Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters?

Species concepts and biodiversity in Trichoderma and Hypocrea : from aggregate species to species clusters? first-author †E-mail:ckubicek@mail.zserv.tuwien.ac.at †E-mail:ckubicek@mail.zserv.tuwien.ac.at ckubicek@mail.zserv.tuwien.ac.at Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Getreidemarkt 9/E1665, A-1060 Vienna, Austria Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Getreidemarkt 9/E1665, A-1060 Vienna, Austria Trichoderma / Hypocrea is a genus of soil-borne or wood-decaying fungi containing members important to mankind as producers of industrial enzymes and biocontrol agents against plant pathogens, but also as opportunistic pathogens of immunocompromised humans. Species identification, while essential in view of the controversial properties of taxa of this genus, has been problematic by traditional methods. Here we will present a critical survey of the various identification methods in use. In addition, we will present an update on the taxonomy and phylogeny of the 88 taxa (which occur as 14 holomorphs, 49 teleomorphs and 25 anamorphs in nature) of Trichoderma / Hypocrea that have been confirmed by a combination of morphological, physiological and genetic approaches. Trichoderma / Hypocrea Genus Molecular taxonomy-phylogeny WHY IS TRICHODERMA IMPORTANT? 1 The anamorphic fungal genus Trichoderma ( Hypocreales , Ascomycota ) contains cosmopolitan soil-borne fungi frequently also found on decaying wood (Samuels, 1996 1996 ; Klein and Eveleigh, 1998 1998 ), of which some are economically important producers of industrial enzymes ( Trichoderma reesei = Hypocrea jecorina ) (Kubicek and Penttilä, 1998 1998 ) and antibiotics (Sivasithamparam and Ghisalberti, 1998 1998 ), or have application as biocontrol agents against plant pathogens (i.e. T. harzianum = H. lixii ; T. atroviride = H. atroviridis ; T. asperellum ) (Hjeljord and Tronsmo, 1998 1998 ). More recently, T. longibrachiatum has also become known as opportunistic pathogen of immunocompromised mammals including humans (Kredics et al., 2003 2003 ). Trichoderma has been recognized to comprise a significant amount of fungal biomass in soil (Nelson, 1982 1982 ; Widden and Abitbol, 1980 1980 ) and is frequently present as an indoor contaminant (Thrane et al., 2001 2001 ). These diverse implications of Trichoderma / Hypocrea with human society render an accurate species and strain identification to be an important issue. However, classical approaches based on the use of morphological criteria are, as in several other fungi, difficult to apply to Trichoderma , due to the plasticity of characters. As a consequence, we shall review the current state of perception of taxa and other taxonomic ranks in Trichoderma and Hypocrea in this paper. TOWARDS DEVELOPMENT OF A CONCEPT FOR THE GENUS TRICHODERMA 1 Although originally introduced by Persoon ( 1794 1794 ), the taxonomy and identification of Trichoderma has remained problematic until relatively recently. Until 1969, nearly all strains of Trichoderma were identified in literature as “ T. viride ” (e.g. also all the cellulase-producing strains of T. reesei ) owing to Bisby’s ( 1939 1939 ) concept that Trichoderma consists of a single species. This led to the erroneous statement even in textbooks that “ T. viride is an industrial cellulose producer”. As T. viride sensu stricto is a relatively rare species ( 1 1 Kubicek, unpublished data; 2 2 Samuels, personal communications), most of the taxa sampled and determined before 1969 are probably misidentified. Rifai ( 1969 1969 ) adopted the concept of ‘aggregate’ species, and distinguished nine ‘species aggregates’, and admitted that some of them (particularly T. hamatum ) likely contain two or more morphologically indistinguishable species. Bissett ( 1984 1984 ; 1991a 1991a ; 1991b 1991b ; 1991c 1991c ; 1992 1992 ; Gams and Bissett, 1998 1998 ) revised Rifai’s aggregate species by subtle recognition of continuities of morphological characters, expanded the morphological criteria to accommodate the wider range of morphological variation expressed by some anamorphs of Hypocrea and adopted some forms previously included in Gliocladium . While dissecting several of Rifai’s species aggregates into several defined taxa, Bissett ( 1991a 1991a ) also established a subdivision of the genus into five sections: Longibrachiatum , Pachybasium , Trichoderma , Saturnisporum and Hypocreanum . The advent of molecular tools for investigations in fungal taxonomy prompted research in the mid-nineties to re-assess the morphology-based taxonomy in Trichoderma . The laboratories of G.J. Samuels (Beltsville, MD, USA), T. Börner (Berlin, FRG) and C.P. Kubicek (Vienna, Aust.) collaboratively pioneered a revision of Bissett’s section Longibrachiatum . They combined the use of molecular markers (ITS1 and ITS2 sequence analysis, RAPD), physiological (isoenzyme analysis) and phenetic characters, and also for the first time included an analysis of potential teleomorphs