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Hypocrea/Trichoderma: species with conidiophore elongations and green conidia

     Pennsylvania State University, Department of Plant Pathology, 301 Buckhout Laboratory, University Park, Pennsylvania 16802

Lisa A. Castlebury

     U.S.D.A.-A.R.S., Systematic Botany and Mycology Laboratory, Room 304, B011A, 10300 Baltimore Avenue, Beltsville, Maryland 20705

Barrie E. Overton

     Pennsylvania State University, Department of Plant Pathology, 301 Buckhout Laboratory, University Park, Pennsylvania 16802

Gary J. Samuels

     U.S.D.A.-A.R.S., Systematic Botany and Mycology Laboratory, Room 304, B011A, 10300 Baltimore Avenue, Beltsville, Maryland 20705

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 

Species of Trichoderma and Hypocrea that have green conidia and sterile or fertile elongations of their conidiophores are described or redescribed and their phylogenetic position explored. The described species include T. crassum, T. fasciculatum, T. fertile, T. hamatum, T. longipile, T. oblongisporum, T. pubescens, T. spirale, T. strictipile, T. strigosum, T. stromaticum, T. tomentosum, Hypocrea aureoviridis f. macrospora, H. ceramica. and H. semiorbis. Trichoderma fasciculatum originally was described from cultures from ascospores of an unidentified Hypocrea specimen; it is considered to be a synonym of T. strictipile. The remaining species of Trichoderma considered here have not been linked to teleomorphs, and the Trichoderma anamorphs of H. aureoviridis f. macrospora and H. semiorbis have not been named. Five new species of Hypocrea are described, viz. H. cremea, H. cuneispora, H. estonica, H. strictipilosa and H. surrotunda. The phylogenetic relationships of these species were inferred based on partial RPB2 and EF-1 DNA sequence data and phenotypic characteristics, including teleomorph, anamorph, colony and growth rates. Trichoderma crassum was found to be a sister species to T. virens, based on molecular sequences and phenotypic data. Hypocrea surrotunda and H. cremea, H. cuneispora and T. longipile, T. fertile and T. oblongisporum, T. tomentosum and H. atrogelatinosa, and T. hamatum and T. pubescens, respectively, were found to be closely related phylogenetically, based on RPB2 and EF-1 gene genealogies. Anamorph and teleomorph phenotype, including conidiophore elongations, phialide morphology, conidial morphology, stroma anatomy and ascospore morphology are not useful predictors of relationships. Despite the shared phenotypic characters of these Trichoderma and Hypocrea species, they are distributed between two major clades of Trichoderma/Hypocrea. Redescriptions and a key to species of Hypocrea/Trichoderma with green conidia and conidiophore elongations are presented.

Key words: Ascomycetes, Hypocreaceae, Hypocreales, molecular phylogenetics, RNA polymerase II subunit RPB2, synanamorphs, systematics, teleomorph-anamorph connection, translation elongation factor EF-1, Trichoderma sect. Pachybasium

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 
Bissett (1991b) described 12 species of Trichoderma Pers. : Fr. and one of Hypocrea Fr. that have green conidia and elongations of conidiophores, viz. T. crassum Bissett, T. fasciculatum Bissett, T. fertile Bissett, T. hamatum (Bonord.) Bain., T. longipile Bissett, T. oblongisporum Bissett, T. pubescens Bissett, T. spirale Bissett, T. strictipile Bissett, T. strigosum Bissett, T. tomentosum Bissett and H. semiorbis Berk. The conidiophore elongations in these species can be sterile or can terminate in one or more phialides.

Bissett placed these species in Trichoderma section Pachybasium (Sacc.) Bissett based on the branching pattern and morphology of the conidiophores (Bissett 1991a, b). This section is now known to be paraphyletic (Kindermann et al 1998) and has been divided into two phylogenetic groups "A" and "B". Because there is no morphological hiatus between the two groups, for the purposes of the present discussion we will refer to sect. Pachybasium in its morphological sense. Most species in sect. Pachybasium produce compact conidiogenous pustules with branching in a pyramidal pattern, with or without fertile or sterile conidiophore elongations. Phialides are typically short and swollen, produced in verticils of 2–7, and often are crowded. Hypocrea ceramica Ellis & Everh., H. aureoviridis f. macrospora Doi and Trichoderma stromaticum Samuels & Pardo-Schultheiss are other species in sect. Pachybasium with conidiophore elongations that were not included in Bissett's work.

Most of the members of sect. Pachybasium have not been linked to teleomorphs. The anamorph of H. semiorbis, a species with colorless ascospores, has been described but not named (Bissett 1991b). The anamorph described by Doi (1972) for H. aureoviridis f. macrospora falls within the morphological section Pachybasium, but there are no available cultures of this taxon. Hypocrea aureoviridis f. macrospora has green ascospores and an anamorph that has green conidia and a fertile conidiophore elongation (Doi 1972). Cultures from an unnamed species of Hypocrea with hyaline ascospores produced T. stromaticum, although the ex-type isolate of T. stromaticum was not derived from ascospores (Samuels et al 2000). The names T. fasciculatum and T. strictipile are based on ascospore isolations of unnamed species of Hypocrea. The Hypocrea specimen from which the T. fasciculatum ex-type originated has not been located (CBS, DAOM). The Hypocrea specimen from which T. strictipile was derived is an undescribed species of Hypocrea, with yellowish stromata and green ascospores, and is described here as H. strictipilosa. In addition, five unnamed species of Hypocrea that have green ascospores and conidiophores with sterile or fertile elongations have been found, mostly in temperate regions.

In the present paper the species of Trichoderma that have elongations of conidiophores are more precisely defined through the addition of temperature/growth information and the Hypocrea species that have such anamorphs are described or redescribed. Their phylogenetic relationships are explored through partial sequences of the protein-coding genes RNA polymerase II subunit (RPB2) and translation elongation factor (EF-1). A key to the species of Hypocrea/Trichoderma that have elongations of conidiophores and green conidia is included.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 
Isolates – The isolates used in this study are listed in Table I. The authors isolated the majority of cultures from Hypocrea collections, but others were obtained from Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS), and from Agriculture and Agri-Food Canada, Eastern Cereals and Oilseeds Research Centre, Ottawa, Canada (DAOM). Isolates cultured by P. Chaverri, G. J. Samuels and Barrie E. Overton are preceded by the abbreviations P.C., G.J.S. and B.E.O., respectively. Representative isolates have been deposited in ATCC, CBS and DAOM. Single-ascospore isolations from fresh collections of Hypocrea were made on CMD (Difco cornmeal agar + 2% dextrose + distilled water + 1% antibiotic solution (0.2% Sigma Streptomycin Sulfate + 0.2% Sigma Neomycin Sulfate + distilled water)) with the aid of a micromanipulator. These cultures are maintained in CMA (Difco cornmeal agar) slant tubes at 4 C and in liquid nitrogen. Many of the species of Hypocrea with green ascospores commonly are identified as H. gelatinosa (Tode : Fr.) Fr., H. flavovirens Berk. and H. aureoviridis Phill. & Plowr. For this reason these species were included in the analysis. Often-cited collectors G. J. Samuels and C. T. Rogerson are abbreviated as G.J.S. and C.T.R., respectively.

Morphological observations – Morphological observations of the anamorph were taken from cultures grown on CMD in 9 cm diam vented plastic Petri plates in an incubator at 20 C, with alternating 12 h fluorescent light and 12 h darkness within approximately 1 wk. These standard characters were measured: width of phialide base, phialide width at the widest point, phialide length, and length/width ratio (L/W), conidium length, conidium width and length/width ratio (L/W), width of cell from which phialides arise (= metulae, subtending hypha, phialide axis), presence of chlamydospores and chlamydospore width. Measurements of continuous characters were taken from images using the beta 4.0.2 version of Scion Image (Scion Corp., Frederick, Maryland). Colony appearance also was described from CMD at 20 C and PDA at 25 C, with alternating 12 h fluorescent light and 12 h darkness, including formation and shape of tufts or pustules. The presence of chlamydospores was recorded by examining the reverse of a colony grown on CMD after ca 1 wk at 20 C under 12 h darkness and 12 h cool white fluorescent light with 40x objective of a compound microscope. Color terminology was obtained from Kornerup and Wanscher (1978).

