C: 0.30330, G: 0.24090, T: 0.22990; a gamma-shape parameter of 0.87796; and substitution rates set to 1.0000 (A–C), 5.2773 (A–G), 1.0000 (A–T), 1.0000 (C–G), 8.4309 (C–T), and 1.0000 (G–T). A maximum likelihood (ML) tree was then obtained in PAUP* using 10 random sequence addition replicates and the substitution model suggested by MODELTEST. Bootstrap analysis was performed with 500 replicates and fast stepwise addition. Morphology Anamorph and teleomorph characteristics were measured from isolates and
specimens representative of each phylogenetic species. Cultures of
Hypocrea were grown on PDA, CMD and SNA at 20°C, with 12 h
fluorescent light and 12 h darkness. Observations of anamorphs were made at
ca. 7–10 d post inoculation. Anamorph and teleomorph characters
were measured following Overton et al.
(2006) with the exception that
optimal growth temperatures were not determined. Colour terminology was
obtained from Kornerup & Wanscher
(1981). Important morphological
characters used in species recognition are discussed in the comments section
immediately following each species description. | ||||
RESULTS Phylogeny Except for minor differences, the gene trees are concordant (Figs
1,
2,
3). The gene tree generated
from ITS is slightly different from those obtained from LEtef1 and
rpb2. Hypocrea sulphurea isolate G.J.S 00-172 from Russia grouped
with North American isolates in the ITS tree
(Fig. 1) but grouped with
G.J.S. 95-140 from Europe in rpb2 and LEtef1 gene trees
(Figs 2,
3). This point of discordance
between the gene trees establishes a phylogenetic species limit for isolates
of H. sulphurea. In all three gene trees, isolates of H.
victoriensis from Australia are phylogenetically distinct from isolates
of H. sulphurea. The phylogenetic position of Protocrea
farinosa varies between the gene trees. In ITS
(Fig. 1) and LEtef1
(Fig. 3) gene trees, P.
farinosa is basal to other species in Trichoderma sect.
Hypocreanum. In the rpb2 gene tree, P. farinosa
resides in the H. pseudostraminea clade
(Fig. 2) with no bootstrap
support. Consequently, the exact phylogenetic position P. farinosa in
relation to Hypocrea spp. with anamorphs referable to sect.
Hypocreanum, is unresolved. Nevertheless, P. farinosa is
clearly situated in Hypocrea s.s.
(Fig. 5, clades A2+B2), with a
bootstrap score of 100 uniting the clades, and it will be referred to as
Hypocrea farinosa in the remaining sections of this text (see
Taxonomy).All three datasets have similar homoplasy indices. The heuristic search of the most parsimonious tree for the ITS dataset yielded 3193 trees with 217 steps. The minimal possible tree length is 169; the homoplasy index (HI) is 0.221 (Fig. 1). From 550 total characters, 421 characters are constant: 41 variable characters are parsimony-uninformative and 88 characters are parsimony-informative. The heuristic search of the most parsimonious trees for the rpb2 dataset resulted in a tree with 650 steps with the minimum possible tree length of 408: HI = 0.372 (Fig. 2). From 956 total characters, 651 characters are constant: 88 variable characters are parsimony-uninformative, and 217 characters are parsimony-informative. The heuristic search of the most parsimonious trees for the LEtef1 data set yielded 64 trees with 314 steps with the minimum possible tree length of 178: HI = 0.433 (Fig. 3). From 863 total characters, 705 characters are constant, 35 variable characters are parsimony-uninformative, and 123 characters are parsimony-informative. The combined phylogenetic analysis using ITS, partial sequences of LEtef1 and rpb2 showed that H. sulphurea, H. subsulphurea, H. victoriensis, H. farinosa, and H. corticioides represent phylogenetically distinct species. Hypocrea sulphurea, H. victoriensis, H. subsulphurea, and H. corticioides formed a monophyletic clade C, supported by a bootstrap score of 77 %, with H. sulphurea distinguished from H. victoriensis by a bootstrap score of 100 % (Fig. 4). European and North American isolates of H. sulphurea formed a distinct subclade supported by bootstrap scores of 92 % in the combined analysis (Fig. 4), but more European isolates must be sequenced before determining whether European isolates represent a distinct phylogenetic species. Hypocrea citrina, H. americana, H. pulvinata, and H. protopulvinata formed a strongly supported monophyletic clade B with a bootstrap score of 100 % (Fig. 4). Hypocrea microcitrina and H. pseudostraminea are located in an unresolved clade A, sister to H. citrina, supported by a bootstrap score of 90 %. The heuristic search of the most parsimonious trees yielded three trees with 1202 steps, with the minimum possible tree length of 753: HI = 0.374 (Fig. 4). From 2358 total characters, 1767 characters