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The Trichoderma koningii aggregate species

The morphological concept of Trichoderma koningii is found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Phylogenetic analysis utilizing partial sequences of the translation-elongation factor 1 alpha (tef1), as well as fragments of actin and calmodulin genes, indicate that phenotypic characters typical of T. koningii evolved independently in three well-separated main lineages. Combined molecular and phenotype data lead to the development of a taxonomy with the recognition of twelve taxonomic species and one variety within the three lineages. These lineages include: (1) T. koningii and T. ovalisporum and the new species T. caribbaeum var. caribbaeum, T. caribbaeum var. aequatoriale, T. dorotheae, T. dingleyae, T. intricatum, T. koningiopsis, T. petersenii and T. taiwanense; (2) the new species T. rogersonii and T. austrokoningii, and (3) the new anamorph T. stilbohypoxyli. Trichoderma koningii s. str. is an uncommon species restricted to Europe and eastern North America; T. caribbaeum var. aequatoriale, T. koningiopsis, and T. ovalisporum were isolated as endophytes of trunks of Theobroma species in tropical America, and T. ovalisporum from the woody liana Banisteropsis caapi in Ecuador; T. koningiopsis is common in tropical America but was isolated also from natural substrata in East Africa, Europe and Canada, and from ascospores in eastern North America, and as an endophyte in Theobroma species; T. stilbohypoxyli, originally described as a parasite of Stilbohypoxylon species in Puerto Rico, is found to be more common in the tropics, besides an endophytic isolate from Fagus in U.K. The additional new species are known almost exclusively from their teleomorphs. Isolates of T. ovalisporum and T. koningiopsis may have biological control potential. A morphophenetic key and a set of tools for molecular species identification were developed.


INTRODUCTION
Trichoderma koningii Oudem. is one of the most commonly cited species of Trichoderma Pers., the anamorph genus of Hypocrea Fr. (Hypocreales, Hypocreaceae). Literally hundreds of publications report the involvement of this species in the biological control of plant diseases caused by other fungi. Among these, T. koningii is reported to produce 6-pentyl alpha pyrone, a spore germination inhibitor (Worasatit et al. 1994). Song et al. (2006) characterized trichokonins, which are peptaibols that have antimicrobial activity, in T. koningii. A search of the literature reveals a role of T. koningii in many activities in addition to biological control of fungus-induced plant disease. For example, culture filtrates of T. koningii and T. harzianum killed 100 % of root-knot nematodes in Sri Lanka (Sankaranarayanan et al. 1997). Trichoderma koningii also benefits plant health and nutrient uptake when it was determined to be highly active in biomineralizing calcium oxalate crystals in soil (Oyarbide et al. 2001), the first reference to indicate this species as a biomineral-producing agent.
What is T. koningii? Despite the fact that the genus Trichoderma Pers. was proposed late in the 18 th century, prior to 1984 only 35 species were included in the genus, and before 1969 very few of these were reported in the literature subsequent to their original description. Trichoderma koningii, described in 1902 (Oudemans & Koning 1902), was included by Rifai (1969) as one of the nine "aggregate" or "morphological" species that he recognized. Bissett (1991a) included it in Trichoderma sect. Trichoderma, which includes the type of the genus, T. viride Pers., on the basis of the morphology of the conidiophore. Lieckfeldt et al. (1998) confirmed membership of T. koningii in sect. Trichoderma using ITS1 and 2 sequences of the rDNA gene cluster, and PCR fingerprinting, a result that has been affirmed in additional publications with other genes (e.g. Kullnig-Gradinger et al. 2002). Lübeck et al. (2004) showed that infra-species variation was greater doi:10.3114/sim.2006.56.03 than inter-species in ITS in the T. koningii aggregate species. Essentially, in that study ITS1 and 2 were not helpful in separating closely related species of sect. Trichoderma, but the authors found that UP-PCR fingerprinting could distinguish T. koningii from T. viride and other members of Trichoderma sect. Trichoderma. The first version of an oligonucleotide barcode based on ITS1 and 2 implemented in TrichOKEY program  is able to identify the T. koningii/ T. ovalisporum/H. muroiana species triplet and attribute it to the "Pachybasium A" clade after Kullnig-Gradinger et al. (2002). Bissett (1991a) divided Trichoderma species among several sections. Among them was sect. Trichoderma, which included T. viride.  proposed a move towards the classification based on phylogenetic clades rather than dividing the genus into sections. They referred to the "Rufa Clade," named for Hypocrea rufa, the type species of the genus, which included members of sections Trichoderma and species from the "Pachybasium A" Clade. The latter group includes T. hamatum (Bonord.) Bainier, the type species of Pachybasium Sacc., and other species. It was refered to as the "Hamatum clade" by Jaklitsch et al. (2006a). In the present work we refer to the combined "Rufa Clade" and the "Pachybasium A" Clade as the "Viride Clade." Trichoderma koningii and the species discussed in the current paper belong to that clade. Lieckfeldt et al. (1998) narrowly defined the morphology of T. koningii and linked it to a teleomorph, Hypocrea koningii Lieckfeldt et al. Lieckfeldt et al. (1998) and Lübeck et al. (2004) demonstrated genetic diversity within the T. koningii aggregate species. Lieckfeldt et al. (1998) noted four additional, morphologically similar and phylogenetically closely related species that they identified as H. cf. muroiana or Hypocrea sp. One of the strains identified by Lieckfeldt et al. (1998) as H. cf. muroiana has since been described as H. stilbohypoxyli B.S. Lu & Samuels (Lu & Samuels 2003). Later, in a revision of T. viride, Lieckfeldt et al. (1999) found nine ITS haplotypes among isolates that conformed to the broadly defined morphospecies T. koningii, of which one was true T. koningii in the narrow sense of Lieckfeldt et al. (1998). Holmes et al. (2004) distinguished T. ovalisporum Samuels & Schroers from T. koningii s. str. and other members of the T. koningii morphological aggregate on the basis of sequences of the protein-encoding gene translation-elongation factor 1-α (tef1) and conidium morphology. In addition to these T. koningii-like species, Holmes et al. (2004) designated four clades of Trichoderma collections that have the T. koningii morphology as "Tkon 20," "Tkon 21," "Tkon 22," and "Tkon 3." Since the study of Lieckfeldt et al. (1999) we have received many additional collections from geographically and biologically diverse sources that can be assigned generally to sect. Trichoderma and specifically to the T. koningii aggregate species. In the present work we examine the phenotypic and phylogenetic diversity found within the T. koningii aggregate species, and develop a taxonomy for those fungi by combining results of morphological, cultural, and molecular-phylogenetic analyses.

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 as isolations as endophytes from living stems of Theobroma and related tree species as reported by Evans et al. (2003). A smaller number of isolates was obtained from the American Type Culture Collection (ATCC), Centraalbureau voor Schimmelcultures (CBS) and 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 using a micromanipulator; usually two or more singlespore cultures were combined in a single stock culture and polyspore cultures were used in all subsequent analyses. Representative cultures are deposited in ATCC and CBS. Kornerup & Wanscher (1978) was used as the colour standard. Isolates and their GenBank numbers are listed in Table 1. The name of the most commonly cited collectors are abbreviated as G.J.S. (G.J. Samuels) and C.T.R. (C.T. Rogerson).
Cultures used for study of anamorph micromorphology were grown on CMD or, less frequently SNA (without filter paper, Nirenberg 1976), at 20 or 25 ºC for 7-10 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. Approximately 20 mL of agar was poured into Petri dishes.
We did not observe any difference in anamorph morphology between CMD and SNA but there was a tendency for more reliable conidial production on SNA than on CMD. Conidial pustules of Trichoderma isolates grown on these two media appeared to be more similar to how they appear in nature than conidia formed on other commonly-used media, including potato-dextrose agar, malt agar and oatmeal agar .
Morphological analysis of microscopic characters was undertaken from material that was first hydrated in the case of herbarium material, or wetted in the case of living cultures, in 3 % KOH. The KOH was subsequently replaced by distilled water. Measurements were made from KOH or water; we did not observe any differences between the two reagents. Where possible, 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 will be found. The parameters used for analysis are listed in Table 3. Chlamydospores were measured by inverting a 7-10 d old CMD culture on the stage of a compound microscope and observing with a 40 × objective. Data were gathered using a Nikon DXM1200 digital camera and Nikon ACT 1 software and measured using Scion Image (release Beta 4.0.2;Scioncorp,Frederick,MD).
Five types of microscopy were used, viz. stereo microscopy (stereo), bright field (BF), phase contrast (PC), Nomarski differential interference contrast (DIC) and epifluorescence (FL). The fluorescent brightener     (Miles, Inc., Elkhart, IN) and sectioned at about 15 µm on a Microtome-Cryostat (International Equipment Co., Needham Heights, MA). The sections were first floated in water and then placed on slides to make semi-permanent preparations following Volkmann-Kohlmeyer & Kohlmeyer (1996). Slides are deposited with the specimens.
Growth rate trials were performed in darkness on potato-dextrose agar (PDA, Difco or Sigma) 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 96-120 h. Each growth-rate trial was repeated three times and the results of the three were averaged.
The slope of the growth curve, which reflects rate of growth per hour, is determined by linear regression. Regression is used to characterize the manner in which the colony radius changes (x's) with the time (y's) when measurements of colony radius are made. By revealing how the mean of the y measures changes as the various x measures change, the regression line is understood to describe the regression of y (colony radius) over x (time of measurement). This regression line is the slope of the growth curve; it is the predicted value of each colony radius for each time of measurement and essentially reports growth per hour (see http://www. animatedsoftware.com/statglos/sgregres.htm).
Principal Components Analysis (PCA), a multivariate analysis (Multivariate Statistical Package, version 1.131; Kovach Computing Services, U.K.), was utilized to determine patterns of variation of phenotype within phylogenetically defined groups. The eigenanalysis is shown in Table 2 and graphical output is shown in Fig. 4. The standardized data used in PCA, and other data analyses, were obtained using Systat version 10 (SPSS Inc., Chicago, IL, U.S.A.).
Dry cultures of Trichoderma species were prepared by placing all or part of a culture growing in 9-cm-diam Petri dish in a cardboard two-slide micro-slide holder (e.g. VWR Scientific, West Chester, PA, U.S.A.) and drying them for ca. 2 h over low heat of a fruit dryer. Dry cultures were prepared so as to preserve essential characters of conidiophore branching and phialides.