The herbarium specimens of Hypocrea were rehydrated briefly in 3% KOH. Rehydrated stromata were supported by Tissue-Tek O.C.T. Compound 4583 (Miles Inc., Elkhart, Indiana) and sectioned at a thickness of ca 15 µm with a freezing microtome. These teleomorph characteristics were evaluated: diameter, height, color and shape of the stroma; texture of surface of the stroma; perithecium shape, length and width; reaction to 3% KOH, color, width and KOH reaction of perithecium wall; ostiolar canal length; color and 3% KOH reaction of stroma outer region; shape, diameter and wall thickness of cells of the outer, middle (immediately below the outer region) and inner region (below perithecia) of the stroma; ascus length and width; distal and proximal part-ascospore length and width. Measurements of continuous characters also were obtained with Scion Image beta 4.0.2. Confidence intervals ( = 0.05), minimum and maximum values for the anamorph and teleomorph morphological characters measured were calculated using Systat 8.0 (SPSS Inc., Illinois).

Molecular phylogenetic analysis – To obtain fresh mycelia for DNA extraction, the isolates listed in Table I were grown in potato-dextrose broth (Difco, Detroit, Michigan) in a 5 cm diam Petri plate for 3–5 d. The mycelial mat was dried using clean absorbent paper towels. The entire dried mycelial mat then was placed in a 1.5-mL Eppendorf tube for immediate DNA extraction. Extraction of genomic DNA was done with Puregene^TM Genomic DNA Isolation Kit (Gentra Systems, Minneapolis, Minnesota). Fragments of two protein-coding genes were amplified and sequenced: RNA polymerase II subunit (RPB2) and translation elongation factor 1 (EF-1). The primers used for PCR and sequencing were: fRPB2-5F (5'-GA(T/C)GA(T/C)(A/C)G(A/T)GATCA(T/C)TT(T/C)GG-3'), fRPB2-7cR (5'-CCCAT(A/G)GCTTG(T/C)TT(A/G)CCCAT-3') (Liu et al 1999); and 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 and 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). PCR reactions were set up using these ingredients for each 50 µL reaction: 5 µL of Perkin-Elmer 10x Buffer with MgCl₂ (Applied Biosystems, Branchburg, New Jersey), 10 µL of 1 mM dNTPs, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, 0.5 µL of Perkin-Elmer AmpliTaq Gold® Taq Polymerase (Applied Biosystems), a maximum of 25 ng/µL of genomic DNA, and double-distilled water to complete a total of 50 µL per reaction. The PCR reactions were placed in a Bio-Rad iCycler thermocycler (Bio-Rad Laboratories, Hercules, California) under the temperature-cycling parameters: Step 1) 10 min at 95 C; Step 2) 40 cycles of 30 s at 94 C, followed by 30 s at 50 C for RPB2 or 55 C for EF-1, and 1 min at 72 C; and Step 3) 10 min at 72 C. Resulting products were purified with QIAquick® PCR Purification Kit (Qiagen Inc., Valencia, California) and QIAquick® Gel Extraction Kit, when more than one band was amplified. Sequencing was performed at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland) using Perkin-Elmer Big Dye terminators with dITP (Applied Biosystems) and an Applied Biosystems DNA sequencer model 3100. Sequences were edited and assembled using Sequencher 4.1 (Gene Codes, Madison, Wisconsin). Clustal X 1.81 (Thompson et al 1997) was used to align the sequences, and then the alignment was refined by hand. The sequences and alignment were deposited in GenBank (Table I) and TreeBase (submission number SN 1244, http://treebase.bio.buffalo.edu/trebase/), respectively.

Phylogenetic analyses were performed using PAUP* 4.0 b10 (Swofford 1999) using Hypomyces stephanomatis Rogerson & Samuels and Nectria cinnabarinna (Tode : Fr.) Fr. sequences as outgroups. Neighbor-joining analyses (NJ) were performed with the Kimura-2-parameter model. Bootstrap values were calculated from 1000 replicates. In addition, maximum parsimony (MP) also was performed using a heuristic search, with a starting tree obtained via stepwise addition, with 1000 random addition sequences, tree-bisection-reconnection as the branch-swapping algorithm, and MULTREES off. Bootstrap values from 1000 replicates were calculated using a "fast" stepwise addition search. A consensus tree was calculated using 50% majority rule. The Incongruence Length Difference Test or Partition Homogeneity Test (PHT) in PAUP* was used to test the congruence among datasets (Cunningham 1997). For this test, parsimony-uninformative characters were excluded, gaps were treated as missing and 500 repetitions were performed. A maximum of 100 trees were saved to conserve computer memory.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 
Phenotype analysis – Most of the isolates studied fit the definition of T. strictipile; other isolates did not fit any of the described species of Hypocrea/Trichoderma. The latter isolates are G.J.S. 91-125, G.J.S. 91–93, G.J.S. 88-73 and G.J.S. 96-129 and therefore are described as new: H. cremea, H. cuneispora, H. surrotunda and H. estonica, respectively. In addition, the teleomorph of T. strictipile is a new species of Hypocrea, H. strictipilosa. The specimen of H. ceramica, from which isolate G.J.S. 88-70 originated, is indistinguishable from the holotype of this species (Ellis and Everhart 1892). The anamorph of H. ceramica described and illustrated in Doi (1966) is indistinguishable from isolate G.J.S. 88-70; the only difference is that in G.J.S. 88-70 conidiophore elongations are fertile or sterile, while the anamorph described in Doi (1966) does not have conidiophore elongations. Unfortunately, Doi's description of the anamorph is based on a Japanese specimen deposited in TNS, which was not available for examination.

The Trichoderma members of the morphological section Pachybasium are strikingly similar in their morphology. With the exception of T. crassum, which is characterized by solitary conidiophores, all the species produce conidiophores in more or less discrete pustules. Phialides of all are broad and short; they tend to be clustered at the ends and along the lengths of broad subtending hyphae that often arise from the base of an elongation that is sterile for a distance to the tip, which remains sterile or produces one or a few ampulliform phialides, depending on the species (Table II). Conidia of all are some shade of green, smooth and ellipsoidal to oblong.

Synanamorphs were observed in 11 species, viz. T. crassum, T. fasciculatum, T. hamatum, T. pubescens, T. spirale, T. strictipile, T. tomentosum, H. cremea, H. surrotunda, H. estonica and H. semiorbis (Table II). The synanamorph is a mononematous conidiophore that arises in the aerial mycelium apart from the pustules. These conidiophores tend to be more or less verticillium- or gliocladium-like and with much longer and more slender phialides than those found in the pustules. Conidia of the synanamorph are held in drops of watery, clear green liquid. Most other synanamorphs found in the other species studied have a sect. Trichoderma morphology. There was considerable variation in the conidiophores formed in the ex-type culture of T. crassum. This culture produced macronematous conidiophores that formed aggregated pustules with short and wide ampulliform phialides and conidiophore elongations. In the same Petri dish, in areas of effuse conidiation, T. crassum produced a gliocladium-like synanamorph with conidia held in drops. The conidia from the pustulate macronematous anamorph were significantly smaller than the ones produced from the gliocladium-like synanamorph. Sequences of RPB2 and EF-1 indicate a very close relationship between T. crassum and T. virens (Miller et al) Arx. Despite similar colony appearance, and conidiophore and conidial morphology, we have not seen in any isolate of T. virens the conidiophore elongations and the macronematous synanamorph that characterize T. crassum. Moreover, all five isolates of T. virens that we studied previously (Chaverri et al 2001) grow and sporulate well at 35 C, whereas the ex-type culture of T. crassum is not able to grow at 35 C (Fig. 1).