of the Trichoderma spp. from this section (Kuhls et al., 1996 1996 ; 1997 1997 ; Samuels, 1996 1996 ; Samuels et al., 1998 1998 ; Turner et al., 1997 1997 ). As a result, section Longibrachiatum was recognized to be monophyletic and to contain ten taxa, within which four pairs displayed teleomorph-anamorph relationships: H. schweinitzii / T. citrinoviride ; H. pseudokoningii / T. pseudokoningii ; H. jecorina / T. reesei and H. orientalis / T. longibrachiatum , p/p. They further merged section Saturnisporum , which included only two species, T. saturnisporum and T. ghanense (Doi et al., 1987 1987 ), with section Longibrachiatum , and recognized the synonymy of T. ghanense with T. parceramosum . Yet, as a whole, the concept of section Longibrachiatum as defined by Bissett ( 1984 1984 ) was largely confirmed, suggesting a degree of correlation between morphological and molecular approaches to taxonomy in Trichoderma . Section Longibrachiatum is a comparably small section of Trichoderma , and phylogenetically most distant from the other sections. The relationship between morphological characters and molecular phylogeny became more complex, however, when larger sections of Trichoderma were investigated. Kindermann et al.( 1998 1998 ) attempted a first phylogenetic analysis of the whole genus. Using sequence analysis of the ITS1 region of rDNA they found that the largest section, section Pachybasium , is actually paraphyletic. Although the use of a single gene fragment alone is insufficient by today’s standards, this finding has been confirmed by phylogenetic analysis of several other genes (Kullnig-Gradinger et al., 2002 2002 ; Chaverri et al., 2003b 2003b ). In this context it is noteworthy that the nomenclatural type strain of sect. Pachybasium , Pachybasium hamatum (Bonord.)= T. hamatum , is not a member of the major one of the two Pachybasium clades (clade B) (Kindermann et al., 1998 1998 ), but clusters together with T. pubescens and T. strigosum in a clade otherwise containing almost all taxa (i.e. with the exception of T. aureoviride ) from section Trichoderma . Because of the lack of a distinctive morphological hiatus between clades A and B of Pachybasium (Kindermann et al., 1998 1998 ), researchers have so far maintained the name Pachybasium for both clades, but it is clear that this is an unsatisfactory situation from a taxonomic point of view. Pachybasium B contains all taxa attributed to this section by Bissett ( 1991b 1991b ) with the exception of the three mentioned above. In addition, Pachybasium B also poses the problem of strong genetic variability of several of its species (e.g. T. harzianum ). Since its species (both ana- as well as teleomorphs) account for the majority of taxa found in field investigations (Kullnig et al., 2000 2000 ; Kubicek et al., 2003 2003 ; Wuczskowski et al., 2003 2003 ; Gherbawy et al., 2004 2004 ; Druzhinina et al., 2004b 2004b ), this raises the question of how to identify species in this large heterogenous group. WHAT IS A SPECIES IN TRICHODERMA ? 1 Most of the taxa of Trichoderma have so far been defined on the basis of morphology, and gene sequence analysis was only used as a confirmative or distinctive complement. Thus, members of the genus are primarily defined by the application of the MSR (morphological species recognition) concept. Since strains of Trichoderma spp. apparently cannot be crossed, application of the BSR (biological species recognition) concept is impracticable. The genealogical concordance phylogenetic species recognition (GCPSR) (Taylor et al., 2000 2000 ) is an attractive alternative or complement to the morphological species concept, but has not been widely applied to Trichoderma / Hypocrea . It requires the analysis of trees of several unlinked genes, and implies that the phylogenetic position of a true species will be concordant in at least some of them, and not be contradicted in the others. Kullnig-Gradinger et al.( 2002 2002 ) used four different loci (ITS1 and ITS2, mitssuDNA, short fifth tef 1 α intron, a fragment of ech 42 large exon) to assess a global phylogeny of the genus. However, a stringent clade to clade concordance was not possible for most of the species because of insufficient phylogenetic resolution by the genes used. Chaverri et al.( 2003a 2003a ) analyzed ITS1 and ITS2, large tef 1 intron, and short fragments of the actin ( act 1) and calmodulin ( cal 1) exon sequences in H. lixii / T. harzianum , and identified seven phylogenetic lineages which were concordant in most trees. However, since the isolates within the seven lineages could not be reliably distinguished morphologically, they were not given recognition as a species. Taylor et al.