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Hypocrea rufa/Trichoderma viride: a reassessment, and description of five closely related species with and without warted conidia

¹ Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 9-166.5, A-1060 Vienna, Austria
² United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Laboratory, Rm. 304, B-011A, BARC-W, Beltsville, Maryland 20705, U. S.A.
³ The Pennsylvania State University, Department of Plant Pathology, Buckhout Laboratory, University Park, Pennsylvania 16802, U.S.A. Current address: New Zealand Institute of Crop and Food Research Ltd., Private Bag 4704, Christchurch, New Zealand
⁴ The Pennsylvania State University, Department of Plant Pathology, Buckhout Laboratory, University Park, Pennsylvania 16802, U.S.A. Current address: Agronomy College, Department of Plant Protection, Zhongkai Agrotechnical College, Guangzhou 510225, China.

^* Correspondence: Gary J. Samuels, Gary{at}nt.ars-grin.gov

	Abstract

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 
The type species of the genus Hypocrea (Hypocreaceae, Hypocreales, Ascomycota, Fungi), H. rufa, is re-defined and epitypified using a combination of phenotype (morphology of teleomorphs and anamorphs, and characteristics in culture) and phylogenetic analyses of the translation-elongation factor 1 gene. Its anamorph, T. viride, the type species of Trichoderma, is re-described and epitypified. Eidamia viridescens is combined as Trichoderma viridescens and is recognised as one of the most morphologically and phylogenetically similar relatives of T. viride. Its teleomorph is newly described as Hypocrea viridescens. Contrary to frequent citations of H. rufa and T. viride in the literature, this species is relatively rare. Although both T. viride and T. viridescens have a wide geographic distribution, their greatest genetic diversity appears to be in Europe and North America. Hypocrea vinosa is characterised and its anamorph, T. vinosum sp. nov., is described. Conidia of T. vinosum are subglobose and warted. The new species T. gamsii is proposed. It shares eidamia-like morphology of conidiophores with T. viridescens, but it has smooth, ellipsoidal conidia that have the longest L/W ratio that we have seen in Trichoderma. Trichoderma scalesiae, an endophyte of trunks of Scalesia pedunculata in the Galapagos Islands, is described as new. It only produces conidia on a low-nutrient agar to which filter paper has been added. Additional phylogenetically distinct clades are recognised and provisionally delimited from the species here described. Trichoderma neokoningii, a T. koningii-like species, is described from a collection made in Peru on a fruit of Theobroma cacao infected with Moniliophthora roreri.

Taxonomic novelties: Hypocrea viridescens Jaklitsch & Samuels sp.nov., Trichoderma viridescens (A.S. Horne & H.S. Williamson) Jaklitsch & Samuels comb.nov., T. gamsii Samuels & Druzhinina sp.nov., T. vinosum Samuels sp.nov., T. neokoningii Samuels & Soberanis sp.nov., T. scalesiae Samuels & H.C. Evans sp.nov.

Keywords Bayesian phylogeny / biogeography / biological control / cacao / endophytes / Hypocrea / Hypocreales / Hypocreaceae / molecular identification / morphological key / nomenclature / species identification / systematics / translation elongation factor 1-alpha

	INTRODUCTION

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 
Trichoderma viride Pers. (Hypocreales, Hypocreaceae) is one of the most commonly reported species of fungi. In only the two years 2004 and 2005 T. viride appeared in nearly 200 articles that were abstracted by CAB. The species is encountered in widely diverse contexts; a few examples of activities include organochlorine degradation as a soil fungus (Smith 1995), biological control in fungus-induced plant disease (Brown & Bruce 1999; Brown et al. 1999), and as the cause of disease in button mushrooms in India (Mishra & Singh 2005). It is said to effect seed germination of flowering plants (Celar & Valic 2005), and enhance phosphorus uptake by plants (Rudresh et al. 2005). It produces enzymes (Nobe et al. 2004), degrades cellulosic agricultural waste to alcohol (Baig et al. 2004), colonises leaf litter (Osono 2005) and is a normal inhabitant of soils (Roiger et al. 1991, Hagn et al. 2003). Do all these citations refer to only one species, T. viride? Kullnig et al. (2001) detected a shockingly high level of misidentification of strains that were reported in the literature as T. harzianum. If this experience is representative of the genus, as it is likely, then not all of these reports actually refer to T. viride. One example that is representative of the degree of inaccuracy in identification is that of a biocontrol fungus reported in the literature as T. viride (Bastos 1988, 1996 a, b) that was ultimately described as the new species T. stromaticum Samuels & Pardo-Schultheiss (Samuels et al. 2000); these two species are distantly related and morphologically and biologically highly dissimilar. Obviously, it is important to clarify the identity of T. viride, otherwise the literature is meaningless.

Bisby in 1939 stated that essentially there was only one species of Trichoderma, T. viride. In spite of some discordant indications, that view held sway until 1969 when Rifai (1969) monographed the genus and characterised T. viride as the only species having globose, warted conidia. This immediately raised suspicion about all reports of activity by Trichoderma species prior to 1969. Even with the description of T. saturnisporum and T. ghanense, both having warted conidia and both being members of T. sect. Longibrachiatum Bissett (Samuels et al. 1998), T. viride stood out because its conidia were globose as compared to ellipsoidal in the other species. Scanning electron microscopy (Meyer & Plaskowitz 1989) revealed the existence of two distinct patterns of conidial ornamentation within strains identified as T. viride, viz. more and less strongly warted. Strains having the less strongly warted conidia were segregated as T. asperellum Samuels et al. (Lieckfeldt et al. 1999; Samuels et al. 1999). In a study of variation within the morphological species T. viride, in addition to recognising T. asperellum and T. viride s. str., Lieckfeldt et al. (1999) noted the existence of two additional ITS-defined groups that had warted conidia, which they referred to as Vd and Ve. The group Vd was very closely related to Vb in its ITS1 and 2 sequences and its morphology. The group Ve was more distantly related and was phenotypically diverse, some of the few included strains having smooth conidia and others having warted conidia. They (Samuels et al. 1999) determined that the group Vb was "true" T. viride by comparison with the over two-hundred-year-old type specimen of the species that is preserved in Leiden. Despite differences in ITS sequences, Samuels et al. (1999) could not see consistent phenotypic differences between Vb and Vd that would support recognition of Vd as a separate taxon.

Bissett (1991a) proposed to include H. rufa/T. viride and its relatives in Trichoderma sect. Trichoderma, including also T. koningii Oudem. and T. atroviride P. Karst. The monophyly of this group either as Trichoderma sect. Trichoderma (e.g. Kullnig-Gradinger et al. 2002) or more recently simply as "the viride clade" (Samuels 2006), has been affirmed by DNA sequence analysis. Since the work of Lieckfeldt et al. (1999) we have obtained many additional specimens and cultures referable to the viride clade and are able to propose a revised taxonomy for this clade. In the present work we re-evaluate T. viride groups Vb and Vd and recognise group Vd as a distinct species.

