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

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 1α 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.


INTRODUCTION
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 , 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(Bastos , 1996 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 doi:10.3114/sim.2006.56.04 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. 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  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 cushionshaped; 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
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 ºC 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 × 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 × 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).
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).
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 ºC 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 . A portion of translation elongation factor 1 alpha (tef1) was amplified using the primers EF1-728F (Carbone & Kohn 1999) and TEF1 rev  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 λ = 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 G↔T = 1, C↔T = 3.55, C↔G = 1.28, A↔T = 1, A↔G = 4.68, A↔C = 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.

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 1alpha (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 , 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 wellsupported 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.
Most of the Trichoderma species that have warted conidia fall within one of these two large clades. Exceptions include T. saturnisporum and T. ghanense, both of which are members of the distantly related T. sect. Longibrachiatum (Samuels et al. 1998) and clade Ve ( Fig.  1). Clade Ve will be discussed in a future publication. All members of the "Large Viridescens" clade are characterised by the formation of peculiar, percurrently proliferating phialides that are diagnostic of Eidamia viridescens, the ex-type of which (CBS 433.34) falls in T. viridescens.

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 cm 2 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 × 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 cm 2 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 ). 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.
The "Large Viridescens" clade includes CBS 433.34, which is the ex-type culture of Eidamia viridescens A.S. Horne & H.S. Williamson. This species was described based on conidiophores produced on PDA; the original illustration is highly suggestive of what we have seen in the "Large Viridescens" clade, especially the extreme form described above as Type 3 and illustrated in T. viridescens (Fig. 5k) and T. vinosum (Fig. 10f). Conidiophores produced by this culture on SNA ( Fig.  6f, g) were typical of Trichoderma, with a more or less uniformly branched conidiophore and typical phialides. The culture remained sterile on PDA but produced a coconut odour and a diffusing yellow pigment. On CMD mononematous conidiophores bearing green conidia in appressed phialides developed, but no pustules and no proliferating phialides were seen. These conidiophores were suggestive of the synanamorph conidiophores described by Chaverri et al. (2004) for species of Hypocrea/ Trichoderma having conidiophore elongations.
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 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.
Chlamydospores were inconsistently produced in most clades. Chlamydospores formed in abundance in T. gamsii (Fig. 13i) and T. neokoningii (Fig. 14m). Chamydospores were especially abundant in T. scalesiae (Fig. 15i). Chlamydospores were typical of Trichoderma in being globose to subglobose and terminal at the ends of hyphae or intercalary within hyphal cells.
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 ºC and in reaching no more than 5 mm colony radius after 4 days at 30 ºC.
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.

Biogeography
Most of the clades that included more than one strain did not show strong biogeographic bias. Hypocrea vinosa was originally described from New Zealand and, in this work, it is restricted to New Zealand and Australia. The "Large Viride" and "Viridescens" clades are widely distributed but are more common in North America and Europe. These are not tropical fungi. Trichoderma viridescens has been found in Peru at high elevation. We have seen only one isolate of T. viride from a tropical region, i.e. G.J.S. 92-15, from Brazil. However, two members of the "Large Viride" clade, DIS 328g (Ecuador) and G.J.S. 04-40 (Brazil), originated in South America. These two endophytic isolates apparently represent two distinct species. Trichoderma neokoningii was isolated in a tropical region in Peru. On the basis of our collecting, T. viridescens is far more common and possibly more widespread than T. viride. Trichoderma viride and T. viridescens are common in Europe as anamorphs, but uncommon as teleomorphs if compared to common species like H. minutispora. There is a tendency for isolates originating in a geographic area (e.g. Taiwan or Europe) to cluster together but there was an equally strong tendency for clades to comprise strains of mixed origin (e.g. Japan, United Kingdom and U.S.A.). Trichoderma gamsii includes strains from widely separated locations, viz. the Tyrrhenian island of Sardinia (Italy), U.S.A. (Texas), Russia and Australia. The clade Vd 3 comprises two biogeographically distinct sister clades. Isolates G.J.S. 00-67 and G.J.S. 97-243 are from eastern U.S.A. Isolates G.J.S. 94-9 -G.J.S. 94-11 were collected in Taiwan.
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. 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.

Definition of species
Each of these large clades includes several wellsupported 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. rufalike stroma but it has smaller ascospores than either H. rufa or H. viridescens. We did not observe an eidamialike 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 eidamiamorphology 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.
