Phylogenetic relationships of varieties and geographical groups of the human pathogenic fungus Histoplasma capsulatum Darling

Abstract

The phylogeny of 46 geographically diverse Histoplasma capsulatum isolates representing the three varieties capsulatum, duboisii, and farciminosum was evaluated using partial DNA sequences of four protein coding genes. Parsimony and distance analysis of the separate genes were generally congruent and analysis of the combined data identified six clades: (i) class 1 North American H. capsulatum var. capsulatum, (ii) class 2 North American H. capsulatum var. capsulatum, (iii) Central American H. capsulatum var. capsulatum, (iv) South American H. capsulatum var. capsulatum group A, (v) South American H. capsulatum var. capsulatum group B, and (vi) H. capsulatum var. duboisii. Although the clades were generally well supported, the relationships among them were not resolved and the nearest outgroups (Blastomyces and Paracoccidioides) were too distant to unequivocally root the H. capsulatum tree. H. capsulatum var. farciminosum was found within the South American H. capsulatum var. capsulatum group A clade. With the exception of the South American H. capsulatum var. capsulatum group A clade, genetic distances within clades were an order of magnitude lower than those between clades, and each clade was supported by a number of shared derived nucleotide substitutions, leading to the conclusion that each clade was genetically isolated from the others. Under a phylogenetic species concept based on possession of multiple shared derived characters, as well as concordance of four gene genealogies, H. capsulatum could be considered to harbor six species instead of three varieties.

FIG. 1

Polymorphic sites in combined data of arf (A), H-anti (B), ole (C), and tub1 (D) loci of H. capsulatum. The second column of each panel shows the name of the multilocus genotype, which was named after the isolate. When more than one isolate had same genotype, the isolate name with the smallest number was used for the genotype. The sequence of H88 is used as the master sequence and only nucleotides that differ from H88 sequence are shown; otherwise, nucleotides are shown as dots. A hyphen (-) denotes a gap. The shaded bars represent the locations of exons.

FIG. 1

Polymorphic sites in combined data of arf (A), H-anti (B), ole (C), and tub1 (D) loci of H. capsulatum. The second column of each panel shows the name of the multilocus genotype, which was named after the isolate. When more than one isolate had same genotype, the isolate name with the smallest number was used for the genotype. The sequence of H88 is used as the master sequence and only nucleotides that differ from H88 sequence are shown; otherwise, nucleotides are shown as dots. A hyphen (-) denotes a gap. The shaded bars represent the locations of exons.

FIG. 2

A single MP tree resulting from analysis of DNA sequences of the 33 multilocus genotypes from each of the four gene regions sequenced. Isolates with an identical genotype are shown together with the representative isolate for each of the 33 genotypes. CI, consistency index; RI, retention index; RC, rescaled consistency index. Numbers below branches represent indices of support based on 500 bootstrap replications of the parsimony procedure. Only values ≥70% are shown. Branch lengths are proportional to the numbers of changes in informative characters between nodes (scale at the lower left). SAm Hcc A, South American H. capsulatum group A; SAm Hcc B, South American H. capsulatum group B; Hc farcimi., H. capsulatum farciminosum. Abbreviations of other groups are listed in Table 1.

FIG. 3

Partition homogeneity test in the total data set comparing the observed summed tree lengths with the distribution of summed tree lengths calculated for 500 randomized data sets.

FIG. 4

(a) NJ tree from analysis of combined data of the four loci. Branch lengths are proportional to distance. (b) Strict consensus of 160 MP trees derived from analysis the same data of the four loci. Numbers below branches represent indices of support based on 500 bootstrap replications of the parsimony procedure; only values ≥70% are shown. Trees were rooted using H. capsulatum var. duboisii as the outgroup taxon based on the morphology of pathogenic yeasts.

FIG. 5

Partition homogeneity test in the South American H. capsulatum var. capsulatum group A comparing the observed summed tree lengths with the distribution of summed tree lengths calculated for 4,000 randomized data sets.