Since the middle of the 19^th century (Tulasne & Tulasne 1865), T. viride has been recognised as the anamorph of Hypocrea rufa (Pers.: Fr.) Fr., the type species of Hypocrea Fr. Like T. viride, H. rufa is possibly the most common name used in the identification of Hypocrea specimens. Hundreds of specimens in herbaria throughout the world are labelled "Hypocrea rufa". However, even a quick glance at specimens shows that a plethora of species has been lumped under this name. For example, species such as H. minutispora B.S. Lu et al./T. minutisporum Bissett and H. pachybasioides Yoshim. Doi/T. polysporum (Link: Fr.) Rifai have both been incorrectly identified as the only distantly related H. rufa.

Webster (1964) provided the first modern description of H. rufa. It is a species that has a stroma that starts out semieffused and whitish to tan to reddish brown and pruinose and with age becomes darker and cushion-shaped; the ascospores are hyaline. In our continuing work with the viride clade we have found that especially the young stroma of most members of the clade is distinctive of a number of often sympatric species that are best distinguished by their Trichoderma anamorphs (Samuels et al. 2006a). We have found indistinguishable teleomorphs for both T. viride groups, Vb and Vd. This calls for a redefinition and redescription of H. rufa. In the present work we refine the description of H. rufa and provide an epitype for the species, we describe as new a teleomorph for T. viride group Vd, redescribe Hypocrea vinosa with its new anamorph T. vinosum, and describe the new species T. gamsii, T. neokoningii and T scalesiae.

	MATERIALS AND METHODS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 
Isolates including NCBI GenBank accession numbers of gene sequences investigated in this study are listed in Table 1. The locations in European countries are indicated with coordinates and map sheets (MTB = Messtischblatt).

Collections and analysis of phenotype
The isolates originated from three natural sources: isolations from ascospores of Hypocrea specimens, direct isolations by a variety of means from soil or dead herbaceous tissue, and isolations as endophytes from sapwood of living stems of Theobroma and related tree species, and from Fagus sylvatica. Isolation of the stem endophytes was done as reported by Evans et al. (2003). A smaller number of isolates was obtained from the American Type Culture Collection (ATCC), Biologische Bundesanstalt (Berlin), the Centraalbureau voor Schimmelcultures (CBS), and from individual colleagues. Cultures derived from single part-ascospores that were germinated on cornmeal agar with 2 % dextrose (CMD, Difco cornmeal agar + 2 % dextrose w/v) and isolated by means of a micromanipulator; usually two or more single-spore cultures were combined in a single stock culture, and such polyspore cultures were used in all subsequent analyses. The working set of cultures is maintained on cornmeal agar slants at ca. 8 °C, in 20 % glycerine at -80 °C, or in liquid nitrogen.

Representative isolates are deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS) and the American Type Culture Collection, Manassas, VA (ATCC). Isolates listed as C.P.K. are those maintained in the collection of Christian P. Kubicek, Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Vienna. Kornerup & Wanscher (1978) was used as the colour standard. The name of the most commonly cited collectors, G.J. Samuels and W.M. Jaklitsch, are abbreviated as G.J.S. and W.J.

Cultures used for study of anamorph micromorphology were grown on CMD, PDA or SNA (Nirenberg 1976), at 20 or 25 °C for 5–11 d under alternating 12 h cool white fluorescent light and 12 h darkness; in the descriptions that follow, these alternating light conditions are referred to when the word "light" is used.

Morphological analyses of microscopic characters were undertaken from material that was first hydrated in the case of herbarium material, or wetted in the case of living cultures, in 3 % KOH. Autolytic activity, which is here defined as usually circular excretions at the tips of hyphae, was assessed under direct microscopic observation using a 10 x objective. Coilings, defined as circularly oriented and coiled intercalary or terminal parts of hyphae, were detected in the same way as autolytic excretions.

Measurements were made from KOH or water mounts and we did not observe any differences when the respective reagents were used. Where possible, at least 30 units of each parameter were measured for each collection. Ninety-five percent confidence intervals of the means (CI) are provided; this figure represents the interval within which 95 % of the individuals of the parameter was found in the analysed isolates. The parameters used for analysis are listed in Table 2. Chlamydospores were measured by inverting a 7–15 d old CMD culture on the stage of a compound microscope and observing with a 40 x objective. Data were gathered using a Nikon DXM1200 or a Nikon Coolpix 4500 digital camera and Nikon ACT 1 software and measured either directly on the microscope or by using Scion Image (release Beta 4.0.2; Scioncorp, Frederick, MD).

Five types of light microscopy were used, viz. stereo microscopy (stereo), bright field (BF), phase contrast (PC), Nomarski differential interference contrast (DIC), and epifluorescence (FL). The fluorescent brightener calcofluor (Sigma Fluorescent Brightener 28 C.I. 40622 Calcofluor white M2R in 2 molar phosphate buffer at pH 8.0) was used for FL.

Scanning electron microscopy (SEM) was done by one of two methods. Material for SEM studies was obtained from cultures that were grown on PDA for up to 2 weeks at 20–25 °C. Agar blocks with abundant conidia were prepared for SEM. For Figs 8 a–h all SEM procedures followed the protocols of Meyer & Plaskowitz (1989), and for Fig. 10h those of Carta et al. (2003) and Erbe et al. (2003).

View larger version (141K): [in this window] [in a new window]   Fig. 8. Scanning electron micrographs of conidia of T. viride and T. viridescens. a–e. T. viride. f–h. T. viridescens. a–b. from type specimen (L); c–d: G.J.S. 92-15; e: G.J.S. 90-95; f: G.J.S. 92-11; g: G.J.S. 94-11; h: G.J.S. 89-142. Scale bars = 5 µm except b = 10 µm

View larger version (136K): [in this window] [in a new window]   Fig. 10. Hypocrea vinosa/T. vinosum anamorph on CMD. a–g. Conidiophores; note vesiculose development suggestive of Eidamia viridescens in a, d, f–g; phialides proliferating percurrently to form submoniliform chains in d–f. More or less typical Trichoderma conidiophores in b. h–j. Conidia. h. SEM, i. in optical section, j. in surface view. a–b, h from G.J.S. 99-158; c, e, i–j from G.J.S. 99-183; d, f from G.J.S. 99-156; g from G.J.S. 02-54. Microscopy: a, i–j. DIC; b–c, e phase contrast; d, f–g fluorescence. Scale bars: a–g = 20 µm; h = 6 µm; i–j = 10 µm.

Growth rate trials were performed in darkness on potato-dextrose agar (PDA, Difco, Biolab, or Merck) and SNA following the procedure described by Samuels et al. (2002) with the addition that cultures were also grown at 25 °C under 12 h darkness/12 h cool white fluorescent light for 5–7 d. Each growth-rate trial was repeated three times and the results of the three were averaged. The slope of the growth curve was determined using the mean of the colony radius (see Samuels et al., 2006a).

DNA extraction and sequencing methods
The extraction of genomic DNA was performed as reported previously (Dodd et al. 2002). A portion of translation elongation factor 1 alpha (tef1) was amplified using the primers EF1-728F (Carbone & Kohn 1999) and TEF1 rev (Samuels et al. 2002) or TEF1LLErev (Jaklitsch et al. 2005). The PCR product of approximately 600 bp covers the large 4^th and the short 5^th introns of the gene. A fragment covering the internal transcribed spacers 1 and 2 (ITS1 and 2) of the rRNA gene cluster was amplified using ITS1 and ITS4 as the forward and reverse primers, respectively (White et al. 1990). DNA sequences were obtained using the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California). Products were analysed directly on a 3100 DNA sequencer (Applied Biosystems). Both strands were sequenced for each locus.