Habitat: Anamorph isolated directly from soil, peat, and wood, found also on leaf litter; isolated as an endophyte from sapwood of Fagus sylvatica in the U.K., isolated from water-damaged building in Denmark, not uncommon. Teleomorph uncommon, inconspicuous, found on wood, less commonly on bark of cut branches, tree tops or 2-120 cm thick logs. In Europe found in open coniferous or mixed deciduous forests, grassland with single trees or at shady road sides, often in piles (to 3 m above the ground) of logs at the edge of forests, stored or lying on bare moist soil, in leaf litter, in grass, the wood in little to medium degree of decomposition and often hard.  Anamorphic cultures studied: see Table 1.
Typification of H. rufa: This species was first described by Persoon in 1796 as Sphaeria rufa. The description, comprising just seven words, was enigmatic: "periphaerica, irregularis, applanata, mollis, rufa intus albida", and no collecting location or specimen was cited. He (Persoon 1801) expanded the description, noting that the species was rare in dense forest on branches of Fagus and that the rufous colour and "scarcely prominent sphaerules [ostiola]" distinguished this species from all others. Fries (1822) included S. rufa in his Systema mycologicum and he later (Fries 1849) placed the species in the new genus Hypocrea. The first mention of a specimen was Fries' (1849) citation of Scleromyceti Sueciae 304. Fries created confusion in this exsiccate when he misnumbered specimens 303 and 304. The specimen list gave number 303 as H. gelatinosa and number 304 as H. rufa but the numbers on the actual specimens were reversed, 303 for H. rufa and 304 for H. gelatinosa (Holm & Nannfeldt 1962;Hennig Knudsen, in litt. 7 Oct. 2004). Tulasne & Tulasne (1865) were the first to establish a link between H. rufa and T. viride and their elegant work established the taxonomic identity of this species. Scleromyceti Sueciae 303 (UPS!) was designated as neotype for S. rufa by Rossman et al. (1999).
Scleromyceti Sueciae 303 (Figs 2 a, b, p) comprises a single piece of decorticated wood glued to a piece of cardboard on which is written only "Fries, Scler. Suec. 303. Sphaeria rufa". Stromata are scattered over the surface of the wood. One is young, semi-effused to pulvinate, light tan, and fibrous. The mature stromata are rufous (ca. 8F8), more or less discoidal, with a deeply wrinkled or convoluted, velutinous surface. Ostiolar openings are not visible. Hyphal hairs arise from the stroma surface. Cells of the stroma surface are angular and brown pigment is deposited unevenly in the cell walls. No asci remain. Discharged part-ascospores are hyaline, spinulose, dimorphic. Subglobose to ovoidal parts, which are assumed to be distal part-ascospores, measure 3.7-4.7(-5.5) × 3.5-4.5(-5.0) μm (n = 30). Ellipsoidal to wedge-shaped parts are assumed to be the proximal part-ascospores and measure (4.0-)4.5-5.0(-5.7) × (3.0-)3.2-3.7(-4.5) μm (n = 30). Although these measurements are somewhat smaller than we have found for about 10 collections, the combined attributes of anamorph and teleomorph that we describe here conform well with Fries's specimen and with the taxonomic identity that was established by Tulasne & Tulasne (1865). We have chosen an epitype for H. rufa, WU 24013, collected in the Czech Republic, that agrees well with the historical concept of H. rufa in teleomorph and anamorph.
Notes: Stromata of H. rufa are usually accompanied by the Trichoderma viride anamorph. Conidia found in nature are dark green, 26F5-8 to 27F4-8, and often citrine-to sulphur-yellow, 4A4-6, hairy patches of mycelium are found. Hypocrea rufa cannot be safely distinguished from the sympatric H. viridescens by the morphology of the teleomorph, and intense yellow cottony patches of the accompanying anamorph are found in both species. The dry stroma colour reported in the description above is difficult to determine due to the small size of stromata, which are often solitary rather than aggregated. Perithecial measurements are only approximated due to indistinct differentiation of the peridium from surrounding tissue. Some pulvinate stromata approach those of H. pachybasioides or H. minutispora in shape and colour when their ostiolar openings are clearly visible, but H. rufa differs from these species in the presence of hairs, especially in young stromata.