are constant: 163 variable characters are parsimony-uninformative, and 428 characters are parsimony-informative. The LEtef1 and rpb2 regions distinguished between North American and European isolates of H. sulphurea, whereas ITS did not. The LEtef1 gene region was less variable than tef1 sequences generated by Overton et al. (2006), using the primer pair ef-1/2, for H. citrina and allies. Sequences were generated from the tef1 gene region for selected species of H. sulphurea and allies included in this study and deposited in GenBank (Table 3). The introns of tef1 were highly variable making alignments between species such as H. citrina and H. sulphurea problematic. Consequently, the tef1 region was excluded from this study. Species of Hypocrea with anamorphs assignable to Trichoderma sect. Hypocreanum did not form a monophyletic group. The K-H test on the combined LEtef1 and rpb2 dataset indicated a significantly worse tree (p < 0.0001) when all Hypocrea with anamorphs in Trichoderma sect. Hypocreanum were constrained to monophyly (Monophyletic Hypocreanum, Table 2). When taxa with hyphal stromata were constrained to monophyly (Monophyletic Hyphal, Table 2) the –log likelihood was significantly worse (P < 0.0001) than that of the unconstrained tree. The phylogenetic relationship of Trichoderma sect. Pachybasium s.l. to clades A2, B2, and C2 could not be established. Hypocrea megalocitrina is situated in clade A2, which was supported by a bootstrap score of 93 %. Hypocrea avellanea and H. pezizoides, both of which have a verticillium-like anamorph, reside in the H. rufa clade C2 (Fig. 5) supported by a bootstrap score of 75 %. Hypocrea parmastoi and H. alcalifuscescens are located in the unresolved clades F2 and G2, basal to all species of Hypocrea included in this analysis (Fig. 5), but have verticillium-like anamorphs referable to Trichoderma sect. Hypocreanum. Based on this dataset, it is unclear whether Hypocrea cinereoflava, H. parmastoi, and H. alcalifuscescens should be maintained within Hypocrea, as all three species were basal to other members of the genus (Fig. 5). For the combined LEtef1 and rpb2 dataset, the heuristic search of the most parsimonious trees yielded three trees with 2469 steps with the minimal possible tree length of 875: HI = 0.646 (Fig. 5). From 1588 total characters, 1009 characters were constant, 127 variable characters were parsimony-uninformative, and 452 characters were parsimony-informative. ML analysis of the combined data resulted in two trees with log Likelihood scores of –12767.00537 (not shown). These trees did not significantly differ from the tree generated in the MP analysis (Fig. 5). | ||||
DISCUSSION Species recognition The combined phylogenetic analyses using ITS and partial sequences of
LEtef1 and rpb2 show that Hypocrea sulphurea, H.
subsulphurea, H. victoriensis, H. farinosa, and H.
eucorticioides represent phylogenetically distinct species that are
members of a strongly supported clade C (Figs
4,
5). Dingley
(1956) suggested that
morphology could not be used to distinguish between H. subcitrina and
H. sulphurea and considered these species synonymous. Based on type
studies, we found the ascospores of H. subcitrina to be consistently
shorter and narrower than those of H. sulphurea. In contrast,
Hypocrea egmontensis is considered a facultative synonym of the older
H. subcitrina.Dingley deposited a culture of H. sulphurea (CBS 500.67) from New Zealand in CBS. Specimens recently collected from Australia had the same ITS sequence as CBS 500.67 and represent Dingley's concept of H. sulphurea. Molecular phylogenetic results indicate that the Australian specimens and the New Zealand culture (CBS 500.67) represent a new phylogenetic species, described here as H. victoriensis, that differs morphologically from H. subcitrina and H. sulphurea. The morphological similarities between Australian specimens of H. victoriensis and North American specimens of H. sulphurea are striking, but the part-ascospores of the Australian species are more strongly spinulose than the part-ascospores found in H. sulphurea. In addition, none of the Australian specimens occurred on Exidia spp., which is a common substrate in North America. This suggests that ascospore ornamentation and substratum are informative species characters for members of the H. sulphurea subclade (clade C, Fig. 4). Hyphal versus pseudoparenchymatous stromata Hypocrea species with hyphal stromata and anamorphs assignable to
Trichoderma sect. Hypocreanum are situated in different
clades. Hypocrea megalocitrina resides in clade A2
(Fig. 5) with H.
psychrophila. Hypocrea avellanea has a hyphal stroma and a
verticillium-like anamorph with conidia that are uniform in size and shape.