View larger version (45K): [in this window] [in a new window]   FIG. 1. Radial growth (mm) after 72 h on PDA (—) and SNA (– – –). Tick marks indicate standard error ( = 0.05). Graphs are organized according to the phylogenetic and morphological relationship

The conidiophores and their elongations were as previously described (Bissett 1991b). We did not observe any phenotypic divergence between the ex-type isolates of T. strictipile or T. fasciculatum. Both had nearly identical growth curves (Fig. 1), colony characteristics or anamorph morphology. The only difference found is that the conidial pustules of the ex-type culture of T. fasciculatum are smaller and more loosely organized than those of the ascospore and conidial isolates of T. strictipile. The ex-type culture of T. fasciculatum was found to be situated amid several isolates of T. strictipile in EF-1 and RPB2 sequence analyses.

Molecular phylogenetic analyses – A total of 903 bp of RPB 2 and 698 bp of EF-1 were included in the analyses. Including only the ingroup taxa, the RPB2 dataset contained 351 polymorphic sites (39%), 72 of them unique. Of the 351 polymorphisms, 54 (15%) are in the 1st codon position, 24 (7%) are in the 2nd codon position and 273 (78%) are in the 3rd codon position. The EF-1 dataset contained 182 polymorphic sites (26%), 53 of them unique. Of the 182 polymorphisms, 32 (18%) are in the 1st codon position, 27 (15%) are in the 2nd codon position and 123 (68%) are in the 3rd codon position.

Molecular phylogenetic analyses show that species of Hypocrea and Trichoderma with green conidia and conidiophore apical elongations do not form a monophyletic group (Figs. 2–7). The analyses also confirm that species in morphological sect. Pachybasium have multiple evolutionary origins, as suggested earlier by Kindermann et al (1998).

View larger version (61K): [in this window] [in a new window]   FIGS. 2, 3. EF-1 DNA analyses. 2. EF-1 neighbor-joining tree. 3. EF-1 maximum parsimony consensus tree (50% majority rule). Consensus indices >70% are indicated by the thicker lines. Bootstrap values >50% shown at branches. Species with green conidia and conidiophore elongations are indicated in bold

View larger version (53K): [in this window] [in a new window]   FIG. 6. Combined RPB2 and EF-1 neighbor-joining tree. Bootstrap values shown at each branch. Species with green conidia and conidiophore elongations are indicated in bold

View larger version (58K): [in this window] [in a new window]   FIG. 7. Combined RPB2 and EF-1 maximum parsimony consensus tree. Bootstrap values >50% shown above branches. Consensus indices >70% are indicated by the thicker lines. Bootstrap values >50% shown at branches. Species with green conidia and conidiophore elongations are indicated in bold

The genealogy of combined EF-1 and RPB2 sequences (Figs. 6, 7) shows Kindermann's group B (Kindermann et al 1998), which contains most of the species in sect. Pachybasium and most of the species studied in this paper. Group B is monophyletic in the NJ tree supported by 88% bootstrap value and paraphyletic in the MP tree. Group B contains at least one group with sect. Trichoderma morphology, viz. H. aureoviridis, H. cremea, H. surrotunda and H. cf. flavovirens. Group A (Kindermann et al 1998), supported here by 100% and 95% bootstrap values in NJ and MP, respectively, contains some species in sect. Pachybasium, including the type species, T. hamatum. However, it also contains species in sect. Trichoderma, such as H. rufa/T. viride Pers.

Based on EF-1 and RPB2 gene genealogies, Hypocrea gelatinosa s. str. and H. aureoviridis s. str. are not closely related to each other or to most of the species of Hypocrea/Trichoderma with green conidia and conidiophore elongations considered in this study. Hypocrea aureoviridis is closely related to the group that contains H. cremea, H. surrotunda and H. cf. flavovirens in RPB2 phylogeny. Hypocrea cf. flavovirens is closely related to H. cremea and H. surrotunda and together form a clade supported by 100% bootstrap value.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 
The combined RPB2 and EF-1 phylogeny presented in this study dealing with 38 species, demonstrates once more that morphology, such as conidiophore elongations and synanamorphs, is not always useful in resolving evolutionary relationships in Hypocrea/Trichoderma.

Molecular phylogenetic analyses show that elongations of the conidiophore and the pachybasium-like morphology of conidiophores have evolved or have been lost multiple times within Hypocrea/Trichoderma. This has been shown previously by Kindermann et al (1998) who, using ITS 1 sequences, divided the morphological section Pachybasium into two groups A and B, and group A was further subdivided into A1 and A2. The type species of sect. Pachybasium, T. hamatum, clustered in A2 along with T. viride, the type species of the genus, T. pubescens and T. strigosum. Clade A1 contains H. pilulifera. Sixteen of the species included in our study, including the new Hypocrea species, cluster in group B. However, in the maximum-parsimony analysis group B is paraphyletic. Clade A2 contains T. hamatum, T. pubescens, T. strigosum, and clade A1 contains H. pilulifera. Once again, sect. Pachybasium is shown to be polyphyletic, but we retain this name as a descriptor of the morphology that is shared by all members of this artificial section.

Teleomorph morphology in Hypocrea is not always useful to distinguish species. Hypocrea cremea, H. estonica, H. surrotunda, H. strictipilosa, H. aureoviridis f. macrospora and H. cuneispora have small, yellowish pulvinate stromata. Hypocrea strictipilosa, H. a. f. macrospora and H. cuneispora, all have larger ascospores. They can be distinguished primarily by the anamorph and, in the case of H. cuneispora, by the "scaly" appearance of the surface of the stroma. In contrast, H. ceramica has a reddish-brown stroma that is very distinctive and spinulose ascospores. Hypocrea semiorbis and the teleomorph of T. stromaticum both have colorless ascospores. The teleomorph of H. a. f. macrospora resembles H. aureoviridis sensu stricto; however the anamorph of H. a. f. macrospora morphologically is distinct from that of H. aureoviridis (Lieckfeldt et al 2001).

Trichoderma strictipile was described on the basis of an ascospore isolate from an unnamed Hypocrea species, which is described here as H. strictipilosa. This species is common in eastern North America and Northern Europe, as far east as the Moscow region of Russia. Trichoderma fasciculatum also is derived from a Hypocrea specimen, but we could not locate that collection in CBS or DAOM. The ex-type and only known isolate of T. fasciculatum is situated amid T. strictipile. In its morphological and molecular characters it is indistinguishable from T. strictipile. Bissett (1991b) considered the two species to be closely related and distinguished them only through the morphology of the conidiophore elongation, straight in T. fasciculatum and flexuous in T. strictipile. The isolates that we studied can have both types of conidiophore elongations in the same isolate. Therefore, we conclude that T. fasciculatum is a synonym of T. strictipile.

Sequence data indicate a close relationship between H. cuneispora and T. longipile, but each is represented by only a single culture and the two anamorphs are so different in their morphology, growth rates and colony characters that we cannot consider them to be the same species. A close relationship between these species and H. strictipilosa/Trichoderma strictipile also is indicated.