While the anamorph, T. viride, is met frequently on wood, but less frequently than T. viridescens, it is difficult to find good teleomorph material. Stromata apparently develop slowly and in a narrow range of ecological conditions, mainly regarding moisture, temperature, and age and degree of decay of substrata. Moreover, they often develop in open habitats that are susceptible to quick desiccation. The frequency of long dry periods has increased in recent years, which may contribute to the fact that teleomorphs are collected rarely. On the other hand, if a habitat is too moist, stromata are soon attacked by hyphomycetes, often seen in specimens as white mould on the stromata. These are obvious reasons why specimens mostly contain immature stromata. Anthropogenic influences, particularly cutting of logs and branches, strongly facilitate development of the teleomorph. Webster (1964) refuted Bisby's (1939) contention that H. rufa could not be distinguished from H. gelatinosa (Tode : Fr.) Fr. by characterising the anamorphs and teleomorphs of the two species. We have examined several of the specimens collected in the U.K. and cited by Webster (SHU!, now in K). On the basis of the stromata alone we could not identify them as H. rufa or H. viridescens. However, conidia found on 2540, 2611, 2618 and 2621 are globose and coarsely warted; most likely they are H. rufa. One of those, 2621, has conidiophores typical of what we have described for T. viride. Jaklitsch & Samuels, Etymology: viridescens, turning green, in reference to the anamorph.
Colonies grown on CMD ( Fig. 7k; 25 °C) filling the Petri plate within 5-6 d, thin, hyaline, regularly circular, hyphae loosely arranged radially, no zonation. Autolytic activity inconspicuous, coilings abundant in some isolates. Aerial hyphae often scarce during fast growth, becoming abundant, particularly towards the margin, broad zone at the margin becoming downy. A diffuse greenish yellow pigment (1B2-6, 2A3-3B4 to 29A2-3) often diffusing through the whole culture. A strong coconut-like odour usually formed. At 15 and 30 °C slower development with less conidiation and less strong coconut-like odour formed, coilings more frequent at 30 °C. Conidia forming within 2-10 d at 25 °C under alternating light, typically effuse, conidial pustules uniformly dispersed throughout the colony, developing in a broad marginal   band, or developing in indistinct concentric rings; after ca. 6-7 d (not always) followed by the development of loose, polymorphic tufts (Fig. 5a) of ca. 0.2-1.5 mm diam on thick, thick-walled and verrucose stipes, confluent to 3 × 2 mm, sometimes in up to three concentric rings or more irregularly disposed, never dense or compact; conidial masses light to dark green from 5 d onwards, 28A4-5 to 27F5-8, development slow, final colour often after 14 d; sometimes conidia yellow at first, then becoming green. Inoculation in the middle of the plate often resulting in more regular distribution of tufts. Conidiophores typically visible at the surface of the pustules. Conidiophores (Fig. 5b-k) dimorphic, representing Types 2 and 3 (described on page 144), the two types typically not forming in the same culture (but see e.g. CBS 333.72 or CBS 119322 with both); conidiophores of both types found on CMD and PDA, Type 2 conidiophores alone found on SNA. Conidiophores of Type 2 (Fig. 6) more or less typical of Trichoderma, conidiophores at the surface of the pustule projecting from the pustule, often with a sterile stipe and a few terminal branches or only few phialides produced at the tip, or conidiophores branched along the length with fertile branches widely spaced; conidiophores forming toward the interior of the pustule tending to have a discernable straight to sinuous main axis with progressively longer fertile branches tending to be paired and arising at right angles to the main axis, branches terminating in 1-3 phialides in a cruciate whorl. Phialides from Type 2 conidiophores lageniform, slightly swollen in the middle when more crowded, straight ( Fig. 6d-g), less frequently sinuous or hooked, phialides sometimes appearing to be proliferated percurrently. Conidiophores of Type 3 (Fig. 5) developing in many PDA and CMD cultures, main axis barely or not discernable, branching at acute angles with respect to the parent hypha, branches rebranching or not, each branch terminating in 1 or 2 phialides; phialides appearing to proliferate percurrently (5f-i, k), often resulting in a submoniliform chain of 2-6 cells ( Fig. 5f, g), the terminal cell of which is a phialide; phialides and subtending cells distinctive in being swollen in the middle and more or less conspicuously constricted above and below the middle, phialides sometimes with a long, cylindrical neck. Conidiophores often arising from variously distorted and swollen cells in pustules (Fig. 5 j, k); submoniliform chains of proliferated phialides are often conspicuous in older pustules. Phialides measured from both types of conidiophores, the longer phialides found on Type 2 conidiophores, (4.8-)7.2-11.5(-20.5) μm long, (1.5-)2.5-3.5(-5.0) μm at the widest point, (1.0-)1.5-2.5(-4.0) μm wide at the base, L/W (1.4-)2.2-4.5(-10.4) (n = 1125); arising from a (1.1-)2.1-3.3(-5.0) μm wide cell (n = 1064). Conidia (Fig. 6h-m, 8f-h) subglobose to ovoidal to ellipsoidal, warted. Intercalary phialides present but not common on all media (Fig. 5i, 6h). Chlamydospores (Fig. 6n) present or absent, terminal or intercalary and subglobose, (2.5-)8-12(-18) μm diam (n = 390).
