Molecular evidence for multiple host-specific strains in the genus Rhinosporidium

Abstract

The taxonomic relationship of Rhinosporidium seeberi with other organisms remained controversial for over a century. Recently, molecular studies have shown R. seeberi to be a protistal microbe in the newly described class Mesomycetozoea at the animal-fungal boundary. Phylogenetic analyses of R. seeberi using 18S small-subunit (SSU) rRNA genes from several hosts suggested Rhinosporidium as a monotypic genus. To test this hypothesis, the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 from eight humans, two swans, and a dog with rhinosporidiosis were sequenced. The ITS regions were amplified by PCR using a primer designed from a unique region of R. seeberi's 18S SSU rRNA genes in combination with the ITS4 universal primer. In addition, the universal ITS4 and ITS5 primers were also used. R. seeberi's ITS sequences showed differences in the numbers of nucleotides among strains. For instance, the eight human ITS sequences were uniformly similar with only a few mismatches and approximately 1,060 bp long. In contrast, sequences from one of the swans and the dog were 1,356 bp and approximately 1,147 bp long, respectively. Clustal analysis of all of the ITS sequences showed multiple 50- to 60-bp gaps and several mismatches among them. Parsimony analysis placed the Rhinosporidium ITS sequences in three well-supported sister groups according to the hosts' identities. This analysis strongly suggests that the genus Rhinosporidium may possess multiple host-specific strains. No correlation was found between this finding and the phenotypic features of R. seeberi in the studied samples.

FIG. 1.

Phylogenetic relationship between the ITS1, 5.8S, and ITS2 sequences of eight human strains of R. seeberi from India (four strains; I1 to I4) and Sri Lanka (four strains; SL1 to SL4), one strain of R. seeberi from a dog (D1), and two strains from swans (S1 and S2). Multiple hit correction and a 1,000-bootstrap-resampled data set were used to assess branch support. The ITS sequences of R. seeberi isolated from humans are all clustered in a solid group. In this group, the strains from India and Sri Lanka grouped in two poorly supported subgroups. In contrast, the two R. seeberi strains from swans formed a strongly supported sister group to the human strains (100% bootstrap) and with the inclusion of R. seeberi from a dog, a well-supported sister group to the dog (87% bootstrap). Numbers above the branches are percentages of bootstrap-resampled data sets obtained by neighbor-joining. The ITS sequences of S. destruens were used as out-group. The scale bar represents evolutionary distance in substitutions per nucleotide.