The RPB2 and EF-1 gene genealogies have revealed phylogenetic "sister species" that differ slightly in sequence and share phenotypic characters. For example, the anamorphs of H. cremea and H. surrotunda are almost indistinguishable. The main differences are found in the growth rate on PDA, an average difference of ca 0.5 µm in conidial length and in the morphology of the stroma. Another example is the similarity between T. hamatum and T. pubescens. Bissett (1991b) had mentioned that these two species were closely related but that he could distinguish them on the morphology of the conidiophore elongations, a difference that we found difficult to see. However, it was found that the growth rate on PDA is significantly faster in T. hamatum. Small but significant differences in the length of the phialides and conidia between the two species also were observed. We have not ruled out the possibility that T. pubescens is a synonym of T. hamatum, but to make a definite conclusion more isolates of both species need to be studied. Trichoderma fertile and T. spirale also are difficult to distinguish morphologically; however RPB2 and EF-1 gene genealogies show that they are not closely related. We could distinguish T. fertile from T. spirale by the slightly smaller conidia, slightly larger phialides, fertile conidiophore apical elongations and slower growth rate on PDA. These examples illustrate the necessity of using a combination of molecular and phenotypic characters to delimit species in Hypocrea/Trichoderma.

Phylogenetic reconstruction in Hypocrea/Trichoderma has relied most heavily on sequence analysis of the ITS region of rDNA (Dodd et al 2000, Kindermann et al 1998, Kuhls et al 1997, Lieckfeldt et al 1998a). However, results of these analyses have not sufficiently resolved relationships of species or separated species within closely related complexes. In addition, some of the isolates studied in this paper considered to be the same species have very different ITS sequences (results not shown). Consequently, the ITS sequence data produce a tree topology that is very distinct from RPB2 and EF-1 phylogenies. For example, we observed that certain isolates of T. strictipile and T. crassum, respectively, possessed divergent ITS 1 sequences, placing them in at least three different positions in the tree. In contrast, RPB2 and EF 1- tree topologies were highly congruent and T. strictipile and T. crassum isolates were monophyletic. Although it is not clear why this happened in our study, the presence of different non-orthologous ITS types has been shown previously for Fusarium spp. (O'Donnell and Cigelnik 1997) and a similar situation might be occurring in Trichoderma.

Until now, there were 13 described species of Trichoderma with green conidia and conidiophore apical elongations. In this paper, five more unnamed species were found to be distinct from the named species and therefore are described as new.

KEY TO SPECIES OF HYPOCREA AND TRICHODERMA WITH GREEN CONIDIA AND CONIDIOPHORE ELONGATIONS

1. Growing on PDA at 30 C after 72 h . . . . . 2

1. Not growing on PDA at 30 C; teleomorph with hyaline ascospores; known only from Australia/New Zealand . . . . . 12. H. semiorbis

     2. Stromatic pustules formed of vesicular to pseudoparenchymatous cells arranged in chains that tend to branch dichotomously near the surface of the pustule; known only from Theobroma cacao . . . . . 16. T. stromaticum

     2. Pustules not stromatic . . . . . 3

3. Conidia with L/W 1.6 . . . . . 4

3. Conidia with L/W < 1.6 . . . . . 7

     4. Phialides 7.2–17.0 x 1.5–2.5 µm, L/W > 2.0 . . . . . 1. H. aureoviridis f. macrospora

     4. Phialides < 7.2 µm long, L/W < 2.0 . . . . . 5

5. Phialides 4.2–6.0 x 3.0–4.5 µm; conidia 4.0–5.7 x 2.0–3.0 µm (L/W 1.6–2.0) . . . . . 6

5. Phialides 6.0–7.2 x 3.7–4.0 µm (L/W 1.5–2.0); conidia 5.5–6.0 x 3.2–3.5 µm (L/W 1.6–1.8) (phialides from conidiophore elongation apices 9.7–15.7 x 2.2–3.7 µm, L/W 3.0–5.5) . . . . . 5. H. cuneispora

     6. Conidia with L/W ca 2.0, 4.0–5.7 x 2.0–3.0 µm; phialides 4.2–6.0 x 3.0–4.5 µm; conidiophore elongations flexuous and frequently branched . . . . . 9. T. longipile

     6. Conidia with L/W 1.6, 4.5–4.7 x 2.7–3.0 µm; phialides 5.5–6.0 x 3.7–4.0 µm; conidiophore elongations relatively straight, unbranched . . . . . 10. T. oblongisporum

7. Phialides 8.2 µm long . . . . . 8

7. Phialides < 8.2 µm long . . . . . 10

     8. Phialides from all kinds of conidiophores 8.2–10.5 µm long . . . . . 9

     8. Phialides from gliocladium-like synanamorph 12.5–17.0 x 4.0–5.0 µm (L/W 3.0–3.8); conidia 5.5–6.5 x 4.2–5.0 µm (L/W 1.2–1.4) (macronematous pustulate anamorph sometimes present with phialides 4.5–9.5 x 3.0–4.2 µm, conidia 3.7–5.2 x 2.5–3.7 µm) . . . . . 3. T. crassum

9. Phialides 8.2–9.5 x 3.5–4.0 µm (L/W 2.2–2.6), conidia 4.5–5.0 x 3.7–4.0 µm (L/W 1.2–1.3); radial growth slow, on PDA after 72 h at 25 C: 26–32 mm and at 30 C: 20–22 mm . . . . . 17. H. surrotunda

9. Phialides 9.0–10.5 x 3.2–3.5 µm (L/W 2.7–3.3); conidia 4.0–4.5 x 3.5–3.7 µm (L/W 1.1–1.2); radial growth fast, on PDA after 72 h at 25 C: 63–72 mm and at 30 C: 70–74 mm . . . . . 4. H. cremea

     10. Conidia with L/W 1.1–1.3 . . . . . 11

     10. Conidia with L/W 1.3 . . . . . 13

11. Conidia 3.3–3.7 x 2.7–3.0, L/W 1.1–1.3; stroma reddish-brown . . . . . 2. H. ceramica

11. Conidia 4.0 µm long; stroma yellowish . . . . . 12

     12. Conidia 4.0–4.2 x 3.2–3.5, L/W 1.2–1.3; phialides from synanamorph 14.7–18.2 x 2.5–3.0, L/W 5.4–7.9 . . . . . 6. H. estonica

     12. Conidia 4.5–4.7 x 3.5–3.7, L/W 1.3; phialides from synanamorph 13.0–14.5 x 3.5–3.7, L/W 3.8–4.3 . . . . . 14. H. strictipilosa/T. strictipile

13. Conidia 3.2–3.5 x 2.2–2.5 µm (L/W 1.4); phialides 4.5–5.0 x 3.0–3.2 µm (L/W 1.5–1.6) (phialides from apical conidiophore elongations 8.5–10.0 x 2.5–2.7 µm, L/W 3.2–3.8, conidia 3.5–3.7 x 2.5–2.7 µm, L/W 1.3–1.4) . . . . . 18. T. tomentosum

13. Conidia > 3.6 µm long, L/W > 1.4 . . . . . 14

     14. Conidiophore elongations branched, with long branches, sterile . . . . . 15

     14. Conidiophore elongations mostly unbranched, and when branched then branches short and fertile . . . . . 16

15. Conidiophore elongations thin, flexuous, branching; phialides 5.5–6.0 x 3.5–3.7 µm (L/W 1.5–1.7); conidia 4.0–4.2 x 2.7–3.0 µm, L/W 1.5–1.6; colonies on PDA at 25 C after 72 h 32–33 mm radius . . . . . 11. T. pubescens

15. Conidiophore elongations, generally straight, branching; phialides 5.2–7.2 x 3.2–4.2 µm (L/W 1.6–1.8); conidia 4.2–5.0 x 2.7–3.0 µm, L/W 1.5–1.6; colonies on PDA at 25 C after 72 h 45–52 mm radius . . . . . 8. T. hamatum

     16. Gliocladium-like synanamorph always present. Conidiophore apical elongations straight, unbranched, generally sterile, when fertile with one or two phialides; phialides 4.5–8.2 x 3.0–4.2 µm; conidia 3.7–5.2 x 2.5–3.7 µm (L/W 1.4) . . . . . 3. T. crassum