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING
The extraction of genomic DNA was performed as reported previously .
The PCR for amplification of the internal transcribed spacers 1 and 2 of the rDNA gene cluster (ITS1 and 2 including the 5.8S RNA gene) was performed in a 50 µL reaction volume using 5 µL of 10 × PCR buffer (Applied Biosystems), 200 µM dNTPs, 25 pmole of each primer (ITS1 and ITS4), 1.25 units AmpliTaq Gold (Applied Biosystems), and about 10-50 ng of template DNA. The reaction mixture was placed in a 0.2 mL PCR tube. The PCR was carried out on a PT-200 PCR system (MJ Research, Waltham, MA, U.S.A.) according to the following protocol: initial activation of AmpliTaq Gold at 95 °C for 10 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min; and a final extension period at 72 °C for 10 min. Five µL of the PCR product was analyzed on 1 % agarose gel in TAE buffer. The positive PCR reactions were purified using the Qiagen QIAquick PCR purification kit (Qiagen, California, U.S.A.) following the manufacturer's instructions. The concentration of the PCR products in ng/µL was determined on 1 % agarose gel electrophoresis in TAE buffer with Lambda Hind III DNA as a marker.
Similarly, a portion of translation elongation factor 1 alpha (tef1) was amplified using the primers EF1-728F (Carbone & Kohn 1999) and TEF1 rev , which resulted in a PCR product of approximately 600 bp, and was sequenced in both directions. The primers for amplification of the calmodulin-encoding gene (cal) were CAL-228F and CAL-737R (Carbone & Kohn 1999). Initially a fragment of actin gene (act) was amplified using the primers Fung.ACT.F1 and Fung.ACT.R1 and the conditions described by Wirsel et al. (2002). Based on the sequences obtained, two Trichoderma-specific act primers were designed Tact1 (5'-TGGCACCACACCTTCTACAATGA) and Tact2 (5'-TCTCCTTCTGCATACGGTCGGA). These two primers were used for amplification of act for all the isolates in this study. Additionally two sequencing primers for act were designed called Tact500F (5'-ATTCCGTGCTCCTGAG) and Tact511R (5'-CTCAGGAGCACGGAAT) and were used for sequencing reactions.
The portion of the RNA polymerase subunit B 2 (rpb2) gene was amplified and sequenced as described by  using fRPB2-5F and fRPB2-7cR (Liu et al. 1999) as forward and reverse primers, respectively. DNA sequences were obtained using the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California). Products were analyzed directly on a 3100 DNA sequencer (Applied Biosystems). Both strands were sequenced for each gene.

ANALYSIS OF SEQUENCE DATA
Sequences were edited and assembled using Sequencher 4.1 (Gene Codes, WI). Clustal X (Thompson et al. 1997) was used to align the sequences; the alignment of each locus was manually edited using MacClade and GeneDoc 2.6 (Nicholas & Nicholas 1997). The sequences were deposited in GenBank (Table 1) and alignments were deposited in TreeBase (http://herbaria.harvard.edu/treebase/), submission number SN 1008). The multiple sequence alignment file for the tef1 locus is also available at http://www.isth. info/phylogeny/koningii.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 bestfit model for the investigated data set. The modelblock3. nex which is compatible with the current version of PAUP* v. 4.0b10 was downloaded from http://workshop. molecularevolution.org/software/modeltest/files/ modelblock3. Both hierarchical LRT and AIC output strategies were considered, although the preference was given to the last one. The unconstrained GTR + I + G substitution model was selected for all tree loci.
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 more than 200 sequences. Alignments of the other two loci (cal and act), neither of which exceeded 100 sequences, were analysed using 3 million generations. To check for potentially poor mixing of MCMCMC, each 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 first 2000 trees for tef1 and the first 500 for cal and act using the "burn" command. According to the protocol of Leache & Reeder (2002), PP values lower then 0.95 were not considered significant, while values below 0.9 are not shown on phylograms and radial trees. 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 = 3.33, C↔G = 1.14, A↔T = 1.32, A↔ G = 5.98, A↔C = 1.43; nucleotide frequencies were estimated as 0.19(A), 0.28(C), 0.17(G), 0.36(T); alpha parameter of gamma distribution shape was 0.23. For cal mean substitution values were estimated as G↔T =1, C↔T = 4.43, C↔G = 0.83, A↔T = 1.15, A↔G = 3.55, A↔C = 1; nucleotide frequencies were estimated as 0.26(A), 0.26(C), 0.24(G), 0.24(T); alpha parameter of gamma distribution shape was 0.1. For act mean substitution values were estimated with a high affinity to pyrimidine transitions (C↔T = 81.9); other transitions were G↔T =1, C↔G = 0.3, A↔T = 0.85, A↔G = 0.83, A↔C = 0.61; nucleotide frequencies were estimated as 0.2(A), 0.3(C), 0.24(G), 0.26(T); alpha parameter of gamma distribution shape was 0.09. The genetic distance was computed in PAUP* v. 4.0b10 under the GTR + I model.

RESULTS
Our work leads us to recognize several species, many undescribed. In the following we have anticipated the formal taxonomy by adopting those names in order to facilitate the presentation of the results.

PHYLOGENETIC ANALYSES OF SEQUENCE DATA
The position of T. koningii-like species on the Hypocrea/Trichoderma genus phylogeny is shown on the radial tree obtained after the analysis of partial rpb2 sequences (Fig. 1). This complex species takes the terminal position on the sect. Trichoderma branch, which consists of "Pachybasium A" and "Viride Clades" ("Rufa Clade" in . It is interesting to note the relatively short genetic distances within clades and species on this branch. The neighbouring H. voglmayrii, which was recently described from the Austrian Alps (Jaklitsch et al. 2006a), or species from "Hypocreanum" and "Lutea Clades" are separated by longer evolutionary distances.
Visual inspection of ITS1 and 2 sequences of strains of the T. koningii complex show a very low degree of variability (max. 6 % of variable sites), corresponding to findings in other studies (Lübeck et al. 2004. Therefore, this locus was not used in phylogenetic reconstructions. However, we were able to develop a species-specific oligonucleotide barcode from ITS sequences for some of these species. It was integrated into the upgraded version of TrichOKEY previously published by . The program allows the identification of four individual species with T. koningii-like morphology and one group of seven species (for details see below).
The high degree of similarity of teleomorphs and anamorphs within the T. koningii species complex led us to anticipate the higher level of sequence similarity of protein-encoding DNA sequences. Therefore we chose phylogenetic markers with relatively big introns such as (i) the partial sequence of the translation elongation factor 1-alpha (tef1) covering the fourth (large) and fifth (short) introns , (ii) the partial actin (act) and (iii) the partial calmodulin (cal) genes with two introns each. Since tef1 is the most variable locus (>50 % of variable sites) it was selected as a reference phylogenetic marker and, consequently, sequenced for all investigated strains. The cal and act genes were used to apply the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept of Taylor et al. (2000) to representatives of the main groups detected by the phylogenetic analysis of tef1.
In order to examine the phylogeny of T. koningii-like strains with respect to their position to the "Viride Clade" we aligned a portion of the tef1 gene for a large number of isolates. First, we attempted to analyse T. koningii-like strains against a background of few representatives of the nearest clades such as T. viride VD and T. viride VB (data not shown). However, the log probability plotted against the number of up to 5 million generations did not reach a stationarity. This indicated a low reliability of  Chaverri & Samuels (2003),  and the present work. Circles at nodes indicate the posterior probability coefficients higher 0.95 as they were obtained after 3 million generations.  Fig. 2 represents a radial Bayesian phylogenetic tree obtained after 5 million chain generations. Analyses of cal and act sequences did not produce problems during repeated MCMC runs; results are shown in Fig. 3.
All three trees show clear separation with high statistical support between fungi of the "Viride Clade"and species from "Pachybasium A" (Kullnig-Gradinger et al. 2002), of which the latter was represented by T. asperellum and T. hamatum. The monophyletic origin of the entire "Viride Clade" was confirmed by phylogenetic analyses of all three loci. As may be seen in the tef1 radial tree (Fig. 2), the majority of strains with a T. koningii morphology appear on a single proliferating branch, which is well-separated from other large species aggregates such as T. viride VB, T. viride VD, T. atroviride, H. stilbohypoxyli and T. erinaceus. The same tree topology is supported by both cal and act trees. This lineage was named "Large Koningii Branch"(LKB) (Figs 2-3).
In the tef1 tree, the most basal position of the LKB is occupied by the highly supported multifurcating clade of T. koningiopsis. Trichoderma koningiopsis presents a genetically variable species because only few internal nodes within that species are well-supported and are of numerous long intraspecific genetic distances. Although strains of T. koningiopsis have the same position on the LKB on the trees of Fig. 3, its identity as a distinct monophyletic clade is not statistically supported in Fig. 2. Bayesian radial phylogram showing the structure of the 'Viride' Clade as it was inferred based on sequences of two introns of tef1. Grey colour is used to separate specimens which are not discussed in this study but whose sequences were used to produce the multiple sequence alignment. Arrows indicate branches leading to species recognized within T. koningii aggregate species. In the highlighted part of the tree, 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. Clades identified as "PS A-F" in the lower half of the tree represent undescribed phylogenetic species (see Table 1).

Fig. 3.
(Page 77). The concordance between two Bayesian phylograms as inferred based on partial act and cal gene sequences. Black circles at nodes indicate the posterior probability coefficients higher than 0.95 as they were obtained after 3 million generations. Grey circles in the cal tree indicate differences in topology when compared to the same isolates in the act tree. Taxon "PS A" indicates an undescribed phylogenetic species (see Table 1). analyses of the cal and act genes. These findings could indicate the presence of a relatively intensive recombination process due to sexual reproduction, despite the fact that the vast majority of the several strains of T. koningiopsis studied were derived directly from substrata with no known teleomorph.
In the LKB of the tef1 tree, T. ovalisporum and T. koningii s. str. possess two equally supported clades. The insignificant support of nodes where both of these species diverge from the main stem suggests approximately simultaneous speciation even for both taxa. However, the divergence was clearly allopatric, because T. koningii is common in North America and Europe, while T. ovalisporum is an endophyte from South America. The phylogenetic position of these two species is not contradicted by cal or act trees, although these genes did not always provide significant support. Compared to other species such as T. koningiopsis, T. koningii s. str. appears to be a relatively homogeneous taxon represented by strains from North America and Europe. Trichoderma koningii strains have almost identical sequences in the hypervariable large intron of tef1. Only one strain (DAOM 167073 from Québec) appeared distinct from numerous other strains, which had very similar or identical sequences, irrespective of their broad geographic distribution.
The upper part of the LKB on all three trees has a stepped structure with well-supported internal nodes (except the cal tree). Based on the concordance between the three loci trees, it consists of at least six phylogenetic species. In general, their phylogeny may be attributed to allopatric speciation because T. taiwanense is Asian, both T. dorotheae and T. dingleyae are isolated, known only from Australia and New Zealand, while strains of T. caribbaeum var. aequatoriale have a South American origin. The terminal position is occupied by T. petersenii. It consists of strictly North American and European clades. Trichoderma intricatum, which is located basal to the species listed above, may be an exception because it is represented by one Asian and one Caribbean strain.
Results of phylogenetic analysis show that additional species having T. koningii-like morphology have evolved independently from the taxa of the LKB. The majority of these species appear on the "Small Koningii Branch" (SKB), which is segregated from the LKB by taxa of the "Viride Clade" (Figs 2-3). The first species on the SKB is T. rogersonii, which is represented by mainly North American and a few European strains. The terminal part of the SKB (tef1 tree) consists of a number of long lineages that lead to geographically separated strains. The divergence among these strains may be explained by allopatric speciation. Three of these six strains originated from Australia and New Zealand, one from Europe, one from the U.S.A., and one from Taiwan. Based on both tef1 and cal loci, Taiwan and North American strains form the most terminal well-supported clade, although on the act tree this clade also includes a European strain. Thus, there is no concordance between topologies of the act tree and trees inferred from sequences of the other two loci. This finding makes it difficult to draw conclusions about phylogenetic species on the terminal part of the SKB.
The third lineage that is characterized by the koningii morphology is represented by the single species T. stilbohypoxyli. Fig. 4. Principal Components Analysis, scatter plot of Eigen vectors. See Table 2 for statistics.