On SNA (Fig. 7 l; 25 °C) colonies similar to CMD, thin, hyaline, hyphae loosely arranged radially, no zonation, aerial hyphae and coilings often more pronounced, chlamydospores (mostly intercalary and angular to ellipsoidal) more frequent than on CMD, no odour and no pigment formed. Conidiation (Fig. 6a-g) first effuse on long aerial hyphae in proximal and central parts of the colony, spreading across the whole plate, then condensed into small loose tufts or pustules up to ca. 1 mm diam, confluent to 6 × 4 mm at the proximal margin, becoming green from 5 d onwards, later also in a ring at the margin, more effuse, pustules becoming dark green (27F4-8) after ca. 2 wk. At 30 °C autolytic activity conspicuous and submoniliform terminal branches of conidiophores more frequent.
Habitat: Anamorph isolated from bark, decorticated wood, mushroom compost, paper board, apple core, root of Pseudotsuga menziesii infected with Phellinus weirii, leaf litter; isolated as an endophyte from trunks of Fagus sylvatica. Stromata forming on wood, more rarely on bark of Picea abies and Fagus sylvatica, less commonly on Corylus avellana, Salix caprea, or overgrowing fungi such as resupinate polypores, mostly on cut and usually at least partly decorticated branches and logs 2-100 cm thick, stored in piles, lying on bare ground, in grass, in leaf litter, on acidic or calcareous soils, mostly in open forests, at shady roadsides, associated with Ophiostoma spp., Nectria fuckeliana, Diatrype stigma s.l., Chlorociboria (green wood), Capronia cf. pilosella, Hypocrea lixii, Ascocoryne sarcoides, Corticium roseum, Hypoxylon sp.; usually on little to medium-decomposed, often hard wood.
Distribution: Common, especially as anamorph, in north-and south-temperate regions: Europe (Austria, Czech Republic, France, Germany, England, Northern Ireland, Russia, Sweden) and North America (U.S.A.: Oregon, North Carolina, Georgia; Mexico); Australia, New Zealand; found also in Taiwan and Japan.    Stromata when dry (Fig. 15f-i) 1-2 mm diam, 0.5-1.5 mm thick, unchanged or slightly darkened in 3 % KOH; solitary or in groups of 3 or fewer; tuberculate to pulvinate, broadly attached, often erumpent through bark cankers or lenticels; at first almost white to yellowish brown or light brown, becoming darker brown to reddish brown (7-8D-E8); stroma surface scurfy due to short, rust-coloured hairs, plane to wrinkled or tubercular from perithecial contours; ostiolar openings not visible or less frequently appearing as few viscid dots.
Colonies grown on SNA reaching > 7 cm radius within 10 d at 20-25 °C under alternating light and darkness, no diffusing pigment formed, no distinctive odour detected; conidial production variable, abundant to scant, in 1-2 mm diam, hemispherical to pulvinate pustules formed in a broad band around the margin and adjacent to filter paper; conidia dark green; fertile conidiophores protruding terminally from pustules or not.