     16. Gliocladium-like synanamorph absent . . . . . 17

17. Conidia 3.5–4.5 x 2.0–3.0 µm (L/W 1.5–1.6), subcylindrical oblong; phialides 5.0–8.0 x 2.7–3.7 µm; conidiophore elongations straight, rarely undulate, usually branched irregularly just above the fertile part, usually with a single phialide at the tip . . . . . 15. T. strigosum

17. Conidia 3.5–4.5 x 2.5–3.0 µm (L/W 1.4–1.5), ellipsoidal; phialides 4.5–6.5 x 3.0–4.7 µm; conidiophore elongations generally unbranched, when branched then with a short branch near the tip of the elongation . . . . . 18

     18. Conidia 3.5–4.0 x 2.5–3.0 µm; phialides 5.2–6.5 x 3.5–4.7 µm (L/W 1.3–2.0); conidiophore elongations straight, generally with one or two short, fertile apical branches; colonies on PDA at 30 C after 72 h 18–55 mm; no known synanamorph . . . . . 7. T. fertile

     18. Conidia 3.5–4.5 x 2.5–3.0 µm; phialides 4.5–6.5 x 3.0–3.7 µm (L/W 1.3–2.0); conidiophore elongations flexuous, unbranched, sterile; colonies on PDA at 30 C after 72 h 60–70 mm radius; synanamorph formed in aerial mycelium . . . . . 13. T. spirale

	SPECIES DESCRIPTIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SPECIES DESCRIPTIONS LITERATURE CITED

 
1. Hypocrea aureoviridis f. macrospora Doi, Bull. Natl. Sci. Mus. Tokyo 15: 728. 1972.

Notes – We examined the isotype and two paratype specimens of H. aureoviridis f. macrospora (NY). Although an anamorph was described for this taxon, none of the cultures is available. This taxon resembles H. cuneispora and H. strictipilosa in the large ascospores, anamorph with fertile conidiophore apical elongations and yellowish stroma. The forma differs from H. strictipilosa in conidial length/width ratio, respectively ca 1.7 and 1.2–1.3. Hypocrea aureoviridis f. macrospora can be distinguished from H. cuneispora by the slightly longer and more slender phialides and slightly smaller ascospores. In addition, H. a. f. macrospora seems to be restricted to Japan and the H. cuneispora holotype is from the U.S.A. The teleomorph of H. a. f. macrospora resembles H. aureoviridis sensu stricto; however, the anamorph of H. a. f. macrospora is morphologically distinct from that of H. aureoviridis, T. aureoviride Rifai (Lieckfeldt et al 2001). Therefore, we doubt that H. a. f. macrospora is a forma of H. aureoviridis s. str., but in the absence of additional fresh material we do not propose to change its status.

Specimens examined.—JAPAN. GUNMA PREF.: Hontani, Naruhara, Ueno-Mura, Tano-Gun, on decorticated wood, 5 Aug 1967, Y. Doi (TNS.D-332 = TNS-F-191616, PARATYPE: NY). NAGANO PREF.: Near Asama-Sanso, the foot of Mount Asama, Komoro City, on decorticated wood, 13 Aug 1967, Y. Doi (TNS.D-333 = TNS-F-223414, PARATYPE: NY), on decorticated wood, 11 Aug 1966, Y. Doi (TNS.D-148 = TNS-F-191611, ISOTYPE: NY).

2. Hypocrea ceramica Ellis et Everh., North Amer. Pyrenomyc. p. 85. 1892 Figs. 8–15, 140, 148, 168

View larger version (19K): [in this window] [in a new window]   FIG. 8. Hypocrea ceramica anamorph (isolate G.J.S. 88-70). Scale bars = 10 µm

View larger version (82K): [in this window] [in a new window]   FIGS. 140–147. Stromata. 140. Hypocrea ceramica (G.J.S. 88-70). 141. H. cremea (holotype). 142. H. cuneispora (holotype). 143. H. estonica (holotype). 144. H. semiorbis (G.J.S. 99-108). 145. H. surrotunda (holotype). 146. H. strictipilosa (G.J.S. 96-189). 147. H. strictipilosa (holotype). Scale bars = 500 µm

View larger version (143K): [in this window] [in a new window]   FIGS. 148–167. Pustules on CMD at 20 C after 1–2 wk of growth in 12 h darkness/12 h light intervals. 148. H. ceramica. 149. T. crassum gliocladium-like synanamorph. 150. H. cremea. 151. H. cuneispora. 152. H. estonica. 153. T. fertile. 154. T. hamatum. 155. T. oblongisporum. 156. T. pubescens. 157. H. semiorbis. 158. T. spirale. 159–162. H. strictipilosa/T. strictipile. 163. H. surrotunda. 164. T. strigosum. 165, 166. T. stromaticum. 167. T. tomentosum. 148–154, 156, 158, 163, 164, 167 = type isolates, 155 = DAOM 167085, 157 = DAOM 167636, 159 = G.J.S. 98-113, 160 = G.J.S. 98-110, 161 = G.J.S. 94-97, 162 = DAOM 167646 (ex-type isolate of T. fasciculatum), 165 = G.J.S. 00-91, 166 = G.J.S. 00-141. Scale bars = ca 1 mm

View larger version (156K): [in this window] [in a new window]   FIGS. 168–183. Colonies on PDA at 25 C after 1–2 wk of growth in 12 h darkness/12 h light intervals. 168. H. ceramica. 169. T. crassum. 170. H. cremea. 171. H. cuneispora. 172. H. estonica. 173. T. fertile. 174. T. hamatum. 175. T. oblongisporum. 176. T. pubescens. 177. H. semiorbis. 178. T. spirale. 179. T. strictipile. 180. T. strigosum. 181. T. stromaticum. 182. H. surrotunda. 183. T. tomentosum. 168 = G.J.S. 88-70, 169–176, 178–180, 182, 183 = ex-type isolates, 177 = G.J.S. 81-195, 181 = G.J.S. 00-91

Chromocrea ceramica (Ellis et Everh.) Seaver, Mycologia 2: 59. 1910.

Anamorph – Trichoderma sp.

Stromata scattered, pulvinate, nearly circular in outline, (0.6–)0.8–1.7(–2.1) mm diam (n = 30), 0.8–1.0 mm high (n = 10), broadly attached, surface smooth, with perithecial protuberances, reddish brown, not changing color in KOH, ostiolar openings obvious due to the discharged green ascospores. Tissue of the stroma of textura angularis. Stroma surface cells (3.4–)4.8–6.3(–8.3) µm diam (n = 30), walls (0.5–)0.6–0.8(–0.9) µm thick (n = 30). Tissue immediately below the surface layer and between perithecia of textura angularis, cells (3.1–)5.0–6.4(–7.8) µm diam (n = 25), walls (0.3–)0.4–0.5(–0.6) µm thick (n = 20). Internal tissue below the perithecia of textura angularis, cells (5.3–)6.7–8.0(–10.8) µm diam (n = 30), walls (0.5–)0.6–0.7(–1.1) µm thick (n = 20). Perithecia immersed in the stroma, generally closely aggregated, subglobose, 195–260 µm high, 112–230 µm wide (n = 10), wall composed of compacted cells, turning brownish in KOH, (9–)13–15 (–17) µm thick (n = 20), ostiolar canal 40–90 µm long (n = 10). Asci cylindrical, (50–)68–95(–96) x (3.2–)4.5–5.5(–7.7) µm (n = 35), ascospores uniseriate. Part-ascospores green, spinulose, dimorphic, distal part globose to subglobose, (3.5–)3.9–4.7(–5.8) x (2.9–)3.4–4.2(–4.3) µm, proximal part cuneiform, (3.4–)4.2–5.2(–6.2) x 1(2.9–)3.1–3.7(–4.0) µm (n = 60).