Molecular phylogenetic analysis
Sequences were edited and assembled using Sequencher 4.1 (Gene Codes, Wisconsin). Clustal X 1.81 (Thompson et al. 1997) was used to align the sequences; the alignment of each locus was manually edited using MacClade or GeneDoc 2.6 (Nicholas & Nicholas 1997). The sequences were deposited in GenBank (Table 1). The MSA file for the tef1 locus is also available at http://www.isth.info/phylogeny/rufa.php.

The interleaved NEXUS file was formatted using PAUP* v. 4.0b10 (Sinauer Associates, Sunderland, MA) and manually formatted for the MrBayes v3.0B4 program. The Bayesian approach to phylogenetic reconstructions (Rannala & Yang 2005) was implemented using MrBayes 3.0B4 (Huelsenbeck & Ronquist 2001). The MODELTEST3-06 package (http://bioag.byu.edu/zoology/crandall_lab/modeltest.htm) was used to compare the likelihood of different nested models of DNA substitution and select the best-fit model for the investigated data set. Both hierarchical LRT and AIC output strategies were considered, although the preference was given to the latter. The unconstrained GTR + I + G substitution model was selected for the tef1 locus.

Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains (with the default heating coefficient = 0.2, heats for cold chains 1 and heated chains 2, 3 and 4 are 1, 0.83, 0.71 and 0.63, respectively) that were simultaneously run for 5 million generations for the tef1 alignment, which comprised 238 sequences. To check for potentially poor mixing of MCMCMC, the analysis was repeated at least three 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 were also checked. Bayesian posterior probabilities (PP) were obtained from the 50 % majority rule consensus of trees sampled every 100 generations after removing the 2000 first trees using the "burn" command. According to the protocol of Leache & Reeder (2002), PP values lower than 0.95 were not considered significant while values below 0.9 are not shown on the resulting phylogram. Model parameter summaries after MCMC run and burning first samples were collected. For tef1 mean substitution values were estimated as GT = 1, CT = 3.55, CG = 1.28, AT = 1, AG = 4.68, AC = 1.5; nucleotide frequencies were estimated as 0.19 (A), 0.27 (C), 0.2 (G), 0.34 (T); alpha parameter of gamma-distribution shape was 0.29. Genetic distance was computed in PAUP* v. 4.0b10 under the GTR + I model.

	RESULTS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 
Phylogeny
The majority of members of Trichoderma section Trichoderma share the same or very similar alleles of internal transcribed spacers 1 and 2 (ITS1 and 2), rendering this locus inappropriate for recognition of some species within the section. Therefore, to infer genetic diversity of the H. rufa/T. viride group we used intron sequences of the translation elongation factor 1-alpha (tef1), the most powerful phylogenetic marker as yet established in the genus. The resulting Bayesian phylogram (Fig. 1), which was obtained from 238 sequences, corresponds well to the previous analysis of related species with T. koningii-like morphology (Samuels et al. 2006a). Considering the analysis of phenotypes, it is obvious that there are two diverged groups named "Large Viride" and "Large Viridescens" clades, both of them with significant statistical support. Isolates of H. rufa form a compact clade composed of mainly European but also North American, Asian and Pacific strains showing its cosmopolitan nature. The "Large Viride" clade includes additional unresolved lineages that apparently represent unnamed species. The description of these taxa requires further sampling and therefore will be discussed in subsequent publications. In this study we have focused on the single endophytic strain from the Galapagos Islands, T. scalesiae sp. nov., which belongs to the "Large Viride" clade but at the same time occupies the most distant position from H. rufa. The largest group on the tree, the "Large Viridescens" clade, splits into two independent evolutionary lineages. The terminal position of the larger one represents a compact and well defined subclade with significant statistical support that contains isolates of the former Vd group (Lieckfeldt et al. 1999), described as H. viridescens below. Similar to H. rufa, this species has mainly European origin, also nearly all primary European nodes include North American, Central American, Asian and Pacific isolates, suggesting the absence of recent allopatric speciation in this group of isolates. Another well-supported clade in the vicinity of H. viridescens is composed of isolates of H. vinosa. As in the "Large Viride" clade this branch contains representatives of several well-supported speciation nodes composed of strains that are closely related to H. viridescens and H. vinosa and undoubtedly represent yet undescribed species of Hypocrea/Trichoderma. This diversity will be discussed in subsequent publications following further investigations and sampling. The material summarised in this study is sufficient to prove the existence of another phylogenetic species with eidamia-like morphology that occupies the second independent lineage within the "Large Viridescens" clade. The new species T. gamsii forms a homogeneous clade mainly represented by isolates from undisturbed soils in Sardinia and Central Russia. As in the case of H. viridescens and H. rufa, T. gamsii did not evolve as a result of any geographic isolation since we also sampled isolates from North America and Australia. We describe the most distant member of the "Large Viridescens" Clade, once again a single strain, as T. neokoningii. The detailed analysis of the highly variable intron sequences of the tef1 gene has clearly shown that, despite their close relationship, H. rufa, H. viridescens, H. vinosa, and a large group of isolates that we describe here as T. gamsii represent distinct sympatric phylogenetic species.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Bayesian radial phylogram showing the structure of the Trichoderma section Trichoderma inferred from sequences of two introns of tef1. Red colour is used to indicate species described in this study. Arrows show branches leading to species recognised within the section. Dark grey filled circles at nodes indicate posterior probability coefficients higher than 0.90 as they were obtained after 5 million generations; black filled circles at nodes show support higher than 0.95. Font colours correspond to regions of sampling on the schematic map. The dotted line around Vd 3 indicates strong phenotypic similarity despite phylogenetic divergence. * - sequences from John Bissett, collection information may be obtained from Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada

Phenotype: anamorph and cultures
DNA sequences referred eighty-seven strains to the "Large Viridescens" clade and thirty-four to the "Large Viride" clade. All of these fungi are typical of Trichoderma in producing copious amounts of green conidia in pustules or in extensive "lawns" on CMD, PDA and SNA. There was a tendency for conidia to form more quickly on SNA than on CMD or PDA and often conidia did not form on either of the latter media while they did form on SNA. Of the three media, SNA is overall better for the study of fungi in the viride clade in terms of more reliable production of conidia. The endophytic T. scalesiae only produced conidia on SNA to which a 1 cm² piece of sterile filter paper had been added; the conidia only formed at the interface of the paper and the agar and on the paper itself.

There was a tendency for yellow pigment to develop in conidia in colonies of the "Large Viridescens" clade grown on PDA and SNA at 25 °C for two weeks, and a yellow pigment often diffused through CMD. No pigment was noted on SNA. Diffusing yellow pigmentation was not noted in colonies belonging to the "Large Viride" clade. A more or less strong coconut odour was detected in PDA and CMD cultures of most members of the "Large Viridescens" clade.