Conidiophores dimorphic, representing Types 2 and 3 (described on page 144), the two types typically not forming in the same culture; conidiophores of both types found on CMD ( Fig. 10a-g), Type 2 conidiophores alone found on SNA. Conidiophores on SNA (Fig. 11c-i) lacking a well-developed main axis, or main axis short; main axis often nodose, branches tending to be paired and separated by short internodes; branches tending to produce a profusion of phialides directly or at the tip of short secondary branches; branches and phialides tending to arise at right angles with respect to their subtending cells. Protruding conidiophores unbranched or sparingly branched along the length, producing one or few cylindrical phialides at the tip. Phialides narrowly lageniform, more swollen in the middle when crowded than when widely spaced, straight, (6.0-)7.7-11(-14.5) μm long, (2.0-)2.2-3.0(-3.7) μm at the widest point, (1.2-)1.5-2.2(-2.7) μm wide at base, L/W = (0.4-)2.4-4.6(-6.5) (n = 90). Intercalary phialides not common. On CMD Type 3 conidiophores forming in compact pustules, comprising vesiculose cells branching in an irregular fashion, each branch terminating in one or a few phialides; phialides lageniform to greatly swollen, often proliferating percurrently to form a phialide, often submoniliform chains of proliferated phialides ( Fig.   10c-g) found in pustules. Conidia subglobose, warted ( Fig. 10h- Notes: As was noted by Cooke (1879), H. vinosa differs from H. rufa in having slightly larger ascospores. The type specimen of this species is in poor condition. It consists of three pieces of decorticated wood with stromata on one of them. The stromata are dark reddish brown, discrete, discoidal, 1-2 mm diam, broadly attached to the substratum, with a slight tendency to have free margins; the surface is plane, perithecial contours or ostiolar openings are not visible. The stroma becomes light orange-brown in KOH. Ascospores are hyaline, conspicuously spinulose, dimorphic. Distal part-ascospores (n = 10) are globose to subglobose, 5.0-6.7 × 5.0-5.5 μm; proximal part-ascospores are oblong, 5.7-7.2 × 4.5-5.2 μm. Webster (1964) attributed a Trichoderma anamorph to a New Zealand collection (PDD 10466) and Rifai (1969) assigned that anamorph to the T. aureoviride aggregate. According to Webster, the specimen that he examined fits the type of H. vinosa well. However our study of PDD 10466 reveals it to be distinct from the type specimen of H. vinosa. PDD 10466 is a poor specimen, with only one old stroma. That stroma is light brown (ca. 6D8). Ascospores are hyaline and dimorphic. Thirty ascospores in asci were measured; they are smaller than in the type collection. The distal parts are (3.7-)4.0-5.0(-6.0) × (3.2-)3.5-4.2(-5.0) μm; the proximal parts are (3.5-)4.0-5.5(-6.3) × (2.7-)3.0-4.0(-4.5) μm. The specimen includes a dry PDA culture. Conidia are green, ellipsoidal to narrowly ovoidal, (3.0-)3.2-3.5(-3.7) × 2.2-2.7 μm, smooth. Conidiophores in the dry culture have collapsed but we did not observe an eidamia-like development. The specimen PDD 10466 was collected in the Auckland region of the North Island of New Zealand, which is biologically distinct from the type locality of H. vinosa. Thus we are convinced that PDD 10466 is not H. vinosa, although we cannot identify it to any known species.   However, conidia of PDD 10466 are smaller than in these species. As far as we are aware, the culture that Webster obtained from that specimen is no longer available. The illustrations provided by Rifai are consistent with many species of Trichoderma in sect. Trichoderma. Smooth conidia were illustrated.
The specimen that we have designated as epitype also agrees well with the type of H. vinosa. This is perhaps not surprising given the relative non-specificity of Hypocrea specimens. The type of H. vinosa was collected in Waitaki, which is located on the east coast of Otago, on the South Island of New Zealand, where Nothofagus is common. It is at least possible that the type specimen was collected on Nothofagus. The epitype and one additional collection were made from N. menziesii in Westland, a region on the east coast of New Zealand that is slightly north of Otago. Thus, the epitype was collected close to the type locality. Samuels & Druzhinina, sp. nov. MycoBank MB501050. Figs 12-13, 16 a-d.