Colonies on CMD at 20 C after ca 10 d flat, with discrete tufts 2–3 mm diam (n = 3) forming in concentric rings from the point of inoculum outward, no odor, no pigmentation of agar. Conidiophores irregularly branched, phialides ampulliform, broader in the middle, and constricted at the tip, formed in whorls of (1–) 3 (–4), (5.0–)6.5–8.0(–13.0) µm long, (2.5–)3.5–4.0(–4.5) µm at the widest point, (2.0–)2.7–3.1(–3.7) µm at the base, L/W (1.2–)1.7–2.2(–4.4) (n = 30), elongations of the conidiophore rare, fertile or sterile, generally terminating in one phialide; subtending hyphae cylindrical, (3.0–)3.5–4.0(–4.3) µm wide (n = 15). Conidia green, smooth, ellipsoidal, (2.8–)3.3–3.7(–4.2) x (2.1–)2.7–3.0(–3.1) µm, L/W (0.9–)1.1–1.3(–1.7) (n = 20). No chlamydospores observed.

Colonies on PDA at 25 C after ca 2 wk flat, aggregated pustules forming from the point of inoculum outward, young conidia pale yellow, no pigmentation of agar, and no distinctive odor. Colony radius after 3 d on PDA at 15 C: 5–19 mm, 20 C: 16–27 mm, 25 C: 35–50 mm, 30 C: 41–57 mm, and 35 C: 0 mm (n = 3). Colony radius after 3 d on SNA at 15 C: 3–7 mm, 20 C: 9–16 mm, 25 C: 17–24 mm, 30 C: 23–32 mm, and 35 C: 0 mm (n = 3).

Habitat – Decorticated wood.

Known distribution – United States and Japan.

Holotype – UNITED STATES. CONNECTICUT: West Haven, on decaying limb of Juniperus, Nov 1888, R. Thaxter (NY).

Additional specimen examined.—UNITED STATES. NORTH CAROLINA: Transylvania County, North of Brevard, Pisgah National Forest, fish hatchery, on decorticated wood, 29 Sep 1988, K. F. Rodrigues, C.T.R., G.J.S., E. Parmasto, R. H. Petersen (NY, culture: G.J.S. 88–70).

Notes – Hypocrea ceramica can be distinguished by the reddish brown to brick-red stroma, a color that is rare in Hypocrea, and the small conidia and short phialides. The conidiophore elongation is a rare trait in this species. Hypocrea ceramica phylogenetically is closely related to H. estonica based on RPB2 and EF-1 molecular sequence data. Both species have small conidia compared to the other species studied in this paper.

3. Trichoderma crassum Bissett, Can. J. Bot. 69: 2376.1991. Figs. 16–24, 149, 169

View larger version (167K): [in this window] [in a new window]   FIGS. 16–24. Trichoderma crassum. 16–18. Conidiophores. 19, 20, 23, 24. Gliocladium-like synanamorph. 21. Conidia. 22. Long conidiophores. 16–19, 21–23 = DAOM 164916 (ex-type), 20 = G.J.S. 98-179, 24 = G.J.S. 95-157. Scale bars: 16–20 = 10 µm, 21 = 5 µm, 22, 23 = 15 µm, 24 = 20 µm

Colonies on CMD at 20 C after ca 1 wk flat, with fasciculate pustules formed near the point of inoculum. Conidiophores on CMD at 20 C after ca 1 wk, in minute pustules or effuse, branching irregularly, with clusters of short ampulliform phialides. Phialides mainly arising in crowded whorls of 2–5, less frequently in pairs, straight, short, ampulliform, base constricted, swollen in the middle, attenuate at the tip, 4.4–9.5 x 3.0–4.2 µm, arising from a cell (3.2–)4.6–5.0(–5.7) µm wide (n = 20). Conidiophore elongations sometimes present, fertile, rarely sterile. Synanamorph conidiophores arising singly in the aerial mycelium, conidiophores (6.0–)20.0–28.5(–49.0) x (4.5–)5.2–6.0(–8.0) µm (n = 15) with each branch terminating in a penicillus of (2–)3–4 closely appressed phialides; phialides (8.5–)13.5–15.7(–28.0) µm long, (3.3–)4.3–4.6(–5.7) µm at the widest point, (2.2–)3.0–3.3(–4.8) µm at the base, L/W (1.8–)3.1–3.6(–7.3) (n = 60). Long unbranched conidiophores with one or two phialides at the tip sometimes present in the aerial mycelia, ca 200–300 µm long (n = 10). Phialides arising from unbranched long conidiophores 24.8–29.3 µm long, 2.9–3.0 µm at the widest point, 3.0–3.5 µm at the base, L/W 8.2–10.0 (n = 30). Conidia green, smooth, broadly ellipsoidal to obovoid, 3.7–5.3 x 2.6–3.7 µm, L/W ca 1.4. Conidia from gliocladium-like synanamorph, in areas of effuse conidiation, (5.1–)5.9–6.4(–8.0) x (3.9–)4.7–4.9(–5.7) µm, L/W ca 1.3 (n = 60). Chlamydospores present in some isolates, terminal and intercalary, subglobose to ellipsoidal, 6–17 µm diam (n = 15).

Colonies on PDA at 25 C after ca 1 wk slightly cottony, conidiation effuse or aggregated in minute flat pustules, <1 mm diam, formed around the point of inoculum, diffusing yellow pigment sometimes found, no distinctive odor detected. Colony radius after 3 d on PDA at 15 C: 10–20 mm, 20 C: 22–33 mm, 25 C: 35–45 mm, 30 C: 36–49 mm, and 35 C: 0–5 mm (n = 9). Colony radius after 3 d on SNA at 15 C: 8–19 mm, 20 C: 17–35 mm, 25 C: 34–53 mm, 30 C: 33–57 mm, and 35 C: 0–6 mm (n = 9).

Habitat – Wood and soil.

Known distribution – North America, New Zealand.

Specimens examined.—CANADA. QUEBEC: Lacolle, from Norway spruce plantation soil, May 1977, P. Widden (culture: DAOM 164916, ex-type). NEW ZEALAND. Tokoroa, Maiden Tram Rds., from soil under Pinus radiata, 24 Sep 1997, A. Chee (culture: G.J.S. 98-179). UNITED STATES. NEW YORK: Tompkins County, Ringwood-Lloyd Preserve, on decorticated wood, 13 Sep 1995, K. T. Hodge (culture: G.J.S. 95-157, as Hypocrea sp., specimen lost).

Notes – The ex-type isolate of T. crassum exhibits two types of anamorphs: one pustulate with macronematous conidiophores that has clusters of short phialides that form aggregated, irregular pustules on PDA and CMD, and another anamorph that has gliocladium-like conidiophores that do not form pustules and are rather spread out through the plate. Other cultures examined did not exhibit the macronematous synanamorph or it was rare. One of the isolates examined, G.J.S. 95-157, originated from ascospores from an unidentified species of Hypocrea with green ascospores. Unfortunately this specimen has been lost and therefore the teleomorph of T. crassum could not be described. Trichoderma crassum phylogenetically is closely related to T. virens based on RPB2 and EF-1 molecular data. The gliocladium-like synanamorph of T. crassum is almost indistinguishable from that of T. virens. Trichoderma crassum can be separated easily from T. virens by the inability of the former species to grow at 35 C.