Anamorphs in Trichoderma sect. Hypocreanum typically produce
conidia that are variable in size and shape. Hypocrea avellanea
resides in the H. rufa clade (Fig.
5) with species having pseudoparenchymatous stromata. Anamorphs in
the Hypocrea rufa clade generally produce conidia that are typically
more uniform in size and shape than those found in Trichoderma sect.
Hypocreanum. Hypocrea alcalifuscescens and H. parmastoi have
hyphal stromata and verticillium-like anamorphs and are basal to other major
clades of Hypocrea/Trichoderma. Species found in clade B2
(Fig. 5), have effused,
extensive stromata, with pseudoparenchymatous tissue, except one, H.
subsulphurea, which is hyphal. Anamorphs in clade B2 produce conidia
variable in size and shape, typical of Trichoderma sect.
Hypocreanum. Hypocrea pezizoides, known to have a
pseudoparenchymatous stroma and a verticillium-like anamorph also resides in
the H. rufa clade (C2, Fig.
5), a finding consistent with Chaverri et al.
(2003). The anamorph of H.
pezizoides produces conidia that initially are light green, but become
hyaline after repeated transfers. Species with pseudoparenchymatous stroma and
anamorphs that produce hyaline conidia variable in size and shape are located
in clade B2 (Fig. 5). Species
with hyphal stromata and anamorphs that produce uniform conidia (of similar
size and shape) are polyphyletic.Petch (1937) established the genus Protocrea Petch for species that have simple ascomata immersed or seated upon a byssoid stroma with ascospores that disarticulate into part-ascospores. Rossman et al. (1999) described the anamorph of Protocrea as acremonium- or verticillium-like. Protocrea farinosa resides in clade B2 (Fig. 5) with other species with acremonium- and verticillium-like anamorphs. A well-defined layer of pseudoparenchymatous tissue was observed below the perithecia in specimens of P. farinosa. Although the teleomorphs of specimens examined varied in the degree of pseudoparenchymatous tissue present, the part-ascospore measurements obtained are identical to those published for P. farinosa by Rossman et al. (1999) and the anamorph characteristics are identical to those described by Doi (1972) for P. farinosa. Trichoderma sect. Hypocreanum and classification The phylogeny of the major clades in Trichoderma/Hypocrea
is essentially unresolved based on the genes used in this study. However,
Hypocrea spp. with well-defined pseudoparenchymatous stroma tissue,
and acremonium- or verticillium-like conidiophores (hypocreanum-like), that
produce hyaline conidia variable in size and shape, can be accommodated in a
large monophyletic assemblage of species B2
(Fig. 5). Kullnig-Gradinger
et al. (2002)
suggested that Trichoderma sect. Hypocreanum and sect.
Pachybasium should be merged as they are phylogenetically
indistinguishable. The present study, which included 17 taxa morphologically
belonging to sect. Hypocreanum, shows that the phylogenetic
relationship of Trichoderma sect. Hypocreanum to sect.
Pachybasium could not be resolved in the combined LEtef1 and
rpb2 dataset. The anamorphs of H. megalocitrina, H.
parmastoi, and H. alcalifuscescens are morphologically similar
to anamorphs typical of Trichoderma sect. Hypocreanum;
nevertheless, these fungi do not belong to the major Hypocreanum
clade B2 (Fig. 5), nor are they
phylogenetically related to members of Trichoderma sect.