( 1999 1999 ) proposed basing phylogenetic species concepts on the concordance between five or more gene trees. This requirement is not easily fulfilled in Trichoderma . In the past, most researchers made heavy use of ITS1 and/or ITS2 (Kuhls et al., 1997 1997 ; Kindermann et al., 1998 1998 ; Lieckfeldt et al., 1998 1998 ; 2001 2001 ; Dodd et al., 2000 2000 ), because this gene cluster is present in > 90 copies per genome and can thus be easily amplified. However, the use of ITS1 and ITS2 has meanwhile become discredited by the fact that some fungi, notably some sections in the closely related Fusarium , and plants have been shown to contain paralogous copies of the parts of the rDNA gene cluster (O’Donnell, 1992 1992 ; Buckler et al., 1997 1997 ; O’Donnell et al., 1998 1998 ; Lieckfeldt and Seifert, 2000 2000 ). Also, Chaverri et al.( 2003b 2003b ) reported unpublished data for the presence of divergent ITS1 and ITS2 sequences in T. strictipile and T. crassum . In contrast, we have so far not obtained evidence for the presence of paralogous ITS1 or ITS2 copies in most species of Trichoderma / Hypocrea , even though we specifically tested for it ( 1 1 Mach et al., unpublished data). On the other hand, a serious drawback of the use of ITS1 and ITS2 is that it provides only poor phylogenetic resolution in some clades, particularly Pachybasium B (Kullnig-Gradinger et al., 2002 2002 ; Chaverri et al., 2003a 2003a ). Among 11 tested loci/fragments (Table 1 1 ) the most promising loci seem to be different fragments of translation elongation factor 1-alpha (EF-1α= tef 1) different fragments of which were sequenced by different research groups (Fig. 1 1 ). The gene has been cloned from H. jecorina and exceeds 2 kb in length, and consisted of several relatively large and variable introns and exons. Also, the coding portions of endochitinase 42 ( ech 42) and RNA-polymerase subunit 2 ( rpb 2) have displayed significant intra- and interspecific variability while other genes, such as the D1 and D2 regions of the 28S rDNA, or the small subunit of the mitochondrial ribosomal DNA (ssu-mDNA) have been used with limited success. Fragments of the calmodulin- and the actin-encoding genes ( cal 1, act 1) had also been used in T. harzianum / H. lixii , but exhibited less powerful resolution than the large exon of tef 1 (Table 1 1 ) (Chaverri et al., 2003a 2003a ). It is important to mention that two different unalignable intron fragments of act 1 are available in GenBank (Table 1 1 ). Portion of parsimoni informative sites from the length of fragment Only tested for closely related taxa Data for section Longibrachiatum are not available N indicates number of species investigated Locus 2 Fragment Length (kb) N Var . (%) * * Reference Internal transcribed spacer 1 and 2 ITS1 and ITS2 0.4 50 25 Kullnig-Gradinger et al., 2002 2002 RNA coding genes Small subunit of the mitochondrial ribosomal DNA ssu-mDNA 0.4 50 5 Kullnig-Gradinger et al., 2002 2002 28S rDNA gene 28S rDNA 0.5 51 8 Kullnig-Gradinger et al., 2002 2002 Protein coding genes Calmodulin cal 1 Intron 0.45 35 ** ** 15 Chaverri et al., 2003a 2003a Actin act 1 1st intron 0.35 35 ** ** 5 Chaverri et al., 2003a 2003a 2nd intron 0.8 18 ** ** 6 1 1 Samuels and Ismael, personal communication RNA polymerase B subunit 2 rpb 2 0.4 97 40 Chaverri et al., 2004 2004 Endochitinase 42 ech 42 Last large exon 0.6 44 *** *** 33 Kullnig-Gradinger et al., 2002 2002 Translation elongation factor 1-alpha tef 1 5th (small) intron 0.1 47 30 Kullnig-Gradinger et al., 2002 2002 4th (large) intron 0.35 125 ** ** 29 2 2 Druzhinina, unpublished data Last large exon 0.7 84 29 Chaverri et al., 2004 2004 Notwithstanding the fact that both regions exhibit an extremely weak phylogenetic signal it is important to reach an agreement between workers regarding which fragment of this gene should be sequenced. We have also attempted to use histon 3A and β-tubulin gene sequences, which have proven worthwhile for phylogenetic analysis in Fusarium and other genera, but while the variation in the histon 3A gene was not high, several Trichoderma spp. contained multiple heterologous copies of the tub 1 gene, thus rendering both genes not applicable for this purpose ( 2 2 Kullnig-Gradinger and Kubicek, unpublished data). Unfortunately, none of these genes is optimal for phylogenetic resolution of the whole genus, or of large clades such as Pachybasium B: while the largest intron of tef 1 provides excellent resolution and high clade support for closely related taxa such as the T. harzianum / H. lixii species clade ( H. lixii , T. harzianum , T. aggressivum , T. tomentosum , T. cerinum , T. velutinum , H. tawa ) or the group of H. rufa ( T. viride , T. atroviride , T. koningii ). However, the large introns in tef 1 are less suited for resolving the phylogenetic elations of more distantly related species due to ambiguous alignments ( 2 2 Druzhinina, unpublished data). On the other hand, the last large exon of tef 1 contains only limited phylogenetic signals for analysis of diverse clades, and thus e.g. in a combined analysis of Pachybasium A and B resulted in lack of support for almost all basal branches, whereas the terminal branches had good support (Chaverri et al., 2003b 2003b ; 2004 2004 ). The same problem was even more apparent with rpb 2. Therefore, the optimal combination of