4. Hypocrea cremea Chaverri et Samuels, sp. nov. Figs. 25–29, 141, 150, 170

View larger version (22K): [in this window] [in a new window]   FIG. 25. Hypocrea cremea anamorph (holotype). Scale bars = 10 µm

Stromata albolutea, modice perlucida, (0.8–)0.9–1.2(–1.5) mm diam. Asci cylindrici, (99–)104–110(–120) x (4.7–)6.0–6.5(–7.0) µm. Ascosporae bicellulares, verruculosae, verrucis magnitudine dissimilibus, ad septum disarticulatae, atrovirentes; parte distali globosa ad subglobosa, (4.2–)5.5–6.0(–6.7) x (4.5–)5.2–5.5(–7.0) µm, parte proximali cuneiformi ad cylindrica, (5.0–)5.7–6.2(–7.2) x (4.0–)4.7–5.2(–6.2) µm. Anamorphosis Trichoderma sp. Phialidis (7.2–)9.0–10.5(–15.2) x (2.7–)3.2–3.5(–4.5) µm; conidii viridia, glabra, (3.5–)4.0–4.5(–4.7) x (3.2–)3.5–3.7(–4.0) µm, ratio longitudo/crassitudo (1.0–)1.1–1.2(–1.4). Incrementum in agaro "PDA" dicto post 72 h 15 C = 14–19 mm, 20 C = 47–50 mm, 25 C = 63–72 mm, 30 C = 70–74 mm, 35 C = 4–5 mm. Holotypus BPI 1112894.

Anamorph – Trichoderma sp.

Stromata scattered, pulvinate, nearly circular in outline, (0.8–)0.9–1.2(–1.5) mm diam (n = 10), ca 0.6 mm high (n = 10), broadly attached, surface smooth, with perithecial protuberances, light yellow, somewhat transparent, not changing color in KOH, ostiolar openings obvious due to the discharged green ascospores. Tissue of the stroma formed of hyaline angular cells that increase in size progressively from the surface to the internal tissue below perithecia. Stroma surface cells (5.2–)8.5–10.2(–14.7) µm diam (n = 30), walls 0.5–0.7(–1.0) µm thick (n = 25). Internal tissue below perithecia formed of cells (8.5–)13.7–17.2(–28.0) µm diam (n = 30), walls 0.7–1.0(–1.2) µm thick (n = 10). Perithecia completely immersed in stroma, generally closely aggregated, subglobose, 233–267 µm high, 138–198 µm wide (n = 5), wall composed of compacted cells, KOH-, 13–17(–19) µm thick (n = 10), ostiolar canal 50–65 µm long (n = 5). Asci cylindrical, (99–)104–110(–120) x (4.7–)6.0–6.5(–7.0) µm (n = 20), ascospores uniseriate. Part-ascospores green, warted, with warts of different sizes, dimorphic, distal part globose to subglobose, (4.2–)5.5–6.0(–6.7) x (4.5–)5.2–5.5(–7.0) µm, proximal part cuneiform to cylindrical, (5.0–)5.7–6.2(–7.2) x (4.0–)4.7–5.2(–6.2) µm (n = 30).

Colonies on CMD at 20 C after ca 1 wk flat, with discrete tufts 2–3 mm diam (n = 6) forming on the edges of the plate, no distinctive odor, no pigmentation of the agar. The branching pattern of the conidiophores irregular, branches not paired and generally longer toward the base, phialides cylindrical, broader in the middle, and constricted at the tip, formed in whorls of (1–)3(–5), (7.2–)9.0–10.5(–15.2) µm long, (2.7–)3.2–3.5(–4.5) µm at the widest point, (1.7–)2.0–2.5(–3.2) µm at the base, L/W (1.6–)2.7–3.2(–5.1) (n = 30), elongations of the conidiophore not common, fertile, terminating in 1 or 2 phialides (9.7–)10.5–12.7(–13.7) µm long, 2.7–3.2 µm at the widest point, (1.7–)2.2–2.5(–3.2) µm at the base, L/W (3.3–)3.5–4.2(–4.4) (n = 10); subtending hyphae cylindrical, (2.7–)3.2–3.7(–5.0) µm wide (n = 22). Conidia green, smooth, obovoid to subglobose, (3.5–)4.0–4.5(–4.7) x (3.2–)3.5–3.7(–4.0) µm, L/W (1.0–)1.1–1.2(–1.4) (n = 30). No chlamydospores observed.

Colonies on PDA at 25 C after ca 1 wk cottony, with no conidia formed until ca 2 wk, no pigmentation of agar and no distinctive odor. Colony radius after 3 d on PDA at 15 C: 14–19 mm, 20 C: 47–50 mm, 25 C: 63–72 mm, 30 C: 70–74 mm, and 35 C: 4–5 mm (n = 3). Colony radius after 3 d on SNA at 15 C: 2–21 mm, 20 C: 27–48 mm, 25 C: 42–55 mm, 30 C: 55–63 mm, and 35 C: 2–7 mm (n = 3).

Habitat – Decorticated wood.

Known distribution – United States (New York).

Holotype – UNITED STATES. NEW YORK: Hollyhock Hollow Sanctuary, Rarick Road, 3 miles South of Feura Bush, on decorticated wood, 21 Sep 1991, R. Lowen (BPI 1112894; cultures: G.J.S. 91-125 = ATCC MYA-2862 = CBS 111146 = DAOM 231312).

Additional specimen examined.—UNITED STATES. NEW YORK: Ulster County, Ashokan Camp, on decorticated wood, probably on a black pyrenomycete, 27 Sep 1998, P. Chaverri (culture: P.C. 14).

Notes – This species is characterized by the semitransparent pale yellow stromata, ascospores with variable size warts, fast growth on PDA at 30 C and very slow or no growth at 35 C. Hypocrea cremea is a sister species to H. surrotunda based on RPB2 and EF-1 sequence data. Their anamorphs are almost indistinguishable: both have phialides that are attached to the subtending hyphae at wide angles, thus they both have morphology of sect. Trichoderma. Hypocrea cremea is distinguished from H. surrotunda by the faster growth rate and smaller conidia. In addition, the stroma of H. cremea is semitransparent and somewhat glossy whereas the stroma of H. surrotunda is opaque.

5. Hypocrea cuneispora Chaverri et Samuels, sp. nov. Figs. 30–34, 142, 151, 171

View larger version (24K): [in this window] [in a new window]   FIG. 30. Hypocrea cuneispora anamorph (holotype). Scale bars = 10 µm

Stromata albolutea, subsquarrosa, KOH+, (0.6–)0.7–1.3(–1.8) mm diam. Asci cylindrici, (109–)121–139(–149) x (5.2–)5.5–6.0(–6.5) µm. Ascosporae bicellulares, verruculosae, ad septum disarticulatae, atrovirentes; parte distali cuneiformi ad subglobosa, (5.5–)6.5–7.0(–8.0) x (4.2–)5.0–5.2(–6.0) µm, parte proximali cuneiformi, (6.0–)7.0–7.7(–10.0) x (3.7–)4.5–4.7(–5.5) µm. Anamorphosis Trichoderma sp. Phialidis (4.0–)6.0–7.2(–10.5) x (3.0–)3.7–4.0(–4.5) µm; conidii viridia, glabra, oblongata ad ellipsoidea, (4.5–)5.5–6.0(–7.2) x 2.5–)3.2–3.5(–4.0) µm, ratio longitudo/crassitudo (1.4–)1.6–1.8(–2.3). Holotypus BPI 1112864.

Anamorph – Trichoderma sp.