PHENOTYPE: ANAMORPH
For summary of continuous characters see Table 3.
A total of eighty-six strains were studied. Typical of Trichoderma, very little aerial mycelium forms on CMD or SNA, and mycelial production on PDA is typically lush. There is variation among the strains of individual clades as to whether conidiophores form in aerial mycelium or in complex cottony pustules on CMD as well as in relative amounts of conidial production. Conidial production on CMD and SNA tends to occur at the margin of the colony. Discrete conidial pustules sometimes form on CMD and SNA, but on PDA pustules are not formed, rather, conidia form in dense, effused areas. On CMD and SNA pustules are at most 1.5 mm diam and usually smaller, hemispherical, uniformly cottony. Entirely fertile, somewhat plumose conidiophores can often be seen within pustules (e.g. Figs 176,197). Usually projecting sterile hairs or conidiophores that are only fertile at the apex are absent, but occasionally long, apically fertile conidiophores are seen in T. austrokoningii (Figs 55,97), T. dingleyae (Fig. 138), and T. koningiopsis (Fig. 212). Conidiophores also form in the aerial mycelium. One isolate of Trichoderma dorotheae (G.J.S. 99-202) formed hemispherical pustules in addition to conidiophores in the aerial mycelium. Conidiophores in the pustules were not easily discerned (Figs 156,161). In older cultures of this species phialides appeared to proliferate percurrently to form a second phialide (Fig.  158). The newly formed phialides were often abruptly swollen in the middle. This aspect is also seen in Eidamia viridescens A.S. Horne & H.S. Williamson (1923), which is T. viride VD of recent publications (e.g. Lieckfeldt et al. 1999, Dodd et al. 2003, Holmes et al. 2004) and distinct from "true"T. viride (VB in Figs 2-3). Pustules generally are compact, formed of intertwined hyphae that tend to branch dichotomously near the surface and to produce short branches that sometimes act as phialides, or for the cells near the surface of the pustules to swell and produce two or more short cells. In addition, verticillium-like conidiophores arise from near the surface of the pustules. Conidia are dark green (27E-F7-8). There is variation in the time and temperature at which conidia appear. Most of the isolates of T. dingleyae and T. dorotheae lost their ability to produce conidia after storage on cornmeal agar slants at ca. 8 °C.
None of the isolates produces sterile hairs, although, as noted above, occasionally long conidiophores that are fertile only at the tip form in some cultures of some strains, as is described for Trichoderma sect. Pachybasium (Bissett 1991b). Often individual branched conidiophores can be seen within pustules when viewed with the stereo microscope. Conidiophores reaching the surface of pustules in members of all clades form a discernable major axis, from which primary lateral branches arise. Primary branches arise at or near 90° with respect to the main axis often singly but also often they arise in pairs or three at a node, with the members at a single node equal in length and progressively longer with distance from the tip of the main axis. Primary (1°) branches rebranch to form secondary (2°) branches. 2° branches follow the same pattern of branching as the 1° branches with longer side branches closer to the main axis and short branches more distal. Phialides arise singly, directly from the main axis near its tip and the 1° branches; they also terminate 1° and 2° branches in whorls of 3 or 4. 1° and 2° branches, respectively, of conidiophores reaching the surface of the pustule tend to be widely spaced from each other. Branches arising from conidiophores found in the interior of the pustule tend to be crowded, with short internodal distances, and phialides tend to be held in dense heads of several.
Conidiophores of T. taiwanense were unusual in often being conspicuously enlarged and verrucose at the base (Figs 290-293).
Phialides were nearly cylindrical, only slightly swollen in the middle, when formed on widely spaced branches and shorter and conspicuously swollen in the middle when crowded. Often phialides were densely clustered with a very short internode between phialides (e.g. T. koningii Fig. 201, T. koningiopsis Fig. 214), we have termed these dense clusters pseudowhorls. Within any culture there can be considerable variation in the size and shape of phialides but there was no difference among the clades in the degree of variation in any of the continuous attributes of the phialides, the mean variation of phialide length for all collections being 6.1 ± 1.8 µm. The mean continuous measurements for phialides in the 86 strains studied was 7.9 ± 1.0 µm long, 3.0 ± 0.3 µm at the widest point, 2.0 ± 0.2 µm at the base, L/W = 2.7 ± 0.6. The cell from which phialides arise was 2.8 ± 0.4 µm and the ratio of the phialide length to the cell from which it arose was 2.8 ± 0.6. Mean phialide length in most collections ranged ≤7.5-8.5 µm. In two species, T. dingleyae and T. caribbaeum var. aequatoriale, mean phialide length was 9.5-10 µm and in T. intricatum and T. stilbohypoxyli mean phialide length was ca. 7.0 µm.
Conidia of all collections included in this study were oblong to ellipsoidal or ovoidal and smooth; the mean length was 4.0 µm, the mean width was 2.7 µm, and the mean L/W was 1.4. The mean length of conidia in most collections ranged 3.8-4.0 µm; conidia in collections of T. dingleyae and T. koningii were longest, 4.1-4.3 µm, while the mean length of conidia of T. austrokoningii, T. ovalisporum, T. intricatum and T. stilbohypoxyli ranged 3.4-3.5 µm. The mean width of conidia of most collections ranged 2.7-3.0; conidia of T. dingleyae, T. dorotheae, T. intricatum and T. ovalisporum were somewhat wider, the mean ranging 3.0-3.2 µm, while conidia of collections of T. koningii, T. koningiopsis and T. stilbohypoxyli were somewhat narrower, ranging 2.6-2.7 µm. The mean length/width ratio of most collections ranged 1.4-1.5; the mean L/W ratio of conidia in collections of T. koningii was 1.6 and in T. ovalisporum and T. intricatum, which have broadly ellipsoidal to ovoidal conidia, the mean L/W was ca.

1.2.
Chlamydospores are produced sporadically by members of the various species and of the clades, only the two collections of T. intricatum failed to produce chlamydospores. Chlamydospores are typical of Trichoderma, being terminal or intercalary within hyphae, and globose to subglobose.

PHENOTYPE: TELEOMORPH
Most of the strains that we studied were derived from ascospores of Hypocrea specimens. The most notable exception was T. koningiopsis, a common tropical species that was most often encountered as direct isolations from substrata, including as an endophyte from trunks of trees of Theobroma cacao and Th. gileri, and only a few isolates were derived from ascospores. Trichoderma koningii s. str. was most often directly isolated from substrata but three cultures were derived from ascospores of Hypocrea collections made in the United States and one from the Netherlands. Trichoderma ovalisporum is known only from four isolations, all from natural substrata. Trichoderma caribbaeum and its variety aequatoriale (DIS 320c) are represented by three strains; of these, two were isolated from ascospores of Hypocrea specimens collected in, respectively, Guadeloupe (G.J.S. 97-3) and Puerto Rico (G.J.S. 98-43), where they were growing on fructifications of black ascomycetes; the variety (DIS 320c) was isolated from the trunk of a live tree of Theobroma gileri in Ecuador and may be an endophyte. Despite strong phylogenetic similarity between DIS 320c, on one hand, and G.J.S. 97-3/ G.J.S. 98-43 on the other, DIS 320c is phenotypically quite different from the other two and we regard it as a variety of T. caribbaeum.
Stromata (when dry, Figs 24-50) were typically 6C-D8, brownish orange to light brown, but in T. petersenii stromata are darker, 7-8E-F8, reddish brown; stromata were typically pulvinate and broadly attached or at most slightly free at the margins. Perithecial elevations, or mounds, were not visible; the stroma surface was plane or wrinkled. Ostiolar openings were not visible, at least in dry specimens, or were barely visible as viscid circular areolae or dots on the stroma surface. There was no reaction to KOH in any tissue. When young, stromata were semi-effused, light brown or tan and villose; the developing stroma retained the villose aspect, which eventually was lost. The villose aspect is the result of short hairs that arise from the cells of the surface of the stroma; these are 5-10 µm long, 2.5-3.5 µm wide, septate, unbranched, often spinulose. The stroma surface, seen in face view, appeared mottled with unevenly deposited brown pigment in the cell walls. The cells at the surface of the stroma, when seen in face view, were angular, 3-7 µm diam, with walls slightly thickened. The stroma comprised three anatomically distinct regions. The surface region was 15-25(-35) µm thick and pigmented, in section appearing yellow when mounted in lactic acid. Cells of the surface region were angular, 2.5-5 µm diam, with slightly thickened walls. The tissue immediately below the stroma surface consisted of compact to loosely disposed hyphae. The tissue below the perithecia was pseudoparenchymatous, the cells measured 5-10(-15) × 3-7(-10) µm; their walls were slightly thickened or not visibly thickened; cells were oriented perpedicular to the surface of the substratum. The stromata of T. taiwanense (G.J.S. 95-93) are atypical in the group because they are luteous, lack hairs and have conspicuous ostiola; however this specimen is old and possibly has lost the traits that are typical of this group.
Asci were cylindrical, 60-70 × 4-5.7 µm, completely filled with ascospores; there was a thickening at the tip of each ascus. Ascospores were bicellular; they disarticulated at the septum into two part-ascospores early in development.