Conidia tended to form in pulvinate to hemispherical pustules < 1–3 mm diam. Distinct pustules measuring 1–5 mm were formed in T. viride/H. rufa on CMD. While pustules formed in T. viridescens/H. viridescens reached 3 mm diam, most often they measured less than 1 mm and often no pustules were formed, the conidiophores arising in more or less continuous cottony lawns. Often conidiophores formed apart from the larger pustules in the aerial hyphae and in minute tufts. Pustules in both groups tended to be cottony, and individual fertile branches could be seen; often conidiophores protruded beyond the surface of a pustule, producing a single phialide or a few fertile branches near the tip, the rest of the conidiophore remaining sterile or nearly so. The pustules of T. viridescens were usually less compact than in T. viride, and transparent under a 10 x objective. In pustules of T. viride produced on CMD, conidia often appeared to form at the surface of the pustule. In all cases, after one week at 20–25 °C, conidia were deep green to dark green 27–28D–F6–8, although lighter green conidia were observed in younger cultures. In some cultures of T. viridescens grown at 25 °C under alternating light on CMD and SNA, conidial masses were yellow. Conidia of T. neokoningii on PDA often were yellow at first. Often conidia of members of the "Large Viridescens" clade became greenish yellow when mounted in 3 % KOH.

Most of the fungi discussed in this work produce colonies that are recognizable as typical of Trichoderma in producing green conidia in abundance on most media. The exception is T. scalesiae, which only produced conidia sparingly on SNA to which a 1 cm² piece of sterile filter paper had been added. Conidiophores in this species were irregularly branched, similar to what was described for T. paucisporum Samuels et al. (Samuels et al. 2006b) and for synanamorphs of pustulate species of Trichoderma (Chaverri et al. 2004). Conidia were held in drops of clear liquid, which appeared yellow to pale green because of the conidia, at the tips of the phialides.

The following results pertain to the remaining species discussed in this work. It is difficult or impossible to define a conidiophore in Trichoderma. Conidiophores are mostly formed in pustules. As was noted above, pustules tend to be composed of intertwined hyphae that terminate in fertile branching systems. For the purposes of the present discussion, the conidiophore is referred to as the terminal branching system of intertwined hyphae that form the pustule. Various types of conidiophores were encountered in this study, and these were largely related to the medium and to the clade. In Type 1 (Fig. 3d, e, i), a well-developed main axis was not readily visible, or it was short and sometimes sinuous. Branching was highly irregular; branches were not paired and phialides tended to arise singly from the main axis. Phialides were often hooked or sinuous (Fig. 3d, e, j, k), cylindrical or somewhat swollen at or below the middle. This type of conidiophore was only found in the "Large Viride" clade, especially in T. viride. The Type 2 conidiophore (e.g. Figs 6, 11, 13, 14) was formed by all clades. In the Type 2 conidiophore there was a more or less readily discernable, well-developed main axis, from which lateral branches arose at or near 90°; the lateral branches were longer with distance from the tip and secondary branches were shorter with distance from the point of departure of the branch from the main axis. Branches often arose in pairs and produced secondary branches in pairs. Phialides tended to terminate branches in cruciate whorls of 3–4. The phialides were straight, cylindrical or somewhat swollen at or below the middle. In Type 3, which was common in the "Large Viridescens" clade, including T. vinosum, T. gamsii and T. neokoningii, the most distinctive characteristic is the production of percurrently proliferating phialides (Figs 5f, g, i, k; 10d–g; 12c, e, f, g; 14h–j), the branching system itself is highly variable in extent and form. At its simplest, a single phialide percurrently produces a second phialide (Fig. 14 h). What appears to be continuing percurrent proliferation of phialides results in a submoniliform chain of five or more cells (Figs 5g, 10 e, 12e, f), each cell of the chain being often abruptly swollen in the middle and separated by the cell above and below by a conspicuous septum. A main axis was discernable or not and was often reduced to a few, short, verticillately disposed branches or a reticulum of branches (e.g. Figs 5i–k; 10d, 12f–h, 14i–j). The most extreme form of the third branching type was observed in old pustules on CMD and PDA, where chains of percurrently proliferating phialides having subglobose bases and extremely long, cylindrical beaks arose from swollen, subglobose cells (Figs 5k, 10f, 12f). Percurrently proliferating phialides having this morphology were also seen occasionally on more typically branched conidiophores (Figs 5d; 12e), on conidiophores that produced typical, non-proliferating, phialides. Proliferating phialides were rarely seen on SNA. Conidiophores of DIS 328g (Vb 1) arose within well-developed pustules; they formed a reticulum with short fertile branches. The branches tended to be sinuous or curved and to be broader than is found in other clades that are studied here. The conidiophores produced often unicellular lateral branches, each of which terminated in 2–4 phialides. The phialides in DIS 328g are shorter than in any strain included in this study and have a smaller L/W ratio; they were often hooked or sinuous.

View larger version (145K): [in this window] [in a new window]   Fig. 3. Hypocrea rufa/Trichoderma viride anamorph. a–b. Conidial pustules formed on CMD. c. Short sinuous elongations arising at the margin of pustules. d–l. Type 1 conidiophores from CMD. (d–e, h = "Type 1" conidiophores). Arrows indicate curved, hooked or sinuous phialides. Note especially solitary phialides in d and e. m–n. Conidia. Surface view of conidia shown in m, optical section shown in n. a–b from CBS 101526. Microscopy: a–b stereo; c bright field; d–h, j–l phase contrast; i, m–n DIC; c, k from CBS 119326; d–e from G.J.S. 89-127; f, m–n from Tr 8. g; l from G.J.S. 04-372; h from G.J.S. 05-463; i from G.J.S. 05-104; j from G.J.S. 99-16 9. Scale bars: a–b = 0.5 mm; d–l = 20 µm; m–n = 10 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 6. Hypocrea/Trichoderma viridescens anamorph. a–h. from SNA. a–b. Pustules. c. A long terminally fertile conidiophore extending beyond the surface of the pustule. d–g. Conidiophores typical of "Type 2," Trichoderma branching and phialides. h. inercalary phialide. i–m. Conidia, showing variation in shape; in i surface view in optical section. i–k from SNA; l–m from CMD. n. Chlamydospores on CMD. Microscopy: a–d stereo. e–g phase contrast. h–m DIC. a–b from G.J.S. 05-115; c–d, g–i, l–m from CBS 433.34 (ex-type of Eidamia viridescens); e–f from G.J.S. 04-232; j from G.J.S. 05-115; k from G.J.S. 99-142. Scale bars: a = 1 mm; b–d = 0.5 mm; e–g, n = 20 µm; h–m = 10 µm.

View larger version (136K): [in this window] [in a new window]   Fig. 11. Hypocrea vinosa/T. vinosum anamorph on SNA. a–b. Pustules. c–i. Conidiophores. All from G.J.S. 02-54. Scale bars: a = 1 mm, b = 0.5 mm; c–i = 20 um.