Pachybasium.The multigene phylogeny of Kullnig-Gradinger et al. (2002) should serve as an example for future phylogenetic analyses to determine sectional relationships, but future studies should include a larger number of taxa and exclude ITS rDNA sequences. The ITS region proved useful in distinguishing between closely related species (Overton et al. 2006) and has been used for the revision of sections Longibrachiatum and Trichoderma (Kuhls et al. 1996, 1997; Kinderman et al. 1998; Samuels et al. 1998, 1999). Overton et al. (2006) demonstrated that ITS rDNA, rpb2, and the tef1 region could establish phylogenetic species limits, but the introns found in the tef1 region, delimited by the primers ef-1 and ef-2, were highly divergent among morphologically similar species. In this study partial sequences of the large exon (LEtef1) were generated for the seven species, including several of those treated by Overton et al. (2006). The LEtef1 region also resolved all major clades established by these authors using the tef1 gene region and distinguished between North American and European isolates of H. pulvinata; therefore LEtef1 is better suited for phylogenetic studies than the tef1 region previously sequenced by Overton et al. (2006). Comparatively few of the approximately 200 named species of Hypocrea have been sequenced to date, with published accounts placing an over-reliance on ITS rDNA sequence data. The LEtef1 and rpb2 sequences generated in this study, work by Chaverri et al. (2003), and data from other gene regions published by Kullnig-Gradinger et al. (2002), have helped to clarify our understanding of the sectional relationships of Hypocrea/Trichoderma. Additional taxa from other genera such as Sarawakus Lloyd, with Trichoderma anamorphs, need to be sequenced before a complete phylogeny of Hypocrea/Trichoderma can be established. The evolution of anamorphs referable to Trichoderma sect.
Hypocreanum There has been considerable speculation published on the evolution of the
anamorphs in Trichoderma sect. Hypocreanum. Samuels
(1996) hypothesized that the
anamorphs referable to Trichoderma sect. Hypocreanum may be
synanamorphs or spermatial states, suggesting that Hypocrea with
acremonium- or verticillium-like anamorphs with hyaline conidia have lost the
ability to produce a primary trichoderma-like anamorph, with pyramidally
branched conidiophores and green conidia. Kullnig-Gradinger et al.
(2002) presented molecular
data suggesting that the more typical trichoderma-like anamorph with green
conidia may have evolved from genera having verticillium-like anamorphs, in
particular Aphysiostroma Barrasa and Arachnocrea Z.
Moravec.Results based on molecular data have not conclusively established the evolution of the Trichoderma anamorph, including those referable to sect. Hypocreanum. Two species are of particular interest when considering the hypotheses promulgated by Kullnig-Gradinger et al. (2002) and Samuels (1996). Hypocrea pezizoides has light green conidia that become completely hyaline in subsequent transfers, suggesting an incomplete reversal to the primitive verticillium-like form with hyaline conidia. This species resides in the H. rufa clade C2 (Fig. 5) based on combined rpb2 and LEtef1 gene sequences and based on ITS sequence data, a finding consistent with Kullnig-Gradinger et al. (2002). Hypocrea cinereoflava produces a primary synnematous anamorph and a verticillium-like synanamorph. This species of Hypocrea is important when considering the hypothesis of Samuels (1996) that the verticillium-like anamorphs found in Trichoderma sect. Hypocreanum represent spermatial states, in which the primary trichoderma-like anamorph was lost. Hypocrea cinereoflava is located in an unresolved basal clade of Hypocrea s.s. (Fig. 5) and, based on the molecular results of this study, it could not be excluded from the genus Hypocrea. The phylogenetic placement of this species basal to all other Hypocrea species sequenced in this analysis could suggest that the ability to produce a synnematous primary anamorph has subsequently been lost. The data obtained in this study provide some support for the hypotheses of Samuels (1996) and Kullnig-Gradinger et al. (2002), leaving room for speculation. Additional taxa need to be sequenced before the evolution of Trichoderma anamorphs can be more accurately determined. | ||||
TAXONOMY
| ||||
KEY TO THE SPECIES TREATED
| ||||
Acknowledgments We wish to thank Dr Walter Gams for rendering the Latin diagnoses and his editorial comments on the manuscript. We wish to acknowledge Dr E. Parmasto for the identification of polypores in our study and Dr Kadri Põldmaa for sharing specimens from Estonia. We thank Dr Gary J. Samuels for providing research notes and sharing collections. Prof. C.P. Kubicek kindly reviewed a previous draft of the paper. This study was supported by the United States National Science Foundation (PEET) grant 9712308 “Monographic Studies of Hypocrealean Fungi: Hypocrea and Hypomyces”. | ||||
Notes Taxonomic novelties: Hypocrea eucorticioides Overton, nom.
nov., Hypocrea victoriensis Overton, sp. nov., Hypocrea
parmastoi Overton, sp. nov., Hypocrea alcalifuscescens Overton,
sp. nov. | ||||
References
| ||||