PHENOTYPE: COLONY MORPHOLOGY AND GROWTH RATE
Colony morphology is described from PDA at 25 and 30 °C in light or darkness after 72-96 h. Colony morphology is more or less consistent within a species. The cultures illustrated in Figs 6-23 are representative of the respective species. There is a tendency for conidia to form in concentric rings that are more or less pronounced; this is especially clear in T. petersenii. With the exception of T. dingleyae and T. dorotheae, conidia tended to form in abundance and to be dark green; conidial production in these two species is poor.
A summary of growth rate curves is shown in Fig.  5. In general, these are rather slow-growing species of Trichoderma.The colony radius is typically less than 50 mm and none reaching a colony radius of 70 mm on PDA, and most less than 40 mm on SNA, when grown for 72 h at optimum temperature of 25-30 °C in darkness (Fig. 5). The temperature optimum for most species is 25-30 °C; the temperature optimum for DIS 203c (T. ovalisporum) and T. dingleyae is lower, 20-25 °C. There is little (radius typically < 5 mm) or no growth at 35 °C for any of the species. On SNA, only T. caribbaeum, T. koningiopsis and T. ovalisporum reach a radius of 40 mm at the optimum temperature; the rest of the species reach a radius of 20-30 mm. On PDA after 72 h darkness, the mean colony radius of most species was < 50 mm; the radius of T. caribbaeum var. caribbaeum and T. ovalisporum was 55-60 mm and the mean radius of T. koningiopsis was 60-65 mm. Most species grow faster at 30 °C than at 20 °C. However, T. dingleyae grew very poorly at 30 °C on both PDA and SNA (radius < 5 mm after 72 h in darkness), whereas at 20 °C colony radius was ca. 20-25 mm, and T. caribbaeum var. aequatoriale grew considerably more slowly at 30 °C (10 mm) than at 20 °C (35 mm).
Green conidia were first observed in PDA cultures of most species within 48-72 h on PDA at 25-30 °C, although individual isolates of a species varied in this regard. In T. caribbaeum var. caribbaeum, T. ovalisporum and T. dorotheae conidia of most isolates were first observed at 20 °C within 48-72 h. All but a few isolates of all species, except T. caribbaeum var.
aequatoriale and T. dingleyae, formed conidia within 96 h. There was a correlation between species and temperature and time of the first appearance of green conidia, with the exception of T. austrokoningii, in which conidia overall appeared after 72 h; but there was considerable variation among the isolates as to the time of first appearance of conidia, which ranged from 48-96 h. In T. dorotheae, T. ovalisporum, and T. petersenii first green conidia were seen beginning after 48 h at 20 °C.
No distinctive odour was detected in any cultures, or rarely a coconut odour in T. koningii.

BIOGEOGRAPHY
With few exceptions, a biogeographic bias was seen in the respective clades (Fig. 2, Table 3). The many reports in the literature of wide distribution not withstanding, distribution of T. koningii is limited to eastern North America and Europe. Trichoderma koningiopsis is a common and cosmopolitan species, but it is more common at tropical than at temperate latitudes.
Most of our isolates originated in the American tropics, but the species occurs in Canada (Ontario) and Germany, and its teleomorph has been found in the U.S.A. (Kentucky). It was also found in the rhizosphere of Coffea arabica from the main coffee-growing area in Ethiopia, where sampling was done from elevations of 1300-2000 m (T. Belayneh, pers. comm.). Trichoderma stilbohypoxyli was also revealed in this work to be a common tropical species, being widespread in tropical America and found in one location in Ghana, but it also occurs in the U.K.
Trichoderma petersenii and T. rogersonii are common and sympatric in eastern North America and central Europe; we have only seen T. rogersonii as isolations from ascospores but we have a single soil isolate of T. petersenii; stromata of T. petersenii have been collected also in Costa Rica. Trichoderma intricatum is known only from two ascospore-derived cultures that originate, respectively, in Puerto Rico and Thailand.
The most problematic clade from the point of biogeography is the clade comprising strains identified here as T. austrokoningii. This clade includes six isolates with unclear phylogenetic position because topologies of corresponding branches are nonconcordant among three loci. Most divergent are two strains, respectively, from Florida and Taiwan. The basal lineage on the tef1 tree comprises two strains (only one shown in Fig. 2) from the South Island of New Zealand. There is a single lineage/isolate from Russia and two closely related collections from tropical Australia. The sequence divergence in this clade suggests that additional sampling would resolve it into two or more species. Although collections in the two Australasian clades are physically relatively close to each other, their actual locations are climatically very different, tropical in the case of the two Australian cultures and south-temperate in the case of the collections from New Zealand.   We cannot say that any geographic region is more diverse than any other as regards the genetic diversity represented in Fig. 2. As was noted above, T. petersenii and T. rogersonii, T. koningii and T. koningiopsis are sympatric in eastern North America. Three species are found in Australia and New Zealand, viz. T. dingleyae, T. dorotheae and T. austrokoningii, although the latter species was found on the northern, tropical Queensland coast of Australia, whereas the other two were collected in south-temperate Nothofagus forests of Australia (Victoria) and New Zealand (S. Island).

SUBSTRATUM
The Hypocrea specimens from which most of the cultures were derived were found either directly on ascomata of, often, members of the Xylariaceae or on indeterminate black fungi on rotting decorticated wood or bark of rotting trees. In only a few cases was a fungal substratum not seen. The isolates taken directly from the substratum were taken from soil, less frequently from fallen leaves and mushroom casing. Several isolates were recovered as endophytes from the sapwood of stems of Theobroma species or, in one case (T. ovalisporum, DIS 70a), a liana. The Trichoderma endophytes of Theobroma gileri were reported by Evans et al. (2003). Five isolates of T. koningiopsis from Ecuador, represented in the cladogram by G.J.S. 01-07, were isolated directly from pods of Theobroma cacao infected with Moniliophthora roreri that had been placed on cacao leaf litter in a search for parasites of the Moniliophthora. Perhaps most interesting of the Trichoderma endophytes of woody plants was T. stilbohypoxyli. We have found (Samuels, unpubl.) that Trichoderma stem endophytes tend strongly to be specific to host genus and to biogeography, but T. stilbohypoxyli was isolated as an endophyte from trunks of ancient Fagus sylvatica in the United Kingdom and Theobroma species in Ecuador and Brazil.

RESULTS OF PRINCIPAL COMPONENTS ANALYSIS (PCA)
PCA was performed to determine the correlation between phenotype traits and clades; the phylogenetic clades were used as the grouping factor. Only characters of the anamorph and of colony morphology and growth rates and geographic distribution were used in the analysis because they were common to all isolates. Characters of the teleomorph were not utilized in PCA because those characters were not common to all strains. Analysis of the teleomorph characters did not resolve groups. In the PCA of the geographic and phenotype characters listed in Table 3, 51 % of the variation is accounted for by the first three axes. While the results of the Eigen analysis (Table 2) do not indicate a strong fit of the data to the model, a scatter plot of the Eigenvalues reveals that isolates of the same clade/species tend to group together (Fig. 4). The two geographically distinct isolates of T. intricatum were separated because of the slow growth of G.J.S. 97-88. Slow growth also pulled DIS 94c from the rest of the isolates of T. koningiopsis, and CBS 979.70 from the rest of T. koningii. The four Puerto Rican gatherings of T. stilbohypoxyli clustered together and distant from the other 8 cultures; this may be because the Puerto Rican strains have, on average, slightly longer and wider conidia than the others. The phylogenetic diversity of T. koningiopsis and, especially, T. austrokoningii is reflected in their wide dispersion on the scatter plot of eigenvalues.

IDENTIFICATION OF SPECIES
Three methods of Hypocrea/Trichoderma species identification based on the analysis of DNA sequences have been developed. Most recently, an automated identification system using oligonucleotide DNA barcodes of ITS1 and 2 sequences was developed. If it is already available for the group under investigation, a barcode is the easiest method to obtain an absolute result. The second possibility is to perform a similarity search (BLAST) against a pool of voucher sequences. This method is very useful because a search can be made using multiple loci; however, results from this technique are unavoidably uncertain, because the user must subjectively weigh every mismatch in the resulting sequence alignment. Moreover, since gene evolution does not always reflect the speciation process, it is highly recommended to obtain a concordant result of several unlinked loci. The third method of molecular species identification is the most reliable one but, at the same time, the most laborious because it implies phylogenetic analyses and the application of the Gene Concordance Phylogenetic Species Recognition (GCPSR) concept of Taylor et al. (2000). A detailed description of the application of each of these methods to the T. koningii aggregate species is given below. Molecular identification is available via a dedicated online "T. koningii morphological species project", which is located at www.isth.info/phylogeny/koningii. Trichoderma and all species from the "Viride Clade" that were known prior to this study. Thus, the barcode distinguished the T. koningii aggregate species as a triplet of T. koningii/T. ovalisporum/H. muroiana. We have investigated the inter-and intraspecific variability of ITS1 and 2 sequences from the complex based on the present larger sample size. Unique speciesspecific oligonucleotide hallmarks for T. petersenii and T. rogersonii and T. koningii s. str. have been discovered. Because all three species are known from many specimens, all of which were considered in the development of the barcode, the resulting identification is reliable ("standard" in TrichOKEY v.1.1). In addition, a characteristic DNA signature that is common to both isolates of T. intricatum is incorporated in the program.
Although, due to the low number of available isolates, the barcode identification is of low reliability and needs to be confirmed by other methods of sequence analysis. Other species from the T. koningii aggregate species such as T. koningiopsis, T. caribbaeum, T. ovalisporum, T. dingleyae, T. dorotheae, T. taiwanense and T. austrokoningii are not distinguishable based on ITS1 and 2 sequences, at least based on the observed diversity. Therefore, they will be identified as "T. koningiopsis or 6 rare species with T. koningii morphology" because T. koningiopsis is the most abundant and cosmopolitan species, known from more isolates than the total number of specimens from other species with ITS1 and 2 haplotype identical to it. Help involving biogeography is provided for distinguishing these species. Thus, the updated version of the ITS1 and 2 barcode (TrichOKEY v. 1.1) is able to distinguish all sympatric species from the complex of the T. koningii aggregate species.

Using tef1 and sequence similarity search program TrichoBLAST
The TrichoBLAST tool for Hypocrea/Trichoderma sequence identification installed on www.isth.info ) determines the sequence from the database to which the query sequence is most similar. The main TrichoBLAST database consists of sequences of five phylogenetic markers (two introns and one exon of tef1, partial exon of rpb2 and ITS1 and 2). With respect to the present group, the first version of this database included only two sequences of each tef1 intron from two strains of T. koningii s. str. and one strain of T. ovalisporum. The remaining biodiversity was not considered. In order to facilitate the identification of species from the T. koningii morphological species, we have extracted both most variable tef1 phylogenetic markers (forth large and fifth short introns) from the type sequences of each new species and inserted them in the main database, which is named "Nucleotide DB of Phylogenetic Markers" (http://isth.info/tools/blast/ blast.php). Because of the high degree of intraspecific variability, we assume some difficulty in species identification based on tef1 introns and similarity search, because the user would need to charge the weight of multiple mismatches on the pairwise alignment. Therefore, in order to minimize the possible dissimilarity between the query and subject sequences in a blast result, we have composed a separate database of both tef1 introns for all available sequences from the T. koningii morphological species. This database is named "koningii tef1" and it is directly available from the T. koningii project page (www.isth.info/phylogeny/koningii) or as a selectable database in the main TrichoBLAST. Thus, the user has a possibility to perform the primary round of species identification using the default, main, database. A positive result ("koningii-positive") leads to the possibility of searching for the most similar haplotype among highly homologous sequences and making allowance in the final identification according to it. The conclusion about species identification may be drawn if the query sequence is significantly more similar (significantly higher bit score) to sequence(s) of one species compared to the similarity to others and if all precautions explained in Kopchinskiy et al. (2005) are taken into account.