genes allowing the application of the GCPSR concept on the whole genus Trichoderma has not yet been applied. Detailed analysis of various loci available in GenBank (Table 1 1 ) may suggested that the simultaneous usage of (i) tef l arge intron and last large exon, (ii) rpb 2 gene, (iii) ech 42 last large exon and (iv) ITS1 and 2 as diagnostic regions may lead to the most reliable phylogeny. However a search for new phylogenetic markers is strongly recommended. A further important point in the use of the GCPSR concept is the justified use of the correct phylogenetic approach, so far, most workers employed the maximum parsimony method to analyze sequence data, which does not employ any modelling of evolution and therefore poses several problems when dealing with either very closely or very distantly related taxa (Salemi and Vandamme, 2003 2003 ). Maximum likelihood methods would be adequate, but suffer from the long computation time they usually require. For this purpose, the Bayesian approach to phylogenetic inferences represents the most recent advance in phylogenetic analysis (Rannala and Yang, 1996 1996 ; Huelsenbeck and Ronquist, 2001 2001 ; Lutzoni et al., 2001 2001 ). It shifts statistical interference away from an emphasis on hypothesis testing ( P -values) and finding the single optimal tree, toward obtaining adequate estimates of uncertainty. Uncertainty is characterized through the use of the posterior distribution of a parameter, which is defined as the conditional probability of observing a particular parameter value given in the data. This involves the use of Markov chain Monte Carlo (MCMC) methods (Metropolis et al., 1953 1953 ), which implements the simulation of a random walk through parameter space, which, if run long enough, will eventually converge on the stationary distribution of parameters (Lewis, 2001 2001 ). Bayesian approaches have been introduced in the analysis of phylogenies of other genera, and had also been applied to Trichoderma / Hypocrea (Chaverri et al., 2003b 2003b ; Druzhinina et al., 2004a 2004a ). When combined with rigorous testing of evolutionary models, this approach yields excellent resolution even for difficult to resolve clades in Trichoderma ( 1 1 Druzhinina et al., manuscript in preparation). HOW CAN SPECIES OF TRICHODERMA BE IDENTIFIED? 1 While this review so far treated the concepts and advances in the recognition and definition of species of Trichoderma , nothing has been said about how an unknown isolate can be identified as one of these species. Morphological analysis is highly prone to error, and consequently roughly 50% of the Trichoderma spp. deposited in culture collections under names obtained by morphological analysis alone are wrong ( 2 2 Kubicek, unpublished data)! Obviously, subjecting every new strain to a thorough GCPSR analysis would result in a safe identification, but most workers who study the occurrence of Trichoderma or Hypocrea in soil or other habitats will not invest such a massive use of money and time. In fact, several researchers are still mainly using morphological methods for identification of Trichoderma , although the use of ITS1 and ITS2 sequence analysis is becoming more and more popular. This is not in contradiction to the statement above that ITS1 and ITS2 do not provide sufficient phylogenetic resolution, because phylogeny and diagnosis underlie two different principles, i.e. even though the change in 2–3 nts may not provide sufficient phylogenetic information, it may be indicative of a given species if it is known that this species always contains these three nucleotides in the respective positions. Hence the intra- and interspecific variability must be fully known in order to confirm or refute species identity. Unfortunately, the preferred process used by some workers is to submit sequences to a BLAST, and then accept the best hit as species identity. This must be criticized for several reasons: first, the GenBank databases contain many sequences of Trichoderma isolates which had been incorrectly identified and thus occur under a false name (Table 2 2 ); second, many researchers without solid bioinformatic training are inexperienced with the principles of BLAST and take an E -value of 0.00 as identity without scrutinizing whether the sequence is identitical or only highly similar. Even if it is identical, it is imperative to know that no other species with the same sequence exists. Sequence AF 3362109 was used as query ( T. harzianum , wrongly deposited as “ T. aureoviride ”), using BLASTN 2 Sequences producing significant alignments: Score (bits) E -value gi|19880152|gb|AF362109.1| Trichoderma aureoviride strain T... 1132 0.0 gi|19880083|gb|AF359399.1| Trichoderma aureoviride strain T... 1116 0.0 gi|32394933|gb|AY154947.1| Trichoderma aggressivum Ir. 560 ... 1112 0.0 gi|19880067|gb|AF359267.1| Trichoderma aureoviride strain T... 1108 0.0 gi|19032416|gb|AF345948.1| Trichoderma harzianum isolate GJ... 1100 0.0 gi|19880081|gb|AF359397.1| Trichoderma aureoviride strain T... 1100 0.0 gi|21239369|gb|AF501330.1| Trichoderma aggressivum f. europ... 