Modern taxonomy has developed towards the establishment of global authoritative lists of species that assume the standardized principles of species recognition, at least in a given taxonomic group. However, in fungi, species delimitation is frequently subjective because it depends on the choice of a species concept and the criteria selected by a taxonomist. Contrary to it, identification of fungal species is expected to be accurate and precise because it should predict the properties that are required for applications or that are relevant in pathology. The industrial and plant-beneficial fungi from the genus Trichoderma (Hypocreales) offer a suitable model to address this collision between species delimitation and species identification. A few decades ago, Trichoderma diversity was limited to a few dozen species. The introduction of molecular evolutionary methods resulted in the exponential expansion of Trichoderma taxonomy, with up to 50 new species recognized per year. Here, we have reviewed the genus-wide taxonomy of Trichoderma and compiled a complete inventory of all Trichoderma species and DNA barcoding material deposited in public databases (the inventory is available at the website of the International Subcommission on Taxonomy of Trichodermawww.trichoderma.info). Among the 375 species with valid names as of July 2020, 361 (96%) have been cultivated in vitro and DNA barcoded. Thus, we have developed a protocol for molecular identification of Trichoderma that requires analysis of the three DNA barcodes (ITS, tef1, and rpb2), and it is supported by online tools that are available on www.trichokey.info. We then used all the whole-genome sequenced (WGS) Trichoderma strains that are available in public databases to provide versatile practical examples of molecular identification, reveal shortcomings, and discuss possible ambiguities. Based on the Trichoderma example, this study shows why the identification of a fungal species is an intricate and laborious task that requires a background in mycology, molecular biological skills, training in molecular evolutionary analysis, and knowledge of taxonomic literature. We provide an in-depth discussion of species concepts that are applied in Trichoderma taxonomy, and conclude that these fungi are particularly suitable for the implementation of a polyphasic approach that was first introduced in Trichoderma taxonomy by John Bissett (1948–2020), whose work inspired the current study. We also propose a regulatory and unifying role of international commissions on the taxonomy of particular fungal groups. An important outcome of this work is the demonstration of an urgent need for cooperation between Trichoderma researchers to get prepared to the efficient use of the upcoming wave of Trichoderma genomic data.

Fungal evolutionary biology is impeded by the scarcity of fossils, irregular life cycles, immortality, and frequent asexual reproduction. Simple and diminutive bodies of fungi develop inside a substrate and have exceptional metabolic and ecological plasticity, which hinders species delimitation. However, the unique fungal traits can shed light on evolutionary forces that shape the environmental adaptations of these taxa. Higher filamentous fungi that disperse through aerial spores produce amphiphilic and highly surface-active proteins called hydrophobins (HFBs), which coat spores and mediate environmental interactions. We exploited a library of HFB-deficient mutants for two cryptic species of mycoparasitic and saprotrophic fungi from the genus Trichoderma (Hypocreales) and estimated fungal development, reproductive potential, and stress resistance. HFB4 and HFB10 were found to be relevant for Trichoderma fitness because they could impact the spore-mediated dispersal processes and control other fitness traits. An analysis in silico revealed purifying selection for all cases except for HFB4 from T. harzianum, which evolved under strong positive selection pressure. Interestingly, the deletion of the hfb4 gene in T. harzianum considerably increased its fitness-related traits. Conversely, the deletion of hfb4 in T. guizhouense led to the characteristic phenotypes associated with relatively low fitness. The net contribution of the hfb4 gene to fitness was found to result from evolutionary tradeoffs between individual traits. Our analysis of HFB-dependent fitness traits has provided an evolutionary snapshot of the selective pressures and speciation process in closely related fungal species.

Cerato-platanins (CPs) form a family of fungal small secreted cysteine-rich proteins (SSCPs) and are of particular interest not only because of their surface activity but also their abundant secretion by fungi. We performed an evolutionary analysis of 283 CPs from 157 fungal genomes with the focus on the environmental opportunistic plant-beneficial and mycoparasitic fungus Trichoderma. Our results revealed a long evolutionary history of CPs in Dikarya fungi that have undergone several events of lateral gene transfer and gene duplication. Three genes were maintained in the core genome of Trichoderma, while some species have up to four CP-encoding genes. All Trichoderma CPs evolve under stabilizing natural selection pressure. The functional genomic analysis of CPs in Trichoderma guizhouense and Trichoderma harzianum revealed that only epl1 is active at all stages of development but that it plays a minor role in interactions with other fungi and bacteria. The deletion of this gene results in increased colonization of tomato roots by Trichoderma spp. Similarly, biochemical tests of EPL1 heterologously produced by Pichia pastoris support the claims described above. Based on the results obtained, we conclude that the function of CPs is probably linked to their surfactant properties and the ability to modify the hyphosphere of submerged mycelia and, thus, facilitate the nutritional versatility of fungi. The effector-like functions do not sufficiently describe the diversity and evolution of these proteins in fungi, as they are also maintained, duplicated, or laterally transferred in the genomes of nonherbivore fungi. IMPORTANCE Cerato-platanins (CPs) are surface-active small proteins abundantly secreted by filamentous fungi. Consequently, immune systems of plants and other organisms recognize CPs and activate defense mechanisms. Some CPs are toxic to plants and act as virulence factors in plant-pathogenic fungi. Our analysis, however, demonstrates that the interactions with plants do not explain the origin and evolution of CPs in the fungal kingdom. We revealed a long evolutionary history of CPs with multiple cases of gene duplication and events of interfungal lateral gene transfers. In the mycoparasitic Trichoderma spp., CPs evolve under stabilizing natural selection and hamper the colonization of roots. We propose that the ability to modify the hydrophobicity of the fungal hyphosphere is a key to unlock the evolutionary and functional paradox of these proteins.