View larger version (140K): [in this window] [in a new window]   Fig. 13. Trichoderma gamsii on SNA. a–g. "Type 2" conidiophores. Intercalary phialide shown in g (arrow). h. Conidia. I. Chlamydospores. a from C.P.K. 2073; b–d from G.J.S. 05-111; e, g from C.P.K. 2079; h from C.P.K. 2075; f, i from C.P.K. 2091. Microscopy: a–c, e–g = PC, d, h = DIC; i = Bright field. Scale bars: a–g, i = 20 µm, h = 10 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 14. Trichoderma neokoningii. a–b. Colonies on PDA (a) and SNA (b) after 1 wk at 25 °C, alternating light. c–d. Pustules from SNA. e–j. Conidiophores. e. Conidiophore protruding from the surface of a pustule with widely spaced branches and nearly cylindrical phialides. f–g. Conidiophore sparingly branched above and densely branched below. h. Percurrently proliferating phialides (arrow). i–j. Eidamia-like conidiophores with conspicuous proliferating phialides. k. Intercalary phialide. l. Conidia. m. Chlamydospore. e–h, k–m from SNA; i–j from PDA. All from G.J.S. 04-216. Microscopy: c–d = stereo; e, i, k = PC; f–h, j–l = DIC; m = bright-field. Scale bars: a–b in 9-cm-diam Petri dishes, c = 1 mm, d = 0.5 mm, e–j, m = 20 µm; k–l = 10 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 5. Hypocrea/Trichoderma viridescens anamorph from CMD. a. pustule. b–k. Conidiophores; arrows show examples of percurrently proliferated phialides. b–c. "Type 1" conidiophores more or less typical of Trichoderma. d. Conidiophore with "Type 1" branching and typical Trichoderma phialides and one branch with elongated and percurrently proliferated phialides. g. showing long submoniliform chains of proliferated phialides. i–k. showing branching typical of the original concept of Eidamia viridescens; vesicles and proliferated phialides in k very similar to the original illustration of E. viridescens. l. Intercalary phialide shown at i. a from G.J.S. 92-11; b, k from ATCC 32630; c from G.J.S. 99-3; d from G.J.S. 98-129; e–f, h from CBS 333.72; g, j from G.J.S. 05-115; i from G.J.S. 98-192; l from G.J.S. 99-142. Microscopy: a. stereo. b, e–g, i–j phase contrast. d, h, k–l DIC. c fluorescence. Scale bars: a = 0.5 mm; b–g, j, k = 20 µm; h–i, l = 10 µm.

View larger version (155K): [in this window] [in a new window]   Fig. 12. Trichoderma gamsii on CMD. a–h. Conidiophores. More or less typical Trichoderma conidiophores in a, e; examples of percurrently proliferating phialides shown at arrows. Note submoniliform chains of percurrently proliferating phialides in b, f; thin arrow in d pointing to intercalary phialide. i. Conidia. a, d, i from C.P.K. 2077; b from C.P.K. 2078; c, e from G.J.S. 04-09; f from G.J.S. 05-111; g–h from C.P.K. 2073. Microscopy: a–h PC, i DIC. Scale bars a–h = 20 µm, i = 10 µm.

Intercalary phialides were seen in some isolates but were neither common nor restricted to any particular clade (e.g. Figs 3k, 12d, 13g, 14k).

Various conidial types were observed in this study. These, like conidiophore types, were largely typical of clades. Most of the strains produced warted conidia. Conidia of T. gamsii (Fig. 12i), T. neokoningii (Fig. 14l) and T. scalesiae (Fig. 15h) are smooth. Trichoderma viride (Figs 3m, n; 8a–e), DIS 328g (Vb 1), G.J.S. 04-40 (Vb 2), and T. vinosum (Fig. 10h–j) have nearly globose conidia that have a length/width ratio 1.0–1.2. Conidia in G.J.S. 03-151/G.J.S. 02-87 (Vd 1) are ellipsoidal, length/width of 1.2–1.4. Conidia of individual collections of T. viridescens vary from subglobose to ellipsoidal (Figs 6i–m, 8f–h); although the mean L/W of all collections in this clade varies from 1.1–1.3, there is considerable overlap between this clade and DIS 328g. Conidia of T. gamsii and T. neokoningii are unusual in being ellipsoidal. Both of these species produce T. koningii-like conidiophores and conidia. Conidia of T. viride are much more coarsely warted than any of the other clades considered here. Warted conidia are also produced by members of clade Ve. Conidia in this clade are subglobose to ellipsoidal. Ornamented conidia were observed for most members of this clade. Conidial warts, while often large, are widely spaced and thus are not as conspicuous as in members of the "Large Viride" and "Large Viridescens" clades that are the focus of the present work.

View larger version (132K): [in this window] [in a new window]   Fig. 15. Trichoderma scalesiae. a–g. Conidiophores formed in the aerial mycelium at the interface of filter paper and agar. h. Conidia. i. Chlamydospores. All from G.J.S. 03-74. Scale bars: a = 0, 5 mm, b, f–g = 20 µm, c-e, h, i = 10 µm, d = 0.5 mm; e–j, m = 20 µm; k–l = 10 µm.

Optimal temperature for growth on PDA for all clades except T. scalesiae and Vd 1 was 25 °C. The optimum for T. scalesiae was 30 °C and the two isolates in Vd 1 exhibited considerable variation at 30 °C (35–70 mm radius after 72 h). Trichoderma vinosum was unusual in having a temperature optimum of 20–25 °C and in reaching no more than 5 mm colony radius after 4 days at 30 °C.

On SNA most isolates reached a radius of no more than 40 mm, and usually less, after 72 h at 25–30 °C. On SNA only DIS 328g (Vb 1), T. viride and T. viridescens demonstrated a clear optimum at 25 °C. On SNA, the optimum for T. scalesiae was 25–30 °C, as it was for T. vinosum. The two isolates of Vd 1 were too variable to show a temperature optimum on SNA. G.J.S. 04-40 (Vb 2) was the fastest growing strain on SNA, reaching 65 mm after 72 h at 30 °C. This temperature differential was not observed on PDA. At 25 °C on PDA DIS 328g (Vb 1), G.J.S. 04-40 (Vb 2), T. gamsii, Vd 1 and Vd 2 reached or exceeded a radius of 45 mm after 72 h. Despite their phylogenetic complexity, both T. viride and T. viridescens showed very little variation in growth rate among their many isolates, both reaching a radius of 30–40 mm after 72 h at 25 °C. Significantly, growth of isolates in both of these clades, as well as in T. vinosum and DIS 328g (Vb 1), was more than 20 mm slower at 30 °C than at 20 °C. Trichoderma scalesiae was the slowest growing, reaching only 10 mm on SNA after 72 h at 25–30 °C and 18 mm on PDA at 30 °C. The fastest growing isolate at 30 °C was G.J.S. 04-40 (Vb 2) reaching 45 mm, although G.J.S. 03-151 (Vd 1) reached a radius of 70 mm after 72 h at 30 °C.

Clade Vd 3, which is a sister to T. viridescens, comprises two distinct groups of isolates. The North American isolates (G.J.S. 00-67, G.J.S. 97-243) cannot be distinguished from T. viridescens in any of their morphs and aspects. The Taiwanese isolates (G.J.S. 94-9 – G.J.S. 94-11) grow significantly more slowly than T. viridescens.