Stromata aggregated or in pairs and adjacent stromata sometimes fusing, pulvinate, circular to irregular in outline, (0.6–)0.7–1.3(–1.8) mm diam (n = 10), (0.8–)0.9–1.0 mm high (n = 10), broadly attached, surface rough, with no perithecial protuberances, orange-brown to buff, becoming darker in KOH, ostiolar openings obvious due to the green ascospores. Outermost layer of stroma composed of dead cells, small, irregularly shaped, compact, that make the surface look rough or scaly. Layer below the outermost layer of stroma composed of angular cells, brownish, (4.7–)6.7–8.0(–12.5) µm diam (n = 30), walls (0.5–)0.7–1.0(–1.2) µm thick (n = 30). Tissue between perithecia and below the second layer composed of hyaline, angular cells, (6.0–)8.5–9.7(–12.2) µm diam (n = 30), walls 0.5–0.7(–1.0) µm thick (n = 30). Internal tissue below the perithecia formed of angular, hyaline cells (11.0–)17.2–20.7(–28.2) µm diam (n = 30), walls (0.5–)0.7–1.0 µm thick (n = 30). Perithecia completely immersed in stroma, generally closely aggregated or with some space in between, subglobose, (262–)271–305(–309) µm high, (156–)162–225(–250) µm wide (n = 10), wall composed of compacted cells, KOH-, (10–)14–18(–24) µm thick (n = 20), ostiolar canal (60–)63–83(–94) µm long (n = 10). Asci cylindrical, (109.0–)121–139(–149) x (5.2–)5.5–6.0(–6.5) µm (n = 10), ascospores uniseriate. Part-ascospores green, warted, generally dimorphic, distal part generally cuneiform or subglobose, sometimes barrel-shaped (5.5–)6.5–7.0(–8.0) x (4.2–)5.0–5.2(–6.0) µm, proximal part generally cuneiform, sometimes barrel-shaped or ellipsoidal, (6.0–)7.0–7.7(–10.0) x (3.7–)4.5–4.7(–5.5) µm (n = 30).

Colonies on CMD at 20 C after ca 1 wk flat, with discrete small tufts <1 mm diam that form on the edges of the plate, conidia produced after ca 2 wk, no distinctive odor, no pigmentation of the agar. Conidiophores generally producing short branches that generally do not rebranch, with some phialides arising directly from the conidiophore. Phialides short, ampulliform, formed in whorls of (1–)2–3(–5), (4.0–)6.0–7.2(–10.5) µm long, (3.0–)3.7–4.0(–4.5) µm at the widest point, (1.5–)2.5–2.7(–3.7) µm at the base, L/W (1.0–)1.6–2.0(–3.4) (n = 30), apical elongations of the conidiophore common, sterile or fertile, with 1 or 2 cylindrical phialides, slightly broader at the middle, constricted at the tip, 11–15 µm long, 2.2–3.2 µm at the widest point, 1.7–2.7 µm at the base, L/W 3.3–5.1 (n = 10); subtending hyphae cylindrical, (2.7–)3.5–4.5(–4.7) µm wide (n = 10); a verticillium-like synanamorph sometimes observed. Conidia green, smooth, oblong to ellipsoidal, (4.5–)5.5–6.0(–7.2) x (2.5–)3.2–3.5(–4.0) µm, L/W (1.4–)1.6–1.8(–2.3) (n = 30). Abundant chlamydospores formed after 3 wk, intercalary or terminal, globose to subglobose, (8.7–)10.2–11.7(–13.2) µm diam (n = 15).

Colonies on PDA at 25 C after ca 1 wk flat, with scant aerial mycelium, conidia not formed until ca 2 wk after inoculation, no pigmentation of the agar and no distinctive odor. Colony radius after 3 d on PDA at 15 C: 14–18 mm, 20 C: 32–35 mm, 25 C: 41–43 mm, 30 C: 29–32 mm, and 35 C: 0 mm (n = 3). Colony radius after 3 d on SNA at 15 C: 9–12 mm, 20 C: 31–34 mm, 25 C: 42–45 mm, 30 C: 19–23 mm, and 35 C: 0 mm (n = 3).

Habitat – Decorticated wood.

Known distribution – United States (Louisiana and Virginia).

Holotype – UNITED STATES. VIRGINIA: Giles County, Cascades Recreation site, 4 miles North of Pembroke, Little Stony Creek, 37° 2' N, 80° 35' W, alt. 840 m, on decorticated wood, 18 Sep 1991, G.J.S., C.T.R., S. Huhndorf, S. Rehner, M. Williams (BPI 1112864; cultures: G.J.S. 91-93 = ATCC MYA-2863 = CBS 111148 = DAOM 231313).

Additional specimen examined.—UNITED STATES. LOUISIANA: East Baton Rouge Parish, Burden Plantation, on hymenium of Phellinus sp., 30 Jul 1991, M. Blackwell, D. Hawksworth, G.J.S. (BPI 1112810, culture: G.J.S. 91-35).

Notes – This species is characterized by large ascospores and large ellipsoidal conidia. The two North American specimens examined sometimes produced stromata on CMD. Hypocrea cuneispora resembles H. aureoviridis f. macrospora, which was described from Japan, in the large ascospores and large ellipsoidal conidia. These two species differ in that H. a. f. macrospora has slightly longer and more slender phialides and slightly smaller ascospores (Doi 1972). Unfortunately, the ex-type culture of H. a. f. macrospora is not available. Hypocrea cuneispora is a sister species of T. longipile based on RPB2 and EF-1 sequence data. Both species have ellipsoidal conidia with a length/width ratio 1.6–2.0 and the conidiophore branching pattern and phialide distribution are very similar. Hypocrea cuneispora anamorph can be distinguished from T. longipile by the longer phialides and the faster growth on PDA and SNA.

6. Hypocrea estonica Chaverri et Samuels, sp. nov. Figs. 35–40, 143, 152, 172

View larger version (23K): [in this window] [in a new window]   FIG. 35. Hypocrea estonica anamorph (holotype). Scale bars = 10 µm

Stromata albolutea, (0.4–)0.6–0.8(–1.0) mm diam. Ascosporae bicellulares, verruculosae, ad septum disarticulatae, atrovirentes; parte distali subglobosa ad oblonga, (3.7–)5.0–5.2(–6.0) x (3.5–)5.0–5.2(–5.7) µm, parte proximali cuneiforme, (4.2–)5.0–5.2(–6.2) x (3.2–)4.2–4.5(–5.2) µm. Anamorphosis Trichoderma sp. Phialidis (4.5–)6.5–7.0(–11.0) x (3.0–)3.5–3.7(–4.5) µm; conidii viridia, glabra, (3.5–)4.0–4.2(–5.0) x (2.5–)3.2–3.5(–4.2) µm, ratio longitudo/crassitudo (1.0–)1.2–1.3(–1.7). Incrementum in agaro "PDA" dicto post 72 h 15 C = 5–16 mm, 20 C = 15–23 mm, 25 C = 25–40 mm, 30 C = 4–13 mm, 35 C = 0 mm. Holotypus BPI 744577.

Anamorph – Trichoderma sp.

Stromata scattered, solitary, pulvinate, circular to irregular in outline, (0.4–)0.6–0.8(–1.0) mm diam (n = 20), (0.2–)0.4–0.5(–0.6) mm high (n = 20), broadly attached, surface smooth, perithecial protuberances visible, pale yellow, becoming brownish in KOH, ostiolar openings obvious due to the green ascospores. Outermost layer of stroma composed of compacted angular cells, (3.5–)5.2–6.0(–8.5) µm diam (n = 30), walls 0.5–0.7(–1.2) µm thick (n = 20). Tissue between perithecia and below the outermost layer composed of hyaline cells, of textura angularis to epidermoidea, (4.2–)6.0–7.5(–11.0) µm diam (n = 30), walls 0.5(–0.9) µm thick (n = 20). Internal tissue below the perithecia of textura angularis to epidermoidea (5.0–)7.5–9.0(–13.2) µm diam (n = 40), walls 0.5–0.7(–1.0) µm thick (n = 30). Perithecia immersed in the stroma, generally closely aggregated or slightly separated, subglobose, (159–)222–281(–348) x (116–)148–199(–237) µm (n = 15), wall composed of compacted cells, turning brownish in KOH, (11–)15–18(–22) µm thick (n = 20), ostiolar canal (46–)54–79(–89) µm long (n = 10). Asci cylindrical, uniseriate, (85–)91–96(–103) x (5.0–)5.7–6.2(–7.2) µm (n = 20). Part-ascospores green, warted, dimorphic, distal part subglobose to oblong (3.7–)5.0–5.2(–6.0)