Using multilocus phylogenetic analysis
As has been shown in this study, the "Viride Clade" is an extremely species-rich group. Therefore, we anticipate the further discovery of new taxa with the T. koningii morphology. In this case, both methods of molecular species identification would provide uncertain results, e.g. the ITS1 and 2 based barcode will lead to an identification at the level of clade and/ or "T. koningiopsis and six rare species" (see above), while the result of similarity search will show the same relation to the group of species instead of only a single taxon. Such a situation may be resolved only by the use of phylogenetic analysis based on several unlinked phylogenetic markers. In order to facilitate the task, we have included type sequences of newly recognized species in the multiloci database of phylogenetic markers (http://www.isth.info/tools/blast/show_all_seq. php). This database is especially designed to assist in retrieving of type sequences for the subsequent phylogenetic analyses. In addition, as has been mentioned above, the T. koningii project page also contains a table listing all the ITS1 and 2, and both tef1 sequences of species from the T. koningii aggregate species, and the geographic origin of the corresponding strain is given. Thus, the final species identification or the detection of the new species may be done based on the phylogenetic analysis, with phylogenetic markers retrieved from www.isth.info/phylogeny/koningii.

Using phenotypic characters
Geography and reproductive isolation have played a large part in our species concept. PCA revealed phenotype-based groups that combine with GCPSR in species delimitation. While there is significant homoplasy in the phenotypic characters, the species that we recognize in most cases are not sympatric.
Although the species that we recognize in this work are characterized to some extent by phenotypic characters, we recognize that often the characters are at best subtle and difficult to observe. It will be difficult to recognize a species if it is out of the currently known geographic range. Nevertheless we have provided a key for species identification based on phenetic characters, which should resolve doubts that may derive from sequence similarities.