Phenotype: teleomorph
The stromata of the species included in this study are morphologically and anatomically so similar that they often cannot be distinguished. The youngest stage, when it could be observed, was semieffused, velutinous to conspicuously hairy and light tan in colour (Figs 4a, d; 2a, c). As perithecial development continued, the stroma became pulvinate to tuberculate or turbinate, and assumed a brown to rufous colour. Occasionally "albino" stromata, off-white to pale yellow, were observed in H. rufa (Fig. 2f) and in H. vinosa (Fig. 16i), in the latter only in an immature state. Often a velvety scurf was also present on mature stromata, the result of short hyphal hairs protruding from the stroma surface (Figs 2k, l; 4 l; 9b, e). Ostiolar openings were usually not visible macroscopically, or were barely visible as lighter areas on the stroma surface, sometimes with darker margins. The stroma surface, when observed in the compound microscope, was composed of small pseudoparenchymatous cells. Typically brown pigment was unevenly deposited in the walls of these cells giving a mottled appearance to the rehydrated stroma (Figs 4j, 9a). The stromata typically have a pigmented cortical layer underlain by a region of loosely arranged hyphae. Asci were cylindrical and had a thin ring in the apex; they typically contained 8 uniseriate ascospores. Ascospores were hyaline, spinulose and disarticulated early to form two halves, or part-ascospores. The part-ascospores were dimorphic, the distal part was subglobose to broadly conical and the proximal part was ellipsoidal or oblong to narrowly wedge-shaped. Ascospore sizes were clade-specific. G.J.S. 02-87 (Vd 1), a teleomorphic member of the "Large Viridescens" clade from Sri Lanka, had the smallest ascospores. Ascospores of H. vinosa were longer in the distal part than in all other species and the width of its proximal and distal parts was greater than in all others. Ascospores of H. rufa and H. viridescens are nearly identical in size. Vb 3 includes two Hypocrea collections from, respectively, Virginia and North Carolina. While these two collections are sympatric with, but phylogenetically distinct from H. rufa, we did not observe any aspect of their teleomorph, anamorph or cultural phenotypes that would serve to distinguish them from that species.

View larger version (125K): [in this window] [in a new window]   Fig. 4. Hypocrea/Trichoderma viridescens teleomorph. a–g. Fresh stromata (a, d–e: immature, b–c, f–g: mature). h–i. Dry mature stromata. j. Surface of stroma reconstituted with water, showing ostioles and unevenly distributed pigment. k. Surface of stroma and ostiolar opening in section. l. Surface of stroma in face view, including unicellular hair. m. Subperithecial tissue in section. n. Palisade of cells above point of attachment of stroma in section. o. Asci with ascospores in cotton blue/lactic acid. Sources: a, l, o: WU 24025. b–c: WU 24027. d, f: holotype WU 24029. e: WU 24024. g, j, m–n: WU 24019. h: WU 24018. i: WU 24028. k: G.J.S. 98-182. Scale bars: a = 2.7 mm, b–c, e = 2 mm, d, g = 1 mm, f = 2.5 mm, h = 0.4 mm, i = 0.5 mm, j = 0.2 mm, k = 30 µm, l = 10 µm, m, o = 15 µm, n = 35 µm.

View larger version (126K): [in this window] [in a new window]   Fig. 2. Hypocrea rufa/Trichoderma viride teleomorph. a–c, e–g. Dry stromata (a, c: immature, downy, b, e–g: mature, f: "albino" stroma). d, h. Fresh stromata. i. Stroma reconstituted with water. j. Ostiolar opening in section. k. Section of stroma with perithecia. l. Hairs on surface of mature stroma. m. Surface of stroma in face view. n. Subperithecial tissue in section. o–p. Ascospores, o: in cotton blue/lactic acid. q. Asci with ascospores. Sources: a–b, p: neotype Scleromyceti Sueciae 303, c: WU 24016; d, g, i–o, q: epitype WU 24013, e: BPI 872089, f: WU 24015, h: WU 24011. Scale bars: a, e, g, i = 0.5 mm, b = 0.8 mm, c–d, f = 1 mm, h = 1.5 mm, j, l, q = 20 µm, k = 0.2 mm, m = 25 µm, n = 30 µm, o = 15 µm, p = 10 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 16. Hypocrea/Trichoderma species. a–d. Trichoderma gamsii. a–b. conidial pustules on SNA (G.J.S. 05-111). c–d. Colonies grown 1 wk at 25 °C alternating light (G.J.S. 05-111). c. PDA. d. SNA. e. T. scalesiae (G.J.S. 03-74), 1 wk at 25 °C alternating light. f–i. H. vinosa stromata. f. From the type collection. g. G.J.S. 99-158; h. G.J.S. 02-54, immature with hairs at stroma surface. i. G.J.S. 99-156, immature. Scale bars: a–b = 0.5 mm; f = 1 mm, g–i = 0.5 mm.

View larger version (156K): [in this window] [in a new window]   Fig. 9. Hypocrea vinosa/T. vinosum teleomorph. a. Cells at surface of stroma in face view. b. Short hairs arising from the surface of the stroma. c. Longitudinal section through a stroma. d. Median, longitudinal section through a perithecium. e. Section through the upper part of a stroma showing short hairs arising from the surface, a pigmented cortical area and intertwined hyphae below. f–g. Ostiolar region of a perithecium. h. cells of the interior of a stroma below a perithecium. i–j. Asci and ascospores; a thin ring can b seen in j. All from G.J.S. 02-54. Scale bars: a–b, e–i = 20 µm; c = 200 µm, d = 100 µm, j = 10 µm.

The isolates G.J.S. 04-40 and DIS 328g were isolated as endophytes from trunks of Theobroma cacao and Th. gileri, respectively, and T. scalesiae was isolated as an endophyte from woody, above-ground tissue of Scalesia pedunculata.

Definition of species
Fig. 1, with T. asperellum as outgroup, demonstrates the considerable known and yet to be described taxonomic diversity in a large part of T. sect. Trichoderma. Despite the existence of several clades that no doubt merit taxonomic recognizion, in the current work we emphasize the "Large Viride" and "Large Viridescens" clades.

Each of these large clades includes several well-supported internal clades, making it difficult to delimit species. In most cases, more or less distinct phenotypic apomorphies lead to our decision as to where to draw species boundaries.

The greatest phylogenetic diversity is found in the "Large Viridescens" clade. At the most distant point of this clade, T. gamsii and T. neokoningii can be distinguished because they both have smooth, ellipsoidal conidia. Trichoderma gamsii is a common species in Europe and North America. Trichoderma neokoningii is only known from a single culture that was collected in Peru as a hyperparasite on a destructive pathogen of Theobroma cacao.

Clade Vd 2 includes European and middle-eastern (Iran) isolates that also have smooth, ellipsoidal conidia. Clade Vd 1 includes isolates from Sri Lanka and Ghana that have ellipsoidal, warted conidia. One of these, G.J.S. 02-87 (Sri Lanka), produces a H. rufa-like stroma but it has smaller ascospores than either H. rufa or H. viridescens. We did not observe an eidamia-like morphology in Vd 1 or Vd 2.

Hypocrea vinosa is distinguished from H. viridescens primarily on the basis of its faster rate of growth and on its larger ascospores. It has a conspicuous eidamia-morphology when grown on CMD.

Clade Vd 3 is phenotypically and biogeographically diverse. We had originally included all of these isolates within T. viridescens. As was noted above, the North American isolates (G.J.S. 00-67, G.J.S. 97-243) cannot be distinguished from H. viridescens, whereas the remaining isolates, all from Taiwan, have a noticeably slower rate of growth than T. viridescens. Their relationship to T. viridescens is indicated by the dotted line in Fig. 1.

Hypocrea/Trichoderma viridescens is a widely distributed species that is common in Europe. It is phenotypically, phylogenetically and geographically diverse, but the phenotypic diversity overlapped to such an extent that we were not able to subdivide the species. Hypocrea/T. viridescens is characterised by north- and south-temperate distribution, relatively slow growth, conidiophores that tend to produce paired branches on SNA, subglobose to nearly ellipsoidal, warted conidia, a coconut odour on PDA and CMD, and the conspicuous eidamia-morphology found on PDA and CMD.