On CMD at 25 °C in light reaching > 7 cm radius within 10 d; conidia forming in a broad band of discrete to confluent pustules around the colony margin; pustules densely cottony, individual conidiophores not apparent, 0.5-1.0(-3) mm diam, green (27-28E-F5-8) or yellow in part, conidiophores also forming in the scantily produced aerial mycelium; less frequently colonies remaining sterile; sometimes with a pale yellow diffusing pigment; typically with a more or less strong coconut-like odour. Colonies grown on SNA with filter paper at 25 °C in light > 7 cm radius within 10 d; conidia forming as in darkness, conidial production often heavy and in larger pustules over the filter paper, at first yellow (1A7), then green (27C-F8), pustules cottony to dense and individual conidiophores not seen. Conidiophores (Fig. 12a-h) comprising convoluted and intertwined, 2-3 μm wide hyphae from which phialides arise. Phialides produced laterally from hyphae and solitary, or terminating branches and solitary or held in pairs, typically swollen in the middle and the tip elongated and cylindrical; percurrently proliferated phialides conspicuous, often forming submoniliform chains. Chlamydospores typically abundant, subglobose, terminal on hyphae, (3.0-)8.5-12(-15.7) × (3.0-)8-11.5(-15.5) μm.
proliferating phialides in T. gamsii on CMD and PDA. Trichoderma gamsii has the largest conidium length/ width ratio that we have yet seen in Trichoderma. Conidiophores of T. gamsii are neither as extensive nor as uniformly branched as they are in T. koningii. Fisher et al. (1993) recovered T. gamsii (as T. koningii) as an endophyte from xylem of Eucalyptus nitens in Australia in low frequency.
Habitat: Isolated from the pseudostroma of Moniliophthora roreri infecting a pod of Theobroma cacao. Notes. There is little morphological distinction between T. neokoningii and T. koningii and T. koningiopsis. The latter two species (Samuels et al. 2006a) produce conidia abundantly on most media in the aerial mycelium or in poorly developed pustules, not in conspicuous pustules as they occur in T. neokoningii. Conidiophores of the former two species are also more regularly branched and do not produce conspicuously percurrently proliferating phialides. Trichoderma koningii is a species of northtemperate regions whereas T. koningiopsis is commonly found in tropical soils. Trichoderma koningiopsis grows faster than T. neokoningii on PDA. Although T. koningii and T. koningiopsis are members of Trichoderma sect. Trichoderma, they are phylogenetically distinct from T. neokoningii. Trichoderma gamsii is closely related to, and morphologically very similar to T. neokoningii; conidia of the common temperate T. gamsii are significantly longer than those of T. neokoningii. Etymology: With reference to Scalesia pedunculata, the host plant from which T. scalesiae was isolated.
Habitat: Endophytic within woody tissues of Scalesia pedunculata (Asteraceae), the "Daisy Tree" of the Galapagos islands. Notes: Trichoderma scalesiae produced conidia only sparingly and then only on SNA to which sterile filter paper squares had been added. In Trichoderma the coconut-like odour is typical of sect. Trichoderma; thus even in the absence of conidia this species was predicted to be a member of that section. Normal Trichoderma species produce conidia abundantly on common mycological media. In the current work we have found that conidial production on CMD and PDA can be unreliable, whereas the carbohydrate-poor SNA can be relied on to stimulate conidial production. In the case of T. scalesiae presumably the cellulose content of filter paper was an absolute requirement for conidium production. We did not attempt to stimulate conidium production on other media by adding filter paper squares to them. Most likely, if this species had been seen in the past it would have been recorded as being sterile. The recently described T. paucisporum Samuels, Suarez & Solis, also a member of sect. Trichoderma, is similar to T. scalesiae in that it produces conidia sparingly on SNA, although it does produce conidia in very low numbers and sporadically on CMD and SNA. Filter paper was not necessary for conidium production on SNA by T. paucisporum. The conidiophores of T. paucisporum are morphologically very similar to those of T. scalesiae (Samuels et al., 2006). Conidiophores in both species are similar to what were described as synanamorphs of some Trichoderma species by . Trichoderma paucisporum was originally isolated from Ecuador, from cacao pods infected with Moniliophthora roreri and lying on the ground.
Scalesia species, which are endemic to the Galapagos Islands, are considered to be the plant equivalents of Darwin's finches. Scalesia pedunculata, a tree, is the largest of the daisy trees. For information concerning S. pedunculata see references found at http://www.arkive.org/species/GES/plants_and_algae/ Scalesia_penduculata/more_info.html. In addition to T. scalesiae, we have also identified T. harzianum Rifai as an endophyte in trunks of S. pedunculata (Samuels, unpubl.).

DISCUSSION
This is the eighth article in our series dealing with members of the phylogenetically and taxonomically complex Trichoderma sect. Trichoderma , Druzhinina et al. 2004, Holmes et al. 2004, Lu & Samuels 2003, 2006a.