1096 0.0 gi|3095175|gb|AF055216.1|AF055216 Trichoderma harzianum str... 1082 0.0 gi|27448757|gb|AF443912.1| Trichoderma harzianum G.J.S. 00–... 1045 0.0 gi|9230639|gb|AF194019.1|AF194019 Trichoderma aureoviride s... 1045 0.0 gi|9230631|gb|AF194011.1|AF194011 Trichoderma harzianum str... 1045 0.0 gi|9230630|gb|AF194010.1|AF194010 Trichoderma aureoviride s... 1045 0.0 gi|27448762|gb|AF443917.1| Hypocrea lixii G.J.S. 91-138 int... 1041 0.0 gi|27448761|gb|AF443916.1| Hypocrea lixii G.J.S. 94-53 inte... 1041 0.0 gi|27448758|gb|AF443913.1| Trichoderma harzianum G.J.S. 00–... 1041 0.0 gi|27448771|gb|AF443926.1| Hypocrea lixii G.J.S. 90-254 int... 1039 0.0 gi|27448769|gb|AF443924.1| Hypocrea lixii G.J.S. 92-110 int... 1037 0.0 gi|27448764|gb|AF443919.1| Hypocrea lixii G.J.S. 92-100 int... 1037 0.0 gi|27448760|gb|AF443915.1| Hypocrea lixii G.J.S. 90-22 inte... 1037 0.0 gi|32394935|gb|AY154949.1| Trichoderma harzianum Ir. 112 C ... 1033 0.0 gi|27448770|gb|AF443925.1| Trichoderma harzianum G.J.S. 92–... 1033 0.0 gi|1813651|gb|U78881.1|THU78881 Trichoderma harzianum isola... 1033 0.0 gi|32394941|gb|AY154955.1| Trichoderma inhamatum Ir. 286 18... 1029 0.0 As a solution to this problem, we have recently developed a DNA-barcode system for quick identification on the basis of defined nucleotide sequence differences in the ITS1 and ITS2 region (Druzhinina et al., 2004b 2004b ). The method relies on the discriminatory power of ITS1 and ITS2, which could be shown to be capable of identifying 70 of a total of 77 investigated species of Trichoderma and Hypocrea . Some taxa could not be distinguished because of ITS1 and ITS2 sequence identity: T. crassum and T. longipile , which however, once delimitated by the barcode, can then easily be distinguished on the basis of morphology (Bissett, 1991b 1991b ; Chaverri et al., 2003b 2003b ); and T. tomentosum and T. cerinum . Although, the latter two are also difficult to distinguish morphologically, phylogenetic analysis has shown that the latter two taxa are the result of a recent allopatric speciation, T. cerinum found only in Eurasia whereas T. tomentosum only in the Americas ( 1 1 Druzhinina et al., manscript in preparation). Therefore, knowledge of the geographic origin of an isolate with a T. tomentosum / T. cerinum sequence may be used to distinguish these two. Otherwise, the two must be distinguished by the aid of additional sequences or phenotype arrays (see below). ITS1 and ITS2 sequence differences were also unable to consistently distinguish between three taxa from the “ H. rufa species complex” namely T. viride , T. koningii and T. atroviride . This is not because no eventually appropriate nt-differences would be detected, but because the species concept of this clade is currently under revision and several additional new taxa will be defined among them ( 2 2 Samuels, personal communication). Sequence analysis of the large tef 1 intron has already been shown to distinguish groups within T. viride and T. koningii , and will probably be the method of choice to complement the ITS based DNA-barcode for identification of these taxa in future. While we think that DNA-barcode will enable many researchers to reliably identify their Trichoderma strains, it is obvious that some researchers will not have access or financial capability to use DNA-based methods, and therefore need alternatives. Samuels and colleagues, advocating the morphologically and physiologically based methods, proposed an interactive key for strain identification in Trichoderma , which, besides subtile differences in morphology ( http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm ), makes use of differences in growth rates on PDA and SNA at 15, 20, 25, 30 and 35 °C (Chaverri et al., 2003b 2003b ). This method is inexpensive, but is time consuming and requires a sufficient number of repetitions ( n > 5) for each sample to be reliable, thus becoming laborious with more than 50 samples being considered as the lowest limit for any ecological investigation. 1 1 Bissett (unpublished) introduced a phenetic method, based on quantification of carbon assimilation patterns using BIOLOG MicroPlates™ (Biolog, Hayward, CA). Generally, phenotype arrays, commercialized by Biolog for identification of food and air-borne fungi, are not as suited for taxonomic purposes in Trichoderma because of the significant strain variation in some species (Kubicek et al., 2003 2003 ). However, we have recently included phenotype arrays into an ‘integrated approach’ when describing the new taxon T. brevicompactum (Kraus et al., 2004 2004 ). Phenotype array analysis of this new species and the phylogenetically closest neighbors method identified a number of carbon sources within the arrays, which were assimilated by T. brevicompactum at statistically different rates. This analysis is rapid and reliable, and even researchers for which phenotype arrays and/or the necessary reader are unavailable can use the published information to design simple agar plates with the respective carbon sources to verify an identification. We have recently used this system for distinguishing a number of species pairs which are morphologically very similar (e.g. T. tomentosum and T. cerinum ; T. harzianum and T. aggressivum ) ( 2 2 Druzhinina and Bissett, unpublished data) and will continue to support by this method new species identified by GCPSR. WHAT DO THE CURRENT DATA TELL US ABOUT THE PHYLOGENETICS OF TRICHODERMA ? 