The most distant point of the "Large Viride" clade is T. scalesiae. This unusual species was isolated as an endophyte from the trunk of an endemic daisy tree in the Galapagos Islands. It only produced few conidia on conidiophores that are atypical in Trichoderma. Even in the absence of conidial development, it is recognizable as a Trichoderma by its strong odour of coconut and also by the production of abundant chlamydospores that are typical of Trichoderma.

A single clade that is sister to H. rufa/T. viride includes Vb 1, Vb 2 and Vb 3. The two isolates of Vb3 were isolated in the mid Atlantic states of the U.S.A. and they cannot be distinguished from T. viride (with which they are sympatric) morphologically. Apart from the phylogenetic difference indicated by sequences of tef1, we cannot observe any way to taxonomically separate them from H. rufa/T. viride. The single strains that comprise Vb 1 and Vb 2 were isolated as endophytes from trunks of, respectively, Theobroma gileri and Th. cacao in Ecuador and Brazil. Both of them have a faster growth rate than H. rufa/T. viride, a difference that is especially marked on SNA, and Vb 2 grows faster than any of the clades included in the present study. Both of these, but especially Vb 2, have somewhat smaller conidia than T. viride. Conidiophores of Vb 2 are Type 1 described on page 144 and typical of T. viride. The unusual conidiophores of DIS 328g (Vb 1) and the short broad phialides distinguish this clade from its closest relatives, Vb 2, Vb 3 or T. viride. The data suggest that these two endophytic strains represent distinct species; their taxonomy will be discussed in a future publication.

As was the case with H./T. viridescens, H. rufa/T. viride is phylogenetically and phenotypically diverse but we did not find any hiatus in the characters that would enable us to recognise more than a single species. The hallmark of T. viride is its remarkably consistent, rather slow rate of growth, strongly warted, globose to subglobose conidia and this is consistent with the type specimen of T. viride (Fig. 8a, b and Samuels et al. 1999). Moreover, the conidiophores found in T. viride, with often solitary, hooked phialides, are consistent with what Tulasne & Tulasne (1865) illustrated for their concept of H. rufa and T. viride.

What we have called T. viridescens could have perhaps been selected as being typical of T. viride, given the overlap in phenotype characters of the anamorph, but conidia in this group are not so strongly warted and the tendency is for ellipsoidal conidia rather than globose. The ex-type culture of Eidamia viridescens (CBS 433.34) was included in our analysis. Thus we name this clade T. viridescens, with Hypocrea viridescens sp. nov. as its teleomorph.

	KEY TO TAXONOMIC AND PHYLOGENETIC SPECIES OF TRICHODERMA SECT. TRICHODERMA DISCUSSED IN THIS PAPER

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 

• 1. Conidia conspicuously warted, warts usually densely disposed and conspicuous............................................ 2
  
• 1. Conidia smooth or warted, warts scattered or inconspicuous............................................................................ 7
  2. Colony radius on PDA after 72 h at 25 °C 50–60 mm........................................................................................ 3
  
• 2. Colony radius on PDA after 72h at 25 °C < 50 mm............................................................................................ 4
  
• 3. Conidia (2.7–)3.0–3.7(–4.2) x (2.2–)2.5–3.2(–3.5) µm; Sri Lanka, Ghana................................................... Vd 1
  
• 3. Conidia (3.0–)3.5–4.0(–4.2) x (3.0–)3.2–3.7(–4.2) µm; endophytic in Theobroma cacao, Brazil.............................................................................................................................. Vb 2
  
• 4. Conidia (3.0–)3.2–4.0(–4.5) x (2.7–)3.0–3.5(–4.0) µm; conidiophores typically sinuous and frequently branched; phialides short and broad, proliferating phialides and/or submoniliform hyphae not formed; endophytic in trunk of Theobroma gileri, Ecuador................................. Vb 1
  
• 4. Conidia larger, (3.0–)3.5–4.5(–8.5) x (2.2–)3.0–4.0(–4.7) µm; not endophytic.................................................. 5
  
• 5. Conidia (3.0–)3.5–4.5(–5.5) x (2.8–)3.4–4.0(–5.0) µm, typically globose, grossly warted, L/W (0.8–)1.0–1.2(–1.5); terminal conidiophores often curved, phialides often widely spaced and solitary, often hooked or sinuous; proliferating phialides usually not formed in pustules................................................................................................................. T. viride
  
• 5. Conidia (2.7–)3.5–4.5(–8.5) x (2.2–)3.0–4.0(–4.7) µm, globose to ellipsoidal, L/W (0.9–)1.0–1.4(–2.0), verruculose; phialides typically forming in whorls, straight; proliferating phialides and/or submoniliform conidiophores often formed............................................ 6
  
• 6. Conidia subglobose, (3.2–)3.5–4.5(–4.7) x (2.7–)3.0–4.0(–4.2) µm, L/W 1.0–1.2(–1.3); mean of distal part-ascospores 5.0–6.5 x 4.7–6.2 µm; mean of proximal part-ascospores 5–7 x 4–5 µm; colony radius on PDA after 72 h at 25 °C typically 25–33 mm; Australia and New Zealand.............................................................................................................................. T. vinosum
  
• 6. Conidia subglobose to ellipsoidal, (2.8–)3.5–4.5(–8.5) x (2.3–)3.0–3.7(–4.7) µm, L/W (0.9–)1.1–1.4(–2.0); mean of distal part-ascospores 4.2–5.5 x 4.2–4.7 µm; mean of proximal part-ascospores 4.5–5.5 x 3.2–4.0 µm; colony radius on PDA after 72 h at 25 °C typically 35–45 mm; cosmopolitan, more common north-temperate...................... T. viridescens
  
• 7. Conidia globose to ovoidal, smooth, finely warted or with larger scattered warts.............................................. 8
  
• 7. Conidia smooth, subglobose, ellipsoidal to ellipsoidal, smooth.......................................................................... 9
  
• 8. Conidia subglobose or ovoidal, finely spinulose (often appearing smooth in light microscope), (2.8–)3.4–3.6(–7.0) x (2.4–)3–4(–6) µm, L/W 1.0–1.7....... T. asperellum Samuels et al. (Samuels et al. 1999)
  
• 8. Conidia subglobose to ellipsoidal, smooth or with large scattered warts; (3.0–)3.2–4.5 (–5.7) x (2.2–)3.0–3.5(–4.0) µm, L/W = 0.9–1.7 (mean = 1.2)......................................................................... Ve
  
• 9. Conidia globose to subglobose or broadly ovoidal........................................................................................... 10
  
• 9. Conidia ellipsoidal to oblong............................................................................................................................. 13
  
• 10. Conidiophores and conidia typical of Trichoderma, green and typically forming in abundance on SNA, PDA and CMD; colonies fast-growing........................................................................ 11
  
• 10. Conidiophores and conidia verticillium-like, forming in wet heads, sparsely formed and very inconspicuous, reliably forming only on SNA.................................................................................... 12
  
• 11. Colonies with a strong coconut-like odour; conidia subglobose to ovoidal, smooth, (2.7–)3.0–3.8(–5.0) x (2.3–)2.8–3.5(–4.0) µm, L/W = (0.8–)1.0–1.3(–1.6); often with a strong coconut-like odor.................................................................... T. atroviride P. Karst. (Dodd et al. 2002)
  