DISCUSSION AND TAXONOMIC CONCLUSIONS
We studied 86 strains of Trichoderma, any of which could have been identified as T. koningii following the schemes of Rifai (1969) or Gams & Bissett (1998).
In the absence of reproductive isolation and clear morphological differentiation for detecting species boundaries, the GCPSR concept (Taylor et al. 2000) remains the only currently applicable choice. It requires the concordant phylogenetic position of a taxon among closely related other taxa based on at least three unlinked loci. The concordant phylogenetic position should also not be contradicted by analyses of other loci. In this study we found few phylogenetic markers that could reliably resolve groups of closely related, apparently recently evolved Hypocrea/ Trichoderma species. The choice of phylogenetic markers for a particular group of fungi is a delicate task.  have listed eleven phylogenetic markers attributed to eight DNA loci used in phylogenetic analyses of Hypocrea/Trichoderma species. The internal transcribed spacers 1 and 2 (ITS1 and 2), which provide considerable diagnostic properties in Hypocrea/Trichoderma , are insufficient for phylogenetic modelling even at the intercladal level. Therefore, for the analysis of T. koningii-like species, we have selected intron-rich fragments of the protein-encoding genes tef1, act, and cal that deliver higher levels of variation. However, sufficiently high resolution was obtained only from both tef1 introns, while the phylogenetic signal of act and cal was moderate. Tree topologies based on act and cal loci are concordant with the selected tree based on tef1, however, overall statistical support of species nodes was relatively low. Therefore, an integrated approach for the identification of Trichoderma species was developed that adopts multivariate analyses of phenotypic characters and patterns of geographical distributions as well as phylogenetic inferences and oligonucleotide barcoding (see also Kraus et al. 2004, Harrington & Rizzo 1999.
The molecular phylogenetic analysis based on three protein-encoding genes revealed several weakly or well supported clades representing taxa that could be characterized and therefore recognized at least partly by phenotypic characters and patterns of geographic distributions. With the help of this integrated approach, a taxonomy was developed that currently accepts twelve species and one variety.
Trichoderma, like many other fungi, suffers from homoplasy of morphological characters, which have been the basis of species descriptions since the genus was described more than 200 years ago. In the absence of characters derived from DNA, all of the isolates that we studied could have been identified as T. koningii in the sense of Rifai (1969). In the present work, geographic distribution and rate of growth in agar culture were among the most significant characters separating species. PCA that included only the few available characters of the anamorph per se (i.e. measurements of conidia and phialides) did not result in a clustering of strains that was consistent with the results of phylogenetic analysis. Certain characters of the anamorph are variable within a phylogenetic species; others suggest homoplasy in different phylogenetic species. There are usually no finite conidiophores; instead, conidiophores are aggregated within more or less well-developed pustules and individual elements cannot be measured and are difficult to characterize. The formation and extent of pustules, or degree of aggregation of "conidiophores", in most species is highly variable and the ability to form pustules may decline with length of time of strain preservation or after successive transfers. Phialides in most groups are longer or shorter, wider or narrower, depending upon whether they are formed near the surface of the pustule, where they are less crowded, or at the interior of the pustule where they are more crowded. However, crowded and less crowded phialides can hardly be analyzed separately from each other. Conidia present the most consistent morphological character because they can be measured and because their morphology remains constant over successive transfers. What compounds the difficulty in finding phenotypic characters is the general lack of pigments in cultures, a character that has been used in taxonomy of Trichoderma sect. Longibrachiatum  or other species-rich genera such as Fusarium.
The teleomorph is not helpful in species recognition and is only of limited use in recognizing phylogenetically distinct clades. In general, individual species within a clade cannot be distinguished on the basis of the morphological characters of the teleomorph. However, clades may or may not have a distinctive teleomorph morphology.  found that among species with green ascospores, the same anatomy of the Hypocrea stroma was found in phylogenetically distant groups, however, all of those teleomorphs were very different from the teleomorphs reported here and from those formed in T. sect. Longibrachiatum . In gross morphology, most of the Hypocrea collections studied here cannot be distinguished from H. rufa (Pers. : Fr.) Fr., the teleomorph of the closely related T. viride, and the type species of Hypocrea. Doi (1972) subdivided Hypocrea of Japan on the basis of ascospore colour and stroma anatomy, but phylogenetic analysis has not upheld those subdivisions.
With the adoption of molecular phylogenetics, it has become obvious that a species delineated by morphological characters is likely to comprise multiple phylogenetic species. If we had limited our study of phenotype characters to those of the conidia, conidiophores and ascospores, we might have concluded (as did Rifai in 1969) that there were potentially more than one "cryptic species" within the morphological species T. koningii. We would not have been able, however, to identify most of them unambiguously. This phenomenon is common in the ascomycetes and led Hawksworth (2001) to revise upward the estimated number of species of fungi. Minute or subtle characters that would have, in an earlier time, been disregarded as insignificant, are now accepted and sought for characterization of clades. For example, the common species T. harzianum was distinguished from the cause of green mould of mushrooms, T. aggressivum Samuels & W. Gams, by the inability of the latter species to grow at 35 °C .
The paucity of phenotypic characters to distinguish clades is not unique to Trichoderma but rather is likely to become increasingly found in the ascomycetes in general as genera are subjected to phylogenetic analyses. In the example of Coccidioides, Fisher et al. (2005) found that salt tolerance was the only phenotypic character to distinguish Coccidioides posadasii from the closely related C. immitis. Botryosphaeria has received much attention recently. Differences in yellow pigment and host distinguished between the phylogenetically closely related Botryosphaeria lutea and B. acaciae (Slippers et al. 2004). The Fusarium solani complex remains an important challenge to taxonomic revision (O'Donnell 2000).
As increasing numbers of members of species-rich genera such as Trichoderma, Fusarium or Botryosphaeria are included in phylogenetic analyses, increasing numbers of morphologically defined species will be found to be paraphyletic or comprise numerous cryptic species. As much as we would wish for a taxonomy that would permit accurate species identification using only the microscope, we must face the reality that there may not be enough characters in morphology and growth to reflect the differences revealed in diverging DNA sequences. Homoplasy of morphological characters may well be the result if genetically distinct lineages occupy the same niche for a long time. This is also confirmed by many cases where data for the overall carbon utilization profiles (Biolog) did not correspond to the phylogenetic analysis, while the analysis of the utilization of certain single carbohydrates did (Kubicek et al. 2003). A similar situation was observed in marine species of the unrelated genus Dendryphiella, collected from different marine sites (Dela Cruz & Druzhinina, unpublished).
One could argue that our species concept in Trichoderma is too narrow, too strongly influenced by phylogeny, but an example from the present study argues against that. Trichoderma rogersonii and T. petersenii are well separated in the phylogenetic analysis (Figs 2-3). They are common and sympatric in the Eastern U.S.A. However, as can be seen from the PCA (Fig. 4), the two species are incompletely separated by a suite of phenotype characters. The characters separating the two species (Table 3) are the L/W and length of conidia and the length of the proximal part-ascospores. In addition, growth rates on SNA are also characteristic of the respective species. While these differences are statistically significant, there will be many cases that cannot be identified on the basis of their phenotype.
In Trichoderma, at least, traditional characters may be too few to provide practical identification of the apparent large number of species. Carbon utilization estimated using a Phenotype MicroArray technique may provide additional characters (Kubicek et al. 2003, Kraus et al. 2004). In Trichoderma we do not yet have the ability to perform in vitro mating experiments, which would help immensely in determining whether strains belong to the same or different biological species.
The species that we have studied in the current work represent a small part of the diversity of the speciesrich "Viride Clade." Work is continuing on taxonomy of this group.
The refined definition of T. koningii given by Lieckfeldt et al. (1998) was reinforced in this study; the species is distinguished by its longer and narrower conidia and slow rate of growth on SNA. Although T. koningii is among the most commonly cited species in the genus, our results suggest that it is an uncommon species of Europe and North America. The far more common species isolated directly from natural substrata and the species often used in biological control applications is the closely related T. koningiopsis.
Trichoderma koningiopsis is essentially a tropical species, known from South America and Africa (Ethiopia), but its Hypocrea teleomorph has been found as far north as New York State. Trichoderma koningiopsis is the most commonly encountered species having a T. koningii-like morphology. This species was reported earlier as "Hypocrea sp. (8)" in part , "T. koningii II" (Dodd et al. 2003) and "T. koningii Tkon 21" (Holmes et al. 2004). Webster (1964) described "Hypocrea sp. 1" from the United Kingdom. The T. koningii-like anamorph described by Webster (1964) strongly resembles T. petersenii in the morphology of conidia, conidiophores and in the formation of concentric rings in agar culture. We have obtained the cultures cited by Prof. Webster from CBS. Based on anamorph morphology as observed in these cultures and sequences of tef1, we can see that Hypocrea sp. 1 was based on a mixture of two species. Webster 2534 = CBS 257.62 = T. harzianum, the anamorph of H. lixii ; the other cultures (Webster 2545= CBS 258.62, 2617= CBS 259.62 and 2644) are all T. minutisporum Bissett, the anamorph of H. minutispora (Lu et al. 2004). Neither of these species is closely related to members of the "Viride Clade" (Samuels 2006). Doi (1974) reported a T. koningii-like anamorph for Japanese collections of H. muroiana Hino & Katumoto. However, the range of conidial types -including those with surface ornamentation -described by him lead us to suspect that more than one species was involved. Moreover, none of the collections cultured by Doi was taken from bamboo, which is the substratum of the type collection of H. muroiana. In our experience, bamboo is an unusual substratum supporting fungi that are not usually found on other substrata, at least not on woody substrata, which was the source of specimens reported by Doi. We have examined the type collection of H. muroiana (YAM) and conclude on the basis of its morphology that it is a member of the "Viride Clade," but there is no material with a living culture available. We are not able to identify to species the T. koningii-like anamorph(s) reported by Doi (1974)  Several isolates of T. koningiopsis, represented by G.J.S. 01-10 and G.J.S. 01-11 but not included in the phylogenetic analysis, were isolated in Ecuador from pods of Theobroma cacao that were infected by the destructive parasite Moniliophthora roreri. These isolates are currently in field trials to protect cacao from the Moniliophthora (C. Suarez, pers. comm.). Trichoderma koningiopsis isolates G.J.S. 04-10 and 04-11 are effective in protecting cotton plants from infection by Thielaviopsis basicola in Texas (C.R. Howell, pers. comm.), and isolate G.J.S. 05-462 (received too late to be included in the present study) is showing potential for control of Fusarium verticillioides in maize (I. Yates, pers. comm.). A single isolate of this species, G.J.S. 97-273 (= BBA 65450), was isolated from soil in Germany. We tested several isolates of T. koningiopsis for their ability to parasitize the cacao pathogen Moniliophthora roreri in vitro (results not shown) following the "preinoculated plate test" described by Evans et al. (2003) and found that several were able to parasitize the mycelium of M. roreri, with the German isolate being especially effective.
Trichoderma koningiopsis is probably cosmopolitan but perhaps more common in tropical regions. In Figs 2-3 the species can be seen to comprise several wellsupported internal branches; however, we were not able to detect any geographic or phenotypic bias to any of the clades. Six strains (designated as "DIS" in Fig.  2) of T. koningiopsis were isolated as endophytes from freshly exposed, living sap-wood of trunks of species of Theobroma in Brazil, Ecuador and Peru. Following the protocol described in Holmes et al. (2004), strains DIS 172ai (from Theobroma grandiflorum) and DIS 229d (from Th. gileri) could be introduced into seedlings of Theobroma cacao and were reisolated from woody tissue but not from the apical meristem. The isolate DIS 339c (from Th. gileri) could be reisolated from all stem sections of Th. cacao seedlings, including the apical meristems, and it could be reisolated from inoculated pods of Th. cacao after 12 weeks, indicating a potential for protecting pods against infection by M. roreri (K. Holmes, pers. comm.). Ecuadorian strains (G.J.S. 01-07-G.J.S. 01-12, Table 1) were isolated from pods of Th. cacao that were naturally infected with the parasite M. roreri, the cause of frosty pod rot, and have been included in a field trial in Ecuador against that pathogen (C. Suarez, pers. comm.).
Trichoderma koningiopsis occupies the most basal position of the Large Koningii Branch (LKB) in the tef1 tree (Fig. 2), although the statistical support of this species on both act and cal trees is particularly low. This finding in combination with confirmed wide distribution of the species in tropical countries may indicate a relatively intensive recombination process due to sexual reproduction. However, the majority of T. koningiopsis strains were isolated as anamorphs from natural substrata. Teleomorph specimens are only known from the Caribbean region and from the U.S.A. Alternately, the paraphyly of T. koningiopsis could be explained if the species were relatively old. Partially sympatric old, clonal lineages could occur sympatrically and, over evolutionary time, accumulated mutations in the introns and other parts of the DNA could explain the variation in the species.
Trichoderma petersenii and T. rogersonii are common and sympatric in eastern North America. Trichoderma petersenii was reported earlier as "Hypocrea sp. (8)" in part , "T. koningii Tkon 3" and "Tkon 22" in part (Holmes et al. 2004). Trichoderma rogersonii was reported as "Hypocrea sp. (4) and (5)" ). The similarity between these two species was noted above. The most obvious difference between these very similar but phylogenetically relatively distantly related species is that T. rogersonii grows more slowly on SNA than does T. petersenii; moreover, conidia of T. petersenii are slightly shorter and broader than those of T. rogersonii (95 % CI of L/ W respectively 1.35-1.39, 1.40-1.46). Their Hypocrea morphs are indistinguishable from each other and, at least in gross morphology, they are indistinguishable from H. rufa (anamorph: T. viride). However, H. rufa is an uncommon species, albeit sympatric with the other two, despite the many reports of its occurrence. It differs from T. petersenii and T. rogersonii in having slightly larger ascospores (distal part-ascospores approx. 4.5-5 × 4-4.5 μm; proximal part-ascospores approx. 5-6 × 3-4 μm). Trichoderma viride is readily distinguished from T. petersenii and T. rogersonii by its subglobose, warted conidia.
Trichoderma koningii is also sympatric with T. petersenii and T. rogersonii. Because these highly similar species are common, despite their phylogenetic distance from each other, it is important that they may be reliably distinguished by the ITS1 and 2 oligonucleotide barcodes.
Trichoderma ovalisporum is distinguished by its subglobose to more ovoidal conidia (Figs 235-236). It was found as an endophyte of Theobroma species and was also isolated from a woody stem of the liana Banisteropsis caapi that was infected by Moniliophthora (Crinipellis) perniciosa, the cause of Witches' Broom disease of cacao in tropical America (Holmes et al. 2004). The fifth isolate was isolated from soil in Panama, where cacao is grown; it was not included in the phylogenetic analysis. The liana isolate (DIS 70a) reinfected and was reisolated from meristematic tissue of Th. cacao, and inhibited radial growth of the frosty pod rot pathogen (Moniliophthora roreri) in vitro. It also persisted on the surface, and within tissues, of cocoa pods in the field for at least 10 weeks. Initial field trials in Costa Rica, where conidia were applied as a spray, indicated an ability to protect pods against infection by M. roreri (Holmes et al. 2004).
With the exception of the one isolate of T. ovalisporum (DIS 70a), all of the DIS isolates studied for this work (Table 1) were isolated as endophytes from woody stems of South American Th. cacao, Th. gileri and other Theobroma species. These and other Trichoderma isolates were reported previously by Evans et al. (2003) and Holmes et al. (2004) to be endophytes of Th. gileri. In the current work we identify several additional endophytes as members of the "Viride Clade" of Trichoderma sect. Trichoderma. Trichoderma koningiopsis was especially well represented in the endophyte isolations but they did not fall into an endophyte-specific lineage in this species. In contrast to T. ovalisporum and T. caribbaeum var. aequatoriale, which are known only as endophytes of cacao and cacao relatives but which are not common even in that niche, it is not surprising that a species that is as common as T. koningiopsis should be found as an endophyte of a common tropical tree. Judging by the large number of isolates of T. koningiopsis that we received from many sources, it would be surprising not to find it as an endophyte of stems of other tropical trees. Equally, it would not be surprising to find additional isolates that have a biological control potential for fungus-induced plant diseases. Interesting is that Arnold & Herre (2003) did not report Trichoderma species as leaf endophytes in Panama. Outside of the T. koningii aggregate species, T. erinaceus (DIS 7, DIS 8 in Fig. 2) was extended to Peru. This species was previously known only from Southeast Asia (Thailand, Cambodia, Malaysia). Trichoderma stilbohypoxyli was originally described as a parasite of the xylariaceous fungus Stilbohypoxylon muelleri in Puerto Rico in 1996. Since then several isolates considerably expand the biological and geographic distribution of this species by discovery of its teleomorph in Costa Rica and Ghana on bark and perithecia of Neonectria jungneri, and endophyte isolations from woody tissue of Theobroma species in Ecuador and Brazil and from Fagus sylvatica in the United Kingdom. The diffusing yellow pigment, especially seen in colony reverse on PDA, and fast growth rate characterize this species.
Trichoderma caribbaeum var. caribbaeum is represented by two collections (G.J.S. 97-3, G.J.S. 98-43); both are derived from ascospores of Hypocrea specimens collected in, respectively, Guadeloupe and Puerto Rico. As can be seen from the PCA (Fig.  4), these two strains are closely similar in phenotype and also genotype (Fig. 2). The isolate DIS 320c, T. caribbaeum var. aequatoriale, forms a highly supported clade with the other two isolates but is phenotypically and apparently biologically distinct. It was isolated as an endophyte from stems of Th. gileri in Ecuador. The considerable differences in phenotype, biogeography and habit, despite its phylogenetic proximity to the ascospore isolates, lead us to recognize the endophyte as a variety, var. aequatoriale. The apparent close relationship between the two varieties is possibly an artifact of sampling; additional sampling could support their separation at the species rank.
At least three species occur in New Zealand and Australia, viz. T. dorotheae, T. dingleyae and T. austrokoningii. Trichoderma dingleyae is the slowestgrowing species in the present study; its temperature optimum is 20-25 °C and the colony radius is < 5 mm after 72 h at 30 °C. The first two species were collected in Nothofagus forests whereas the third, T. austrokoningii, was found in the tropical Queensland coast and in Nothofagus forests of New Zealand. Hypocrea vinosa Cooke was described from New Zealand (Cooke 1879) and is reported often in the literature, or on the World Wide Web, from diverse geographic regions (e.g. Brazil, Bresadola 1896; Japan, Komatsu & Hashioka 1966;New Guinea, Doi 1971). Ascospores in the type specimen of H. vinosa (K!) are unusually large (distal part-ascospores 5.1-6.7 × 5.0-5.5 µm; proximal partascospores 5.7-7.2 × 4.6-5.3 µm), suggesting that most or all of the reports of this species outside of New Zealand are based on misidentifications. CBS 247.63, H. austrokoningii, is derived from a Hypocrea specimen received from New Zealand (J.M. Dingley No. 3, Auckland, Te Aroha). However, we cannot locate that specimen in CBS or PDD to confirm its identity. We have collected specimens in New Zealand that conform to the type collection of H. vinosa and redescribe the species in another publication (Jaklitsch et al. 2006b).
Unlike most clades, the one that includes T. austrokoningii is geographically diverse, including lineages (Figs 2, 3) from tropical Australia (Queensland, Figs 51-59), temperate New Zealand (Figs 80-88), Russia (Figs 71-79), and a single lineage that includes one collection from the United States (Florida) and one from Taiwan (Figs 89-101). Subtle phenotypic differences characterize each clade (e.g. growth rates, Fig. 102, and ascospore measurements). The phylogenetic and phenotypic diversity of the isolates in this "austrokoningii" clade, which occupies the terminal position of the SKB clade (Fig. 2), suggests that more than one taxon could be involved and that additional sampling would resolve this clade.
Some of the species are represented by one or two strains. We would not normally describe a species based on such a small amount of material because there is no way to estimate intraspecific variability. Nonetheless, T. taiwanense, based on a single collection from Taiwan (G.J.S. 95-93), and T. intricatum, based on two collections (G.J.S. 97-88 from Thailand and G.J.S. 96-13 from Puerto Rico), are phylogentically distinct from all other species that we have included. The two strains of T. intricatum are phenotypically distinct as shown by PCA (Fig. 4). Trichoderma intricatum was formerly reported as "H. cf. muroiana/Hypocrea sp. (6)" in Lieckfeldt et al. (1998).