1 Notwithstanding the limitations and caveats outlined above, the use of molecular tools enabled the taxonomy of Trichoderma to advance strongly over the last years, and we will therefore attempt to summarize the outcome. So far, 88 taxa have been recently redefined by combination of molecular and phenetic tools (Table 3 3 ). Four taxa introduced by Chaverri et al.( 2004 2004 ) (Table 4 4 ) still lack phylogenetic analysis. Among the 88, 14 anamorph-teleomorph relationships have been demonstrated and thus represent holomorphs, 49 have been described in Hypocrea , while the remaining 25 were described as Trichoderma . In the latter, two cases, the other (sexual/asexual) form has not been found in nature. It is possible (but unlikely) that many of these Hypocrea species occur naturally only in their teleomorph state and that many of the hitherto described Trichoderma species may lack a teleomorph state due to clonal evolution. Only taxa, which have been verified by molecular analyses are included in the table. The following species were not included, as they have recently been abandoned: T. inhamatum (= T. harzianum ); T. fasciculatum (= T. strictipilis ), T. flavofuscum (= T. virens ) and T. croceum (= T. polysporum ) Clades follow the nomenclature of Chaverri et al.( 2004 2004 ) and define phylogenetic groups of species, which received high statistic support in all investigations performed so far Species names listed in the same line indicate anamorph-teleomorph relationships, and are only given for cases where both forms have been found in nature Kubicek, C.P., 2003 Druzhinina, I., 2004 Section Clade 1 1 Anamorph 2 2 Teleomorph 2 2 Reference Longibrachiatum T. longibrachiatum H. orientalis Samuels et al., 1998 1998 T. citrinoviride H. schweinitzii Samuels et al., 1998 1998 T. reesei H. jecorina Samuels et al., 1998 1998 T. ghanense Samuels et al., 1998 1998 T. pseudokoningii H. pseudokoningii Samuels et al., 1998 1998 T. saturnisporum Samuels et al., 1998 1998 T. konilangbra Samuels et al., 1998 1998 T. effusum Bissett et al., 2003 2003 T. sinensis Bissett et al., 2003 2003 T. sp. MA Wuczskowski et al., 2003 2003 H. andinensis Samuels et al., 1998 1998 H. novazelandia Samuels et al., 1998 1998 H. cerebriformis 3 3 Kubicek, unpublished data H. poronoidea 3 3 Kubicek, unpublished data H. peltata Dodd et al., 2002 2002 Trichoderma H. pezizoides Chaverri et al., 2004 2004 H. avellanea Chaverri et al., 2004 2004 Rufa T. viride H. rufa Lieckfeldt et al., 1999 1999 T. atroviride H. atroviridis Dodd et al., 2003 2003 T. koningii H. koningii Lieckfeldt et al., 1998 1998 T. strigosum Kullnig-Gradinger et al., 2002 2002 T. ovalisporum Samuels, 2004 2004 T. erinaceum Bissett et al., 2003 2003 H. stilbohypoxyli Lu and Samuels, 2004 2004 Pachybasium A T. hamatum Kullnig-Gradinger et al., 2002 2002 T. pubescens Kullnig-Gradinger et al., 2002 2002 T. asperellum Kullnig-Gradinger et al., 2002 2002 H. neorufa Dodd et al., 2002 2002 H. flavoconidia Druzhinina et al., 2004a 2004a Pachybasium B Pachybasioides T. polysporum H. pachybasioides Lu et al., 2004 2004 T. minutisporum H. minutispora Lu et al., 2004 2004 T. piluliferum H. pilulifera Lu et al., 2004 2004 H. parapilulifera Lu et al., 2004 2004 H. stellata Lu et al., 2004 2004 H. laciwombatensis Lu et al., 2004 2004 Hypocreanum H. citrina Chaverri et al., 2004 2004 H. lactea Chaverri et al., 2004 2004 H. sulphurea Chaverri et al., 2004 2004 H. pulvinata Chaverri et al., 2004 2004 Chlorospora H. aureoviridis Chaverri et al., 2004 2004 H. candida Chaverri et al., 2004 2004 H. cremea Chaverri et al., 2004 2004 H. surrotunda Chaverri et al., 2004 2004 H. sinuosa Chaverri et al., 2004 2004 H. chlorospora Chaverri et al., 2004 2004 H. thelephoricola Chaverri et al., 2004 2004 H. costaricensis Chaverri et al., 2004 2004 H. thailandica Chaverri et al., 2004 2004 H. virecentiflava Chaverri et al., 2004 2004 Lixii/catoptron T. harzianum H. lixii Chaverri et al., 2004 2004 T. aggressivum Samuels et al., 2002 2002 T. tomentosum Chaverri et al., 2004 2004 T. cerinum Bissett et al., 2003 2003 T. velutinum Bissett et al., 2003 2003 T. sp. DAOM 175928 4 4 Druzhinina, unpublished data H. tawa 4 4 Druzhinina, unpublished data H. atrogelatinosa Chaverri et al., 2003b 2003b H. ceracea Chaverri et al., 2004 2004 H. cinnamomea Chaverri et al., 2004 2004 H. straminea Chaverri et al., 2004 2004 H. catoptron Chaverri et al., 2004 2004 Virens T. virens H. virens Kullnig-Gradinger et al., 2002 2002 T. crassum H. crassa Chaverri et al., 