Genus paracoccidioides: Species recognition and biogeographic aspects

Abstract

Background: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes.

Methodology/principal findings: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11-32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi.

Conclusions/significance: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides.

Figure 1. Haplotype nets generated by ANeCA and NETWORK programs, for Alpha-tubulin, GP43 and ARF genes.

Each haplotype is indicated by a circle, whose colored slices represent geographic distribution, according to the legend on the right side. mv: median vector = nodes and links which connect the nodes. It is a hypothesized ancestral sequence required to connect existing sequences within the network with Maximum Parsimony.

Figure 2. Real Time PCR with TaqMan probes for SNP detection in isolates belonging to the different species from Paracoccidioidesgenus.

Endpoint fluorescence intensity graphics for: SNP3-ARF (G for S1, PS3 and P. lutzii isolates, and A for PS2 isolates), SNP4-GP43, exon 2 (G for S1, PS2 and P. lutzii, and C for PS3) and SNP5-PRP8 intein (A for S1, PS2 and PS3, and G for P. lutzii). Green dots: “heterozygote” additional controls, using two different DNA samples together; black squares: negative controls;“X”: undetermined samples.

Figure 3. Phylogenetic tree generated with the program BEAST, ancestral areas according to Likelihood Analysis of Geographic Range Evolution.

The tree was generated with the program BEAST. The blue bars illustrated the extent of the 95% highest posterior density intervals for each divergence time. The scale is in years. A, B and C are the areas settled for LAGRANGE analysis. Areas in the branch tips represent the current distribution, and areas in the nodes of the tree are the most probable ancestral areas according to LAGRANGE analysis. The main dispersal events are indicated by red arrows. Tip labels designate the name of the haplotype (same names asin Table 3, for the ARF gene), followed by the species found in this haplotype (S1, PS2, PS3 or P. lutzii).Marine incursion events, which occurred while Lacazia and Paracoccidioides genera diverged, are indicated with blue arrows along the time scale axis.

Figure 4. Geographic distribution of Paracoccidioides genus and L. loboi.

Current distribution of Paracoccidioides species S1, PS2, PS3 and P. lutzii, and their sister species L. loboi, in South America (modified from [53]).