Characteristics in culture:
Optimum temperature for growth on PDA and SNA 25-30 °C. Colonies grown on PDA in intermittent light forming conidia within 48 h at 25 °C; after 96 h in light, conidial production in 2 concentric rings. Conidia on PDA and CMD 26E-F8 (deep green to dark green). No pigment diffusing through the agar; no distinctive odour. Colonies grown on CMD at 20-25 °C under light filling the Petri plate within 1 wk, conidia abundant, continuously dispersed around the colony margin and also forming in few 1-2 mm diam cottony pustules; individual conidiophores visible within the pustules, completely fertile to the tip. Conidiophores more ore less symmetrical, comprising a recognizable main axis, 2-3 µm wide, fertile branches arising along the length of the main axis, often paired, with longer or shorter internodes. Branches arising at an angle of slightly less than 90° with respect to the main axis, longer branches near the base and short branches or solitary phialides arising near the tip; 1° branches rebranching or producing phialides directly; 2° branches producing phialides at the tip and often along the length. Phialides typically straight, lageniform, cylindrical or slightly swollen in the middle, held in whorls of 3 or 4, sometimes crowded when formed within a pustule; intercalary phialides formed but uncommon. Conidia broadly ellipsoidal, smooth. Chlamydospores not observed.
Habitat: Bark of hardwood trees. Notes: The description given above is based on the two cited Australian collections. We specifically did not designate specimens not found in Queensland as paratypes because there is reason to doubt that they are truly collections of T. austrokoningii.
Trichoderma austrokoningii s. lat., T. dingleyae and T. dorotheae occur in Australia and New Zealand. In Australasia, T. austrokoningii is known from a tropical region of Australia, the Queensland Coast, and from subtropical (CBS 247.63) to temperate (G.J.S. 99-116) parts of New Zealand (Figs 80-88). The teleomorphs of these species are indistinguishable. Conidia of T. dingleyae and T. dorotheae are longer and, especially, wider than are those of T. austrokoningii. Conidia in the Australian collections of T. austrokoningii (Figs 51-59) are shorter [(2.5-)3.0-3.5(-4.2) µm, CI = 3.3-3.4 µm] than are those of the other collections of this species. Growth (Fig. 102) of T. austrokoningii at 30 °C on PDA is the same as at 25 °C (radius = ca. 30 mm after 72 h), whereas growth of the two New Zealand isolates is considerably slower at 30 °C (radius = < 15 mm) than at 25 °C (radius = ca. 30 mm). Australian collections of T. austrokoningii grow faster on SNA than either T. dingleyae or T. dorotheae or any of the other cited collections of T. austrokoningii (colony radius for Australian T. austrokoningii at 25 and 30 °C = 25-35 mm as compared to a radius of < 25 mm at 25 °C and < 15 mm for T. dingleyae, T. dorotheae and the other collections of T. austrokoningii).
In Taiwan the occurrence of T. austrokoningii overlaps with that of T. taiwanense. Trichoderma taiwanense has larger conidia and somewhat smaller part-ascospores than does the Taiwanese collection of T. austrokoningii.  (2.5-)3. 5-4.5(-5.5) (2.7-)3. 5-4.5(-5.5) (    Only two asci could be measured from three collections. 4 Continuous measurements from SNA. All measurements pertaining to growth rate report the range observed in three, independent trials. 5 maximum and minimum of all cultures studied. 6 Total range of two isolates. 7 Measured from pustules.       Etymology: In reference to the Caribbean Ocean region where the species has been collected.
Characteristics in culture: Optimum temperature for growth on PDA and SNA 25-30 °C. Colonies grown on PDA with faint concentric rings and poor conidial production after 96 h at 25-30 °C. Colonies grown on SNA producing abundant aerial mycelium, sterile after 96 h. Conidia green (27E7-8), without yellow coloration.
No pigment diffusing through the agar, no distinctive odour. Conidia forming slowly on PDA, after 72-96 h at 20 °C, later at higher temperatures. Colonies grown on CMD at 20 °C under light filling the Petri plate within 1 wk; conidia forming in scattered pustules in concentric rings; pustules to 1 mm diam, tending to coalesce, dense; conidia also forming well apart from the pustules in the scant aerial mycelium. Conidiophores projecting from the pustules, entirely fertile or sparingly branched along the length, sometimes bearing only one or a few phialides at the tip but otherwise sterile. Conidiophores highly intricated within the pustule, a strongly developed main axis not discernable, lateral branches tending to be solitary, paired branches uncommon. Phialides held in cruciate to verticillate whorls of 3 or 4 or arising singly, especially near the tip of the main axis and along the length of 1° branches, straight, lageniform, somewhat swollen in the middle; intercalary phialides present but not common. Conidia ellipsoidal to nearly oblong, smooth. Chlamydospores produced sparingly on CMD, terminal on hyphae, subglobose, (n = 30) (5.5-)6.7-10(-11) µm diam.
Habitat: On pyrenomycetes (incl. Xylariaceae) and decorticated wood. Etymology: "Aequatoriale" in reference to Ecuador, where the species has been found.
Optimum temperature for growth on PDA and SNA 25 °C. Not growing at 35 °C. Colonies grown on PDA and SNA in darkness sterile within 96 h; conidia forming on PDA, CMD and SNA at 20-25 °C within 10 d when grown under light. On PDA and CMD mononematous conidiophores obscure, conidia held in pale green drops of watery liquid. Conidiophores on CMD mononematous and produced in pustules; mononematous conidiophores 75-85 µm long, 3.5-5.5 µm wide at the base, branching verticillium-or gliocladium-like with convergent phialides terminating each branch. Phialides tapering uniformly from base to tip. Conidia from mononematous conidiophores broadly ellipsoidal, (3.5-)3.7-4.2(-4.5) × (2.5-)2.7(-3.0) µm. On SNA conspicuous pustules scattered throughout the colony; pustules hemispherical, 1-2 mm diam, dense, without projecting sterile hairs, slowly producing greyish green conidia (27D5). Conidiophores entirely integrated into pustules, not projecting from pustules; conidiophores near the surface of the pustules with a barely discernable, short main axis producing short 1° branches at 90° and solitary phialides; branches typically unicellular, terminating in 3-5 phialides in a whorl; phialides often arising directly, separated by short internodes and then forming a dense "pseudowhorl". Phialides more or less cylindrical or slightly swollen in the middle, straight or slightly hooked. Conidia ellipsoidal, smooth. Mononematous conidiophores similar to those found on CMD forming on SNA. Chlamydospores not observed on CMD. Notes: Trichoderma caribbaeum var. aequatoriale was originally isolated as an endophyte from the trunk of a Theobroma gileri tree. When inoculated onto leaves of Th. cacao, it could be re-isolated from the lower parts of the plant. The variety is unusual in growing faster at 20 °C than at 30 °C. Holotypus H. dingleyae PDD 83838; holotypus anamorphosis T. dingleyae cultura sicca ex ascospora oriens PDD 83838.

Characteristics in culture:
Optimum temperature for growth on PDA and SNA 20-25 °C. Colonies grown on PDA in darkness or in light for 96 h with abundant white aerial mycelium, sterile. No pigment diffusing through the agar; no distinctive odour. Colonies grown on CMD at 20 °C under light producing scant aerial mycelium; conidia forming in scattered, hemispherical, grey-green pustules 0.5-1.0 mm diam. Pustules very compact; long, terminally fertile conidiophores barely protruding beyond the surface; many small, easily detached pustules forming in the aerial mycelium. Conidiophores within the pustules irregularly branched, typically without a discernable main axis; phialides often held in dense divergent clusters, often arising from swollen nodes. Protruding conidiophores with a long stipe and terminating in 2-4 phialides in a single verticil. Phialides formed within pustules cylindrical to broadly flask-shaped, straight or slightly hooked; those forming at the tips of long conidiophores often narrowly cylindrical or tapering uniformly from base to tip, or slightly swollen in the middle. Conidia ellipsoidal to broadly ellipsoidal, smooth. Chlamydospores few, terminal, subglobose, (3.2-)4.0-6.7(-8.7) µm diam.
Habitat: Bark and wood of Nothofagus spp.