2004 2004 Semiorbis H. semiorbis Chaverri et al., 2004 2004 H. hunua Kullnig-Gradinger et al., 2002 2002 T. fertile Kullnig-Gradinger et al., 2002 2002 T. oblongisporum Kullnig-Gradinger et al., 2002 2002 Strictipilis T. strictipilis H. strictipilosa Chaverri et al., 2004 2004 T. longipile Chaverri et al., 2004 2004 H. cuneispora Chaverri et al., 2004 2004 H. aureoviridis var. macrospora 4 4 Druzhinina, unpublished data Stromatica T. stromaticum Chaverri et al., 2004 2004 T. rossicum Bissett et al., 2003 2003 T. sp. PPRI 3559 Kullnig-Gradinger et al., 2002 2002 Ceramica H. ceramica Chaverri et al., 2004 2004 H. estonica Chaverri et al., 2004 2004 Lutea H. lutea Chaverri et al., 2004 2004 H. megalomagna Chaverri et al., 2004 2004 T. brevicompactum Kraus et al., 2004 2004 Psychrophila H. psychrophila Chaverri et al., 2004 2004 H. megacitrina Chaverri et al., 2004 2004 “ Lone lineages ” T. spirale Kullnig-Gradinger et al., 2002 2002 T. helicum Bissett et al., 2003 2003 H. gelatinosa Chaverri et al., 2004 2004 H. chromosperma Chaverri et al., 2004 2004 H. sulawensis Chaverri et al., 2004 2004 H. nigrovirens Chaverri et al., 2004 2004 H. phyllostachidis Chaverri et al., 2004 2004 Taxon Reference Hypocrea albocornea Doi, 1972 1972 Hypocrea centristerilis Doi, 1972 1972 Hypocrea clusiae Chaverri et al., 2004 2004 Hypocrea cornea Chaverri et al., 2004 2004 Phylogenetic studies of these 88 species showed that Trichoderma and Hypocrea form a single holomorph genus, within which two major clades can be distinguished (Fig. 2 2 , Table 3 3 ): one, which contains all the taxa described in section Longibrachiatum (Samuels et al., 1998 1998 ), T. effusum and T. sinensis (Bissett et al., 2003 2003 ), Trichoderma sp. MA 3239 (Wuczskowski et al., 2003 2003 ), H. cerebriformis and H. poronoidea ( 3 3 Kubicek, unpublished data). H. patella forms a stable basal branch to the Longibrachiatum clade. At the moment it is unclear whether H. peltata should be included in section Longibrachiatum or forms a sister clade. For the sake of simplicity we include it in this section. The second clade forms several well supported subclades: one, leading to members of section Trichoderma including “ Pachybasium A” ( T. viride / H. rufa , T. koningii , T. atroviride , T. ovalipsorum , H. neorufa , H. stilbohypoxyli , T. erinaceum , T. asperellum , T. hamatum , T. pubescens and T. strigosum ), and which also contains H. pezizoides , H. avellanea (Chaverri et al., 2003b 2003b ), and T. flavoconidia (Druzhinina et al., 2004a 2004a ). The branch leading to this clade is frequently accompanied, but usually not strongly supported, by two sister clades: the Pachybasioides clade and the Hypocreanum clade. The former contains H. pachybasioides / T. polysporum (= T. croceum ), H. pilulifera , H. parapilulifera , H. stellata , H. minutispora / T. minutisporum and H. laciwombatensis (Lu and Samuels, 2004 2004 ), whereas the latter includes H. citrina , H. lactea , H. sulphurea and H. pulvinata (Chaverri et al., 2004 2004 ; 4 4 Overton, personal communication). The other, large clade contains one weakly supported branch, but otherwise remains essentially unresolved by all genes or gene fragments used so far (Kullnig-Gradinger et al., 2002 2002 ; Chaverri et al., 2003a 2003a ; 2003b 2003b ; 2004 2004 ). This weakly supported branch leads to H. aureoviridis / T. aureoviride and a group of Hypocrea spp. described recently ( H. aureoviridis var. macrospora , H. candida , H. cremea , H. surrotunda , H. sinuosa , H. chlorospora , H. thelephoricola , H. costaricensis , H. thailandica and H. virecentiflava ) with very similar morphological and genetic characters. Apart from this clade, several terminal subclades can be identifed which contain closely related species, but whose phylogenetic relationship remains unclear: H. lixii / catoptron (containing H. lixii / T. harzianum , T. aggressivum , T. tomentosum , T. cerinum , T. velutinum , T. sp. DAOM 172928, H. tawa , H. atrogelatinosa , H. ceracea , H. cinnamomea , H. straminea and H. catoptron ); H. virens ( H. virens / T. virens , T. flavofuscum , T. crassum ); H. semiorbis ( H. semiorbis , T. oblongisporum , T. fertile ); H. strictipilosa ( H. strictipilosa / T. strictipile , H. aureoviridis var. macrospora , H. cuneispora , T. longipile , T. fasciculatum (synonymized with T. strictipile ); H. ceramica ( H. ceramica , H. estonica ); and H. stromatica ( H. stromatica / T. stromaticum , T. rossicum , T. sp. PPRI 3559). The presence of species of section Hypocreanum as a subclade in clade “ Pachybasium B” deserves some comments: Samuels ( 1996 1996 ) hypothesized that the anamorphs of species of Hypocrea with effused stromata having colourless conidia on irregularly verticillate conidiophores, which Bissett ( 1991a 1991a ) placed in Trichoderma sect. Hypocreanum may be synanamorphs or spermatial states, and therefore inappropriately placed in Trichoderma . Because the anamorphs had not been found in nature, Gams and Bisset ( 1998 1998 ) omitted sect. Hypocreanum from their treatment of Trichoderma . However, phylogenetic data proved that the respective taxa are clearly members of the genus. Kullnig-Gradinger et al.