Joly et al. BMC Genomics 2010, 11:422 http://www.biomedcentral.com/1471-2164/11/422 (Pucciniomycotina; Microbotryomycetes), a free-living saprobic yeast that is phylogenetically close to rusts but differs in its saprophytic lifestyle; and Ustilago (Ustilagi- nomycotina; Ustilaginomycetes), the corn smut, which is phylogenetically more distant from Melampsora but is also a plant pathogen. In a few cases, sequences within HGGs matched different paralogous sequences in one of the comparators (usually Puccinia). Consensus sequences were thus created from sequences having identical BLASTX hits in the three comparators, allow- ing multiple consensus sequences for a single HGG. SimiTri was used to plot 417 consensus sequences from 369 Melampsora HGGs against related species’ gene models (Figure 4). For each HGG, most Melampsora sequences grouped slightly towards Puccinia, its closest relative (Figure 4). Again, the difference between non- secreted and secreted HGGs was striking with 69.8% of non-secreted HGGs exhibiting homologous sequences in the three comparators, while only 33.0% of secreted HGGs had such homologues. Even tough the percentage of secreted HGGs with no hit in the three other species (16.0%) was almost four times the percentage of unique non-secreted HGGs (4.2%), this was not sufficient to explain this difference. The percentage of HGGs unique to Pucciniales was twice higher for secreted HGGs (29.2% compared with 13.8%). Similar secreted/non- secreted HGGs ratios were observed for the percentage of secreted HGGs absent from Ustilago (11.3% Page 8 of 16 compared with 5.1%) or from Sporobolomyces (9.4% compared with 4.5%). While HGGs absent from Ustilago but found in both Puccinia and Sporobolomyces could have been eliminated following the Ustilaginomycotina/ Pucciniomycotina radiation, HGGs absent from the free- living Sporobolomyces but found in the more distant Ustilago could represent genes involved in host-patho- gen interactions and/or biotrophic lifestyles. Similarly, recent observations on the whole genome sequences of Ustilago maydis and Laccaria bicolor suggested that the inventory of certain enzymes underwent massive gene loss as a result of its adaptation to a biotrophic (U. may- dis) or symbiotic lifestyle (L. bicolor) [40,62]. Molecular genetic analysis of plant-pathogen interac- tions includes many layers of antagonistic coevolution. Investigation of molecular evolution at these various levels usually reveals diversifying selection and the selec- tive maintenance of variation, resulting in positive selec- tion at the genomic interfaces of escalating attack and defense systems [63]. Following this idea, one of the most reliable indicators of positive selection at the mole- cular level is a higher non-synonymous nucleotide sub- stitution rate (dN) than the synonymous nucleotide substitution rate (dS) between two protein-coding DNA sequences (ratio ω = dN/dS > 1) [64]. Based on this cri- terion, statistical methods, such as the approximate (counting) and the maximum likelihood (ML) methods, have been developed [65-67]. We calculated the dN and Figure 4 Similarity of Melampsora homologous gene groups (HGGs) to the proteomes of other basidiomycetes. Similarity of Melampsora A) non-secreted and B) secreted homologous gene groups (HGGs) to the proteomes of Puccinia graminis f. sp. tritici, Ustilago maydis and Sporobolomyces roseus. For each of the 369 Melampsora HGGs, a BLASTX search was performed against the proteomes of the other three species and SimiTri was used to plot the sequence similarity relationships between 417 consensus sequences derived from these HGGs and related species. Each tile in the graphics represents a unique consensus sequence and its relative position is computed from the raw BLAST scores derived above (with a cutoff of > 40). Hence, each tile’s position indicates its degree of sequence similarity to each of the three selected databases. Sequences showing similarity to only one database are not shown. Sequences showing sequence similarity to only two databases appear on the lines joining the two databases. The position of positively selected HGGs is indicated by arrows. Tiles are colored by their highest BLASTX score for each of the databases: red ≥ 300; yellow ≥ 200; green ≥ 150; blue ≥ 100; and purple < 100.