Molecular characterization of pathogenic members of the genus Fonsecaea using multilocus analysis

Abstract

Members of the fungal genus Fonsecaea causing human chromoblastomycosis show substantial geographic structuring. Genetic identity of clinical and environmental strains suggests transmission from plant debris, while the evolutionary processes that have led to spatially separated populations have remained unexplained. Sequences of ITS, BT2, ACT1, Cdc42, Lac and HmgA were analyzed, either by direct sequencing or by cloning. Thirty-seven clinical and environmental Fonsecaea strains from Central and South America, Asia, Africa and Europe were sequenced and possible recombination events were calculated. Phylogenetic trees of Cdc42, Lac and HmgA were statistically supported, but ITS, BT2 and ACT1 trees were not. The Standardized Index of Association (I(A) (S)) did not detect recombination (I(A) (S) = 0.4778), neither did the Phi-test for separate genes. In Fonsecaea nubica non-synonymous mutations causing functional changes were observed in Lac gene, even though no selection pressures were detected with the neutrality test (Tajima D test, p>0.05). Genetic differentiation of populations for each gene showed separation of American, African and Asian populations. Strains of clinical vs. environmental origin showed genetic distances that were comparable or lower than found in geographic differentiation. In conclusion, here we demonstrated clonality of sibling species using multilocus data, geographic structuring of populations, and a low functional and structural selective constraint during evolution of the genus Fonsecaea.

Figure 1. Consensus trees of Fonsecaea based on ITS ribosomal DNA, BT2, ACT1, Cdc42, HmgA and Lac of 37 strains, constructed with MEGA5.0 and 500 bootstrap replicates, CBS 109631 was taken as outgroup.

Figure 2. Clustering of amplified fragment-length polymorphism banding pattern of isolates of Fonsecaea spp. analyzed by using unweighted pair group method with arithmetic mean.

Subclusters showed as in Figure.

Figure 3. Laccase activity assay.

Colored metabolite diameters of tested strains were measured after 7-day culture at 25°C on solid MM medium with 5 mM ABTS. Statistical analysis shown as mean ± standard deviation (A). The plates show the generation of colored metabolite compound (B), F. pedrosoi (CBS 273.66), F. monophora (CBS 117236), F. nubica (CBS 121720).