In the introduction to the current work we suggested that reports of T. viride refer to more than one species. We have shown that even the classical morphological concept of T. viride, i.e. a green-conidial species with globose, warted conidia, is paraphyletic. In the present paper we distinguish the two most common species of Trichoderma that have warted conidia and describe a new species that has warted conidia, T. vinosum. We augment the known diversity of morphological expression in Trichoderma to include the peculiar Eidamia morphology. During the course of the study, additional species having warted conidia or that were closely related to T. viride or T. viridescens were revealed to us. In addition we describe a second species, T. scalesiae, that but for DNA sequence analysis and careful examination of cultures on SNA, would be reported as a sterile unknown fungus. Although we present results from sequencing of only a single gene, tef1, the resulting clades conform to phenotypic apomorphies in defining species. Clearly, from Fig.  1 it can be seen that additional species remain to be characterized. These will be discussed in forthcoming publications.
In the present work, as in the accompanying article in this issue that concerns T. koningii (Samuels et al. 2006a), the question that we have had to answer was how to delimit taxonomic species. As we continue to collect new specimens and submit them to DNA sequencing and phylogenetic analysis, we find within clades considerable homoplasy in the few morphological features that are available for analysis. Despite the formation of a teleomorph by several species, this morph is so highly conserved within the viride clade as to be virtually useless in species-level taxonomy while at the same time being diagnostic of the clade. Similarly, while the basic conidiophore morphology in the viride clade, defined here as "Type 2", signals membership in the clade, it varies only little among the species. For this reason, one cannot fault earlier mycologists for having recognized few species of Trichoderma, or even later mycologists for having failed to recognize the differences between T. viride and T. viridescens. In both the T. viride and T. koningii works our strategy for recognition of taxonomic species was to closely integrate phenetic and phylogenetic characters. These two analyses were undertaken independently in two laboratories, and therefore were unbiased. Species were recognized when the concordance between both approaches were found. Although we clearly understand that the phylogeny of one single locus, in this case tef1, is useful to formulate a hypothesis concerning the phylogeny of the fungus, we also understand that use of this single gene region is not powerful enough to falsify a species hypothesis.
Thus, we rely on the concordance between phenotype and genotype. It is also important to note that for some groups the high observed phylogenetic diversity enabled us to recognize a species (e.g. T. gamsii), while in other cases, when the diversity was low and both sets of characters were unclear, we refrained from proposing a new taxonomy (Vd 1-3).
The formation of warted conidia in the viride clade seems to suggest that this character is derived within Trichoderma and that smooth conidia are the primitive state. However, we are not yet in a position to discuss evolution of most phenotypic characters in Trichoderma, and conidium ornamentation is not an exception. Tuberculate conidia are produced by completely unrelated species of Trichoderma, including T. saturnisporum and T. ghanense (both sect. Longibrachiatum, Samuels et al. 1994), andBissett (1991b) illustrated warted conidia in T. virens (sect. Pachybasium) using scanning electron microscopy. On the other hand the eidamia-like morphology of conidiophores produced on rich media is apomorphic for the large viridescens clade defined here, and the coconut-like odour produced by several members of the entire viride clade.
The level of knowledge of Trichoderma species is perhaps greater than for any other genus of fungi. We acknowledge the difficulty of identifying Trichoderma species on the basis of their phenotype, despite the fact that morphology-based keys to species, and illustrations of at least the most common species is available at www.nt.ars-grin.gov. However, every species is represented in GenBank by sequences of two or more genes and by a multiplicity of correctly identified strains (Samuels 2006). Moreover, all known species of Trichoderma may be identified using molecular markers at www.isth.info. The majority of species can be safely identified by the DNA barcoding based on ITS1 and 2 loci (Kopchinskiy et al. 2005). However, since species from Trichoderma section Trichoderma share the same or very similar alleles of ITS1 and 2, they should be identified by an integration of the barcode method (TrichOKEY) and Trichoderma similarity search TrichoBLAST (Kopchinskiy et al. 2005) as described in Druzhinina et al. (2006). If a researcher has access to a sequencing facility, there is no need to misidentify a species of Trichoderma. We strongly recommend that individuals check the identity of strains that are of interest and we urge editors of journals reporting properties of Trichoderma species to require that the identity of the strains be verified by members of the International Subcommittee on Taxonomy of Hypocrea (ISTH) which can be accessed at www.isth.info.