**2.1.1 Cryptic species or phylogenetic species**

Complementary methodologies have been applied to differentiate and characterize *cryptic species* or *phylogenetic species* in a fungi, correlating morphological, biogeographic, biochemical, ecological and, most recently, phylogenetic traits (e.g. refs). Applying the PSC proposed by Taylor et al. (2000), Chaverri et al. (2003) examined the internal transcribed spacer regions of rDNA (ITS1 and ITS2), the large intron of the transcription elongation factor 1-α (*tef1α)*, and short fragments of the actin (*act*1) and calmodulin (*cal1*) exon sequences in *H. lixii/T. harzianum*, to determine seven phylogenetic lineages in *T. harzianum*. However, they declined to recognize the lineages as phylogenetic species since they could not be reliably distinguished morphologically. Similarly applying GCPSR, Samuels et al. (2006) found that the *T. koningii* species aggregate includes three well-separated main lineages defined by phenotypic characters, and further recognized twelve taxonomic species and one variety within the three lineages: (1) T. *koningii, T. ovalisporum* and the new taxa *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 species *T. stilbohypoxyli*. Druzhinina et al (2010b) recently revisited the genetic diversity in *T. harzianum,* examining three unlinked gene loci for 93 strains isolated worldwide. Their data illustrated clearly the complex history of speciation in the *H. lixii*-*T. harzianum* species group, rejecting the anamorph/teleomorph combination in favour of separate species status for *H. lixii* and *T. harzianum*, with the phylogenetic position of most isolates not resolved and attributed to a diverse network of recombining strains lacking strict genetic borders. In a similar study employing multiple gene phylogenies and multiple methods of phenotype profiling, Druzhina et al. (2010a) demonstrated that isolates previously identified as *H. jecorina* comprised four phylogenetic species, including *H. jecorina*/*T. reesei sensu stricto* containing most of the teleomorph isolates and the wild-type strain of *T. reesei* (QM6a) that has subsequently been genetically modified and employed in biofuel production. Conversely, all of the strains isolated as anamorphs from soil were referred to *T. parareesei.* It becomes clear from these studies that

Biodiversity of *Trichoderma* in Neotropics 307

they developed TrichoMARK to detect one or more sequence fragments of these genes as phylogenetic markers. The latter program is capable of distinguishing the five groups of species haplotypes that have identical ITS 1 and 2 sequences, *viz*.: *T. tomentosum / T. cerinum, T. longipile / T. crassum, T. koningii / T. ovalisporum / T. muroiana, H. lutea / H. melanomagna,*  and *T. longibrachiatum / H. orientalis / H. cerebriformis.* In the case of *H. lixii / T. harzianum*, the program detects intraspecific differences accurately in this cluster, which contains several putative phylogenetic species. The ISTH website (www.isth.info) also provides the primer

These may be based on the profiles of particular enzyme classes such as chitinases or cellulases, although other metabolic profiling techniques have been developed to validate new species which can also potentially provide data on the ecological roles of species (e.g. Kubicek et al. 2003, Hoyos-Carvajal et al., 2009a). The latter studies employed Biolog FF ® microplates (Biolog Inc., Hayward CA) comprising 96 cells containing different carbon sources and redox reagents sensitive to the activity of the enzyme succinate dehydrogenase in the citric acid cycle. Photometry at 590 nm and 750 nm provide quantitative measurements of assimilation and growth (measuring mycelial density) and respiratory activity on the different substrates. The metabolic profiles obtained may be characteristic of species, and the assimilation of specific substrates may allow hypotheses on the ecological role of species. For example, the assimilation of polyols such as maltitol and adonitol could

Broad studies on the taxonomy and biodiversity of *Trichoderma* have been carried out in North America and some regions of Europe (*e.g*. Bissett 1991a,b,c, 1992), where the distribution of species is now reasonably well known, particularly for specific taxa or groups (Lieckefeldt et al., 2001). Some regions have been studied in detail, e.g. Wuczkowski et al., 2003, investigated the genetic diversity of a European river-floodplain landscape near Vienna, and Migheli et al., 2009 studied the biodiversity of *Trichoderma* in Sardinia, a Mediterranean hot spot of biodiversity, analyzing the influence of abiotic factors on the distribution of species *Trichoderma.* In the latter study, 482 strains of *Hypocrea/Trichoderma* were identified from undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes), with the finding that most of the strains were pan-European and/or pan-global species. Meinke et al., 2010 described the *Trichoderma* communities in rhizosphere of four varieties and transgenic lines of potato in Germany. They observed a heterogeneous distribution and varying diversity of *Trichoderma* dependent on soil characteristics, climate and

Studies in previously uninvestigated regions or habitats have frequently led to the discovery of new taxa. Kullning et al. (2000) examined 76 isolates from Russia, Nepal and North India, reporting seven species (*T. asperellum, T.atroviride, T. ghanense, T. hamatum, T. harzianum, T. virens* and *T. oblongisporum*) and five new taxa. They also found T. *harzianum* the most genetically diverse speices, with the *T. harzianum* complex representing the majority of isolates. A similar study was conducted by Kubicek et al. (2003) in Southeast Asia, where they reported *T. asperellum, T.atroviride, T. ghanense, T. hamatum, T. harzianum, T. koningii, T. spirale, T. virens, T. viride* and *H. jecorina* (anam: *T. reesei*), along with seven new species

sequences and protocols necessary for sequencing the genes used for identification.

indicate activity of dehydrogenases relevant to survive droughty conditions.

**3.** *Trichoderma:* **distribution and biogeography** 

management practices, in this case not related to the crop variety.

**2.2.3 Metabolic tests** 

phylogenetic structure within these complex species groupings must be taken into account in selecting potential isolates to use in industrial applications. For example, although the name "*T. harzianum*" has been uniformly applied to the biological control agent in the past, there is now increasing evidence that several, genetically diverse species are used in biocontrol (Druzhinina and Kubicek, 2005).
