Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea

Background: Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi. Their main function is to confer hydrophobicity to fungal surfaces in contact with air or during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or themselves resulting in morphogenetic signals. Based on their hydropathy patterns and solubility characteristics, hydrophobins are divided into two classes ( I and II), the latter being found only in ascomycetes. Results: We have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/ Hypocrea, using three draft sequenced genomes ( H. jecorina = T. reesei, H. atroviridis = T. atroviride; H. virens = T. virens) an additional 14,000 ESTs from six other Trichoderma spp. ( T. asperellum, H. lixii = T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, respectively. Ten is the highest number found in any ascomycete so far. All the hydrophobins we examined had the conserved four beta- strands/ one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these hydrophobins ( HFBs) contained an extended N- terminus rich in either proline and aspartate, or glycine- asparagine. Phylogenetic analysis reveals a mosaic of terminal clades containing duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non- synonymous nucleotide substitutions provides evidence for strong purifying selection ( K-S/ K-a >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins ( 2 - 4) from each species, and most were from Sordariomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other Sordariomycetes occurred in shared clades. Conclusion: Our study shows that the genus Trichoderma/ Hypocrea has a proliferated arsenal of class II hydrophobins which arose by birth- and- death evolution followed by purifying selection.