( 2002 2002 ) postulated that the ability to form the Trichoderma -like anamorph may have been lost during evolution of these species. A phylogeny based on 28S-rDNA sequence analysis showed that genera having Verticillium -like anamorphs ( Aphysiostroma , Podocrea , Arachnocrea ) occur in a stable basal position to the genus Trichoderma implying that Trichoderma evolved from them. We thus favour the interpretation that the ability to form the Trichoderma -like anamorph morphology, which may have supported the switch from fungicolous to saprophytic habitats, was subsequently lost in some evolutionary lines. Other Hypocrea species with Verticillium -anamorph morphology such as H. tawa or H. hunua occur nested in other clades (Table 1 1 ) (Kullnig-Gradinger et al., 2002 2002 ), indicating that the loss of genes (or their expression) required form the Trichoderma -like anamorph occured several times during the evolution of the genus. Phylogenetic analysis revealed that three other species ( H. lutea / H. melanomagna / T. brevicompactum ) also cluster together, but their association with any of the large clades is unclear and dependent on the genes and species used ( 1 1 Kubicek, unpublished data). Besides these, there are still a number of species for which no close neighbor is known, and for which the phylogenetic position within Pachybasium B could so far not be identified: H. psychrophila / H. megacitrina , which are found in the vicinity of the Hypocreanum clade; T. spirale , which has phylogenetic affinity to T. virens and T. harzianum ; T. helcum , which has a slight affinity to T. spirale ; and H. gelatinosa , H. chromosperma , H. sulawensis , H. nigrovirens and H. phyllostachidis , which all occur as Lone Lineages of unresolved Pachybasium B. WHAT NEXT? 1 The current information, as summarized above, places Trichoderma among the fungal genera most thoroughly investigated taxonomically today, in view of the fact that at least two but on average more gene sequences are known for every recognized species. While the 88 species recently characterized by molecular methods are phylogenetically well supported, their evolution and phylogenetic relationship has so far been more difficult to resolve. This may either reflect the lack of known ancestors (as with most other fungi), or the occurrence of high selection pressure during the evolution of these clades, consequently leading to dichotomous trees. However, we must emphasize that these 88 species are still probably only a minor fraction of the existing number of taxa. The CABI online database of fungal names ( http://www.indexfungorum.org http://www.indexfungorum.org ) lists 401 Hypocrea spp., and while several of them may be redundant (i.e. synonyms of other described species) or not correctly placed in the genus (e.g. H. pallida ), this may be compensated by others yet to be described. One should bear in mind that many of the currently known Hypocrea spp. had been isolated and described by Yoshimichi Doi (see Samuels, 1996 1996 ) from sampling in Japan, the Western Pacific and South America. In our lab, Walter Jaklitsch has recently initiated a study on the biodiversity of Hypocrea spp. in Central Europe, and his preliminary data indicate the presence of at least 10–15 new undescribed taxa in the samples from the first 18 months. Given the comparatively small area investigated, and the fact that huge areas like Africa or Central Asia have not been studied at all, we expect that 400 will be even too low. The number of Trichoderma spp. may remain lower, but also here a further rise can be anticipated. From our own study on the biodiversity of Trichoderma , at least 10 putative new ‘phylogenetic spp.’ are currently in the pipeline, and will be described in the near future. In addition, screening of so far neglected geographic areas will likely show new taxa (Kullnig et al., 2000 2000 ; Kubicek et al., 2003 2003 ; Wuczskowski et al., 2003 2003 ), and several such investigations have recently been completed or are being undertaken (e.g. New Zealand, S.L. Dodd and coworkers; Iran, D. Zafari; Sardinia, Q. Migheli and I. Druzhinina; China, T. Xu; Rwanda, J. Bissett and I. Druzhinina). Still, areas like most of the African continent and the Pacific have not been investigated. As emphasized above, most of the phylogenetic analyses are incomplete, due to the limited suitability of the gene sequences used so far (Table 1 1 ). To improve this situation, novel gene sequences are needed. 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The authors are grateful to Walter Jaklitsch from their laboratory and to various colleagues for sharing unpublished information. 10.1631/jzus.2005.B0100 Project supported by FWF-grants P-12748-MOB and P-16601 to C.P.K., Austria 2004-09-08 2004-10-07 2005-01-20 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 6B