• 11. Colonies lacking a coconut-like odor; conidia subglobose to ovoidal, finely spinulose (often appearing smooth with light microscope), (2.8–)3.4–3.6(–7.0) x (2.4–)3–4(–6) µm, L/W 1.0–1.7; rarely with a coconut-like odour.............. T. asperellum (Samuels et al. 1999)
  
• 12. Conidia (3.0–)3.5–4.5(–5.0) x 2.5–3.5 µm; colony radius 24–26 mm after 96 h at 25 °C on PDA; growing on Moniliophthora roreri on pods of Theobroma cacao, Ecuador........................................................................... T. paucisporum Samuels et al. (Samuels et al. 2006)
  
• 12. Conidia (2.5–)3.0–3.7(–4.0) x 2.2–)2.7–3.2(–3.5) µm; colony radius < 15 mm after 96 h at 25 °C on PDA; endophyte in stems of Scalesia pedunculata, Galapagos Islands.............................................................................................................................................. T. scalesiae
  
• 13. Conidia (3.2–)3.5–4.0(–4.5) x 2.5–3.0 µm; Peru.......................................................................... T. neokoningii
  
• 13. Conidia larger, (3.2–)3.5–4.5(–4.7) x (2.2–)2.5–3.5(–3.7) µm; Iran or cosmopolitan temperate...................... 14
  
• 14. Conidia (3.5–)3.7–4.5(–5.5) x (2.5–)2.7–3.5(–3.7) µm; L/W (1.0–)1.2–1.5(–1.7); colony radius on SNA after 72 h at 25 °C typically < 35 mm, north- and south-temperate................... T. gamsii
  
• 14. Conidia larger, (3.2–)4.0–5.0(–5.8) x (2.2–)2.5–3.0(–3.2) µm; L/W (1.1–)1.4–1.8(–2.1); colony radius on SNA after 72 h at 25 °C typically ca. 40–45 mm; Iran........................................................ Vd 2
  

	DESCRIPTIONS OF THE SPECIES

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS KEY TO TAXONOMIC AND... DESCRIPTIONS OF THE SPECIES DISCUSSION References

 
Continuous characters not provided in the descriptions are given in Table 2.

1. Hypocrea rufa (Pers.: Fr.) Fr., Summa Veg. Scand., Sectio Post. 383. 1849. Figs 2, 3, 7a–f, 8 a–e.
   
   • Sphaeria rufa Pers., Obs. Mycol. 1: 20 (1796): Fr., Syst. Mycol. 2: 335. 1822.
     
   
   Anamorph: Trichoderma viride Pers., Neues Mag. Bot. [Roemer's] 1: 92. 1794: Fries, Syst. Mycol. 3: 215. 1832.
   
   • = Trichoderma lignorum (Tode) Harz, Bull. Soc. Imp. Natur. Moscou 44: 116. 1871.
     
   • = Trichoderma glaucum E.V. Abbott, Iowa State Coll. J. Sci. 1: 27. 1927.
     
   
   Stromata when fresh (Fig. 2d, h) 1–4(–6) mm long, 0.5–1.5 mm high, solitary to gregarious, or aggregated in small numbers or crowded in lines along wood fibres, at first semi-effused, flat, velutinous, with white mycelial margin; becoming pulvinate, more rarely turbinate or discoid, circular to irregular in outline, surface smooth to slightly uneven to granular, broadly attached, margin often becoming free and concolorous with stroma surface; at first white, remaining white with yellowish ostiolar openings ("albino" form), or more commonly becoming variably coloured from the centre: first yellowish, then pale ochraceous, light brownish or yellow- to orange- to rust-brown (5A4–7, 5B4, 5C6–7, 6CD5–8), later light to dark reddish brown (7–8CD6–8, 8E7–8), sometimes with whitish to rust-coloured scurf; ostioles invisible or appearing as watery, hyaline, or indistinct darker dots, sometimes projecting, convex, often irregularly distributed.
   Stromata when dry (Fig. 2a–c, e–g) (0.5–)0.6–3 (–5.7) mm long, (0.4–)0.6–2(–3.4) mm broad, (0.2–) 0.3–0.6(–0.9) mm thick (n = 31), KOH^–, darker and surface more uneven than in fresh stromata, granular to finely tuberculate, sometimes extremely uneven with perithecial contours visible; ostioles not visible or partly convex or semiglobose, appearing as hyaline or brown dots, generally hyaline after addition of water (Fig. 2i); young stromata velvety to conspicuously hairy (Fig. 2a, c), with diffuse yellowish orange, yellowish brown or (orange-)brown colours, 4B4–5, 5AB5–6, 5CD7–8, 6B5, 6CD5–8, later light-, 7CD5–8, 7E7–8 to deep reddish brown, 8EF5–8 to 7F6–8, 8CD5–6, to dark brown, 7EF5–6; albino form white or becoming pale yellowish, 4A3–4, with numerous, conspicuous light brownish dots (Fig. 2f).
   Stroma anatomy: Cells of stroma surface in face view (Fig. 2m) pseudoparenchymatous, (3.5–)5–9(–14.5) µm (n = 30) diam, walls to 1 µm thick, reddish brown in water, orange-brown in lactic acid, pigment unevenly deposited in cell walls, giving a mottled appearance to the stroma surface. Ostiolar area in dry stromata (32–) 40–84(–126) µm (n = 33) diam. Hairs (Fig. 2l) arising from the stroma surface, yellowish to pale brown, comprising 2–5 cells, apically rounded, rarely branched, sometimes consisting of only one inflated cell, (7.5–)10–29(–62) µm (n = 79) long, (2–)3.5–5(–6.5) µm wide (n = 49), walls 0.5–1 µm thick. Cortical region (12–)17–30(–35) µm (n = 20) thick, cells forming a textura angularis, slightly compressed, reddish brown, in lactic acid orange-brown, (2–)3.5–9(–13.5) µm (n = 60) diam in vertical section, walls up to 1 µm thick. Cells immediately below the cortex comprising a mixture of intertwined hyphae, (2.5–)3–6(–6.5) µm (n = 10) wide, vertical and parallel between perithecia, and few subglobose to angular cells similar to those of the cortex. Perithecia (Fig. 2k) (161–)182–245(–307) x (107–)140–210 (–251) µm (n = 31), flask-shaped, ellipsoidal to globose. Ostiolar canal (70–)75–107(–120) µm (n = 21) long, (32–)33–55(–69) µm (n = 15) wide at the opening (Fig. 2j), cells surrounding the ostiolar opening a palisade of hyaline, narrowly cylindrical, apically slightly expanded cells, plane with the surface or projecting to 14–43 (–69) µm (n = 15). Peridium colourless, consisting of laterally strongly compressed thin hyphae, basally and apically pseudoparenchymatous, indistinct, scarcely differentiated from and merging with the surrounding tissue, apical part flanking the ostioles conspicuously thickened. Tissue below the perithecia (Fig. 2n) of homogeneous, dense textura epidermoidea, of globose to elongate, thin-walled, hyaline cells, (4–)5–19(–26) x (3–)4.5–10(–12.5) µm (n = 30), not layered but cells gradually small