Characteristics in culture:
Optimum temperature for growth on PDA and SNA 25 °C. Colonies grown on PDA in darkness or in light forming conidia only after 96 h or remaining sterile. Colonies grown on CMD at 20 and 25 ºC under light filling the Petri plate within 1 wk, conidia forming in 1-3 mm diam pustules formed around the periphery of the colony or pustules not evident and conidiophores arising in the aerial mycelium. Often long, entirely fertile conidiophores visible in the pustules. Conidiophores lacking a discernable main axis and branches not obviously paired or sometimes arising in whorls of 3, or unilaterally branched; conidiophores arising from pustules often fasciculate (Figs 155,156), internodes between the branches often short, branches increasing in length with distance from the tip of the main axis, rebranching to form 2° branches, which terminate in slightly divergent whorls of 3-4 phialides. Phialides lageniform, only slightly swollen in the middle. With age of culture, phialides tending to proliferate percurrently to form new phialides, the newly formed phialide often abruptly swollen in the middle (Fig. 158). After storage, cultures sterile or forming dense, subglobose, greygreen pustules on the surface of the agar and in the aerial mycelium. Conidia broadly ellipsoidal, smooth. Chlamydospores few, terminal, 2. 7-5.7(-8.5) µm diam.
Habitat: On bark and wood of Nothofagus and Eucalyptus species.
Characteristics in culture: Optimum temperature for growth on PDA and SNA 30 °C; less than 5 mm at 35 °C. Colonies grown on PDA in darkness or under light at 25 °C producing conidia within 8 h; within 96 h conidia abundant in the aerial mycelium in marked concentric rings; on SNA conidia beginning to form in small pustules in a ring around the original inoculum. No diffusing pigment or distinctive odour detected on any medium. Colonies on CMD filling the Petri plate within 1 wk at 20 °C; conidiophores forming around the margin of the colony in a more or less continuous, ca. 1 cm broad band of confluent, poorly-defined, cottony pustules, within which entirely fertile conidiophores can be seen (Fig. 176); conidia dark green (27F8). On SNA conidia forming in more or less discrete, hemispherical pustules. Pustules lacking projecting terminally fertile conidiophores or sterile hairs. Conidiophores with a discernable main axis, more or less symmetrical, often with 2 branches arising on either side of a single node, 1° branches arising at or near 90° with respect to the main axis, progressively longer with distance from the tip, rebranching to form unicellular 2° branches. Phialides arising directly from the main axis, 1° branches and terminating the 2° branches, in whorls of 3-5; dense whorls at the tips of branches not noted; phialides lageniform and somewhat swollen in the middle to cylindrical, straight, rarely slightly hooked or sinuous; supporting cell not significantly different in width from the widest part of the phialide. Conidia broadly ellipsoidal to ovoidal, smooth. Chlamydospores not observed.
Habitat: Decorticated wood inhabited by other ascomycetous fungi including Rosellinia sp.
Trichoderma intricatum is sympatric with T. koningiopsis, T. caribbaeum var. caribbaeum and T. stilbohypoxyli in Puerto Rico. The species can be distinguished with some difficulty. Trichoderma koningiopsis and T. caribbaeum have a faster growth rate on PDA than either T. intricatum or T. stilbohypoxyli, the radius of T. koningiopsis reaching 70 mm at 30 °C and that of T. intricatum 50-60 mm at 30 °C, whereas the colony radii of T. intricatum and T. stilbohypoxyli reach a maximum of only ca. 50 mm. Conidia form slowly, after 96 h at 25°C, in PDA and SNA cultures of T. caribbaeum, but conidia form in abundance within 96 h on these media in the other species. The distal and proximal part-ascospores of T. intricatum (Table  3) (Table 3). Conidia of T. intricatum are wider and the L/W of conidia is smaller than in T. caribbaeum, T. koningiopsis and T. stilbohypoxyli (1.3-1.5). PDA cultures of T. stilbohypoxyli typically produce a diffusing yellow pigment. Oudem. in Oudemans & Koning, Arch. Néerl. Sci. Exactes Nat., Sér. 2, 7: 291. 1902. Figs 14, 196-207 Characters of anamorph: Colonies grown on CMD filling the Petri plate within one wk; no diffusing pigment, no distinctive odour. Conidia forming in a narrow band around the colony margin in confluent, compact to cottony pustules up to 1 mm diam and in the sparingly produced aerial mycelium, sometimes with only a slight tendency to form pustules; individual conidiophores often visible within the pustule (Fig.  197). Conidiophores with a discernable main axis, more or less symmetrical, often with 2 branches arising on either side of a single node, 1° branches arising at or near 90° with respect to the main axis, progressively longer with distance from the tip, rebranching to form unicellular 2° branches. Phialides arising directly from the main axis, the 1° and 2° branches, in whorls of 3 or 4, often dense whorls at the tips of branches and in intercalary positions, lageniform, somewhat swollen in the middle, straight; phialides often densely clustered in "pseudowhorls" (Figs 201, 202). Conidia oblong, smooth, green. Chlamydospores sparingly produced, subglobose, (6.5-)7.5-11.5(-14.5) µm, terminal on hyphae.

Characteristics of cultures:
Optimum temperature for growth on PDA and SNA 30 °C. Colonies grown on PDA in darkness or in light for 96 h often forming conidia after 48 h at 25 and 30 °C in a dense lawn or the centre remaining sterile; conidia also forming in 2 or 3 concentric rings; conidia abundant in light-grown colonies, less abundant and sometimes lacking from colonies grown in darkness. Colonies grown on SNA in darkness or in light for 96 h producing abundant aerial mycelium; conidia tending to be uniformly dispersed in the aerial mycelium in broad concentric rings, sometimes forming cottony pustules. Conidial masses 25E-F8 (deep green to dark green), seldom with yellow coloration (26-27E-F). No pigment diffusing through the agar; no distinctive odour (rarely a faint coconut odour detected). Colonies grown on CMD at 20 °C under light filling the Petri plate within 1 wk, conidial production nearly continuous with a tendency to form highly compact to cottony, 1-2 mm diam pustules; conidial production sometimes  [199][200][201][202]203,205 = PC;204,206 = DIC,207 = BF. Bars: 196 = 1 mm;[197][198][199][200][201][202]205, 207 = 20 µm; 203-204, 206 = 10 µm.  restricted to the margin of the colony. Often long, entirely fertile branches visible in the pustules (Fig.  210). Conidiophores comprising a recognizable main axis, ca. 3 µm wide; fertile branches arising along the length of the main axis, more or less paired with longer or shorter internodes; terminal part of conidiophore often sparingly branched and with long internodes between branches (Figs 212-213); branches formed from the interior of pustules, sometimes pachybasiumlike, with short, crowded phialides . Branches arising at an angle of slightly less than 90º with respect to the main axis, longer branches near the base and short branches or solitary phialides arising near the tip; 1° branches rebranching or producing phialides directly, 2° branches tending to be paired; all fertile branches terminating in a whorl of phialides. Phialides typically straight, sometimes hooked or sinuous, narrowly lageniform or sometimes conspicuously swollen in the middle (especially when crowded at the interior of a pustule), held in whorls of 2-5, sometimes several phialides arising from the same point and crowded (Fig. 214); intercalary phialides forming but not common (Fig. 215). Conidia ellipsoidal, smooth. Chlamydospores abundant to sparse or lacking, terminal to intercalary, globose to subglobose, (n = 168) (3.0-)9-9.5 (-16) µm diam.
Habitat: Isolated from pods of Theobroma cacao and from trunks of Th. grandiflorum, Th. gileri, Theobroma sp. ("cacao de monte"), mushroom compost, soil, twigs and decaying leaves. Teleomorph found on wood, possibly associated with ascomycetes.   Notes: With the exception of the soil isolate DAOM 165782 (North Carolina), we only know T. petersenii from ascospore isolations. It is common in the eastern United States together with T. rogersonii, T. koningii and T. koningiopsis. Trichoderma petersenii is characterized by a moderate growth rate, PDA colonies that are distinctive because of the conspicuous concentric rings and abundant conidia (Fig. 18), and stromata that are darker in colour, tending to be red-brown, than stromata of the other species (Figs 41-43). Samuels, sp  Holotypus H. rogersonii: BPI 870964A; holotypus anamorphosis T. rogersonii: cultura sicca ex ascospora oriens BPI 870964.

Trichoderma stilbohypoxyli
Notes: As is evident from the several specimens and cultures cited here, T. stilbohypoxyli is a common tropical species, the teleomorph of which we have seen in Ghana and Puerto Rico. Additional tef analysis (not shown) has shown T. stilbohypoxyli to be phylogenetically diverse, the diversity corresponding to some extent with the geographic diversity that is indicated in the specimens examined cited above. However, we lack consistent morphological information to use in recognizing species within the phylogenetic diversity. Ascospores of the single Ghanaian collection were somewhat larger than they were in the Puerto Rican collections (respectively: distal part-ascospores: 4.2 ± 0.4 × 3.7 ± 0.2 μm vs 4.0 ± 0.3 × 3.5 ± 0.3 μm; proximal part-ascospores 4.5 ± 0.5 × 3.5 ± 0.2 μm vs 4.7 ± 0.5 × 3.0 ± 0.2 μm). The Puerto Rican cultures grew more slowly than the others on PDA, reaching a maximum radius of 45 mm after 72 h at the optimum temperature of 30 °C; colony radius in the other isolates was 55-70 mm. This difference was not seen on SNA. We were surprised to find T. stilbohypoxyli as an endophyte of trunks of ancient beech trees in the United Kingdom and Theobroma species in South America. Interestingly, the Fagus endophytes (G.J.S. 05-474, G.J.S. 05-475) clustered independently of the Theobroma endophyte (DIS 205b) from Brazil.
In the Caribbean region, T. stilbohypoxyli is found with T. caribbaeum var. caribbaeum, T. intricatum and T. koningiopsis. Trichoderma caribbaeum and T. koningiopsis have the longest conidia and, with T. stilbohypoxyli, they have the longest ascospores and highest L/W for their conidia. Trichoderma stilbohypoxyli is distinguished by having shorter and narrower conidia than T. caribbaeum and T. koningiopsis and by having the slowest growth rate of all these species when grown on PDA, a difference that is especially noticeable at 20 °C; this difference is not seen on SNA. On SNA at 20 °C T. caribbaeum and T. koningiopsis grow considerably faster than T. stilbohypoxyli and T. intricatum, a difference that is not seen at higher temperatures.

Characteristics in culture:
Optimum temperature for growth on PDA 25-30 °C, on SNA 25 °C. Colonies grown on PDA in darkness or in light forming conidia in the centre of the colony with conidial production gradually spreading outwardly in faint concentric rings. Conidia dark green on CMD. No pigment diffusing through the agar; no distinctive odour. Colonies grown on CMD at 20 °C under light filling the Petri plate within 1 wk, conidia forming in minute, ill-defined tufts and in the aerial mycelium. Conidiophores symmetrical, comprising a recognizable main axis, 2-3 µm wide, often arising from an enlarged, verrucose base (Figs 290-293), fertile branches arising along the length of the main axis, more or less paired, with longer or shorter internodes. Branches arising at an angle of slightly less than 90° with respect to the main axis, longer branches near the base and short branches or solitary phialides arising near the tip; 1° branches rebranching to a limited extent or producing phialides directly, 2° branches producing phialides at the tip and often along the length. Phialides typically straight, lageniform, cylindrical or slightly swollen in the middle, held in whorls of 3 or 4, sometimes crowded and penicillate when formed within a pustule; intercalary phialides not seen. Conidia ellipsoidal, smooth. Chlamydospores not observed.
Known distribution: Republic of China (Taiwan). Notes: The Hypocrea specimen from which T. taiwanense is derived is overmature. Few asci remain and the stroma is collapsed. The stroma does not appear to be typical of members of this group in its gross morphology, but this could be the result of overmaturity. We have not described the Hypocrea teleomorph of this species as new pending the discovery of material in better condition.
Two species having the T. koningii morphology and included in the present work are found in Taiwan, T. taiwanense and T. austrokoningii. For distinctions between these two phylogenetically distinct but phenotypically similar species see commentary under T. austrokoningii and Table 3.