Environmental Mapping of <i>Paracoccidioides</i> spp. in Brazil Reveals New Clues into Genetic Diversity, Biogeography and Wild Host Association - Fig 3

<p><b>A)</b> Molecular Phylogenetic Analysis by of ITS locus revealed by Maximum Likelihood and Neighbor Joining methods, using the Jukes-Cantor model parameters with range correction. Replication percentages on the tree are grouped in the bootstrap test (1000 replicates) and shown next to the branches. The sequences related to environmental samples are identified by acronyms SO_GO (Soil of Goiás) and AR_GO (Aerosol Goiás), AR_MG (Aerosol Minas Gerais) and SO_RO (Soil of Rondônia). <b>B)</b> Median-joining network showing the unique haplotypes of the Soil Clades I and II. Circles are proportional to haplotype frequency and numbers of mutations are represented by black dots. Red circles represent hypothetical missing intermediates (median vectors).</p

Detection of <i>Paracoccidioides</i> spp. by Nested PCR and <i>in situ</i> hybridization techniques, both in soil and aerosol samples in the states of Goiás, Minas Gerais and Rondônia, Brazil.

<p>Detection of <i>Paracoccidioides</i> spp. by Nested PCR and <i>in situ</i> hybridization techniques, both in soil and aerosol samples in the states of Goiás, Minas Gerais and Rondônia, Brazil.</p

Brazilian sites and biomes where environmental detection (from soil, aerosol and armadillos) of <i>Paracoccidioides</i> spp. has been carried out.

<p>The collection areas encompass the states of Minas Gerais (MG), Goiás (GO) and Rondônia (RO) states, to São Paulo (SP) and Pará (PA). Circles outside of the map indicate the percentage of positivity in each area for <i>P</i>. <i>brasiliensis</i> (green) and <i>P</i>. <i>lutzii</i> (red) by <i>in situ</i> hybridization for aerosol samples. The circles outside of the map in black and grey indicate the percentage of positivity in each area for <i>P</i>. <i>brasiliensis</i> and <i>P</i>. <i>lutzii</i> by Nested PCR for soil and aerosol samples. The white circle indicates the negative detection in the evaluated areas (N.D. = Not detected). The armadillos indicate the number of collected animals and the positivity for isolation of <i>Paracoccidioides brasiliensis</i> (Pb) in each location of our study (in RO, GO and MG) and from previous works in SP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004606#pntd.0004606.ref016" target="_blank">16</a>] and PA [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004606#pntd.0004606.ref033" target="_blank">33</a>]. The triangles (green), squares (yellow) and circles (red) show the distribution of the clinical isolates for cryptic species (S1, PS2 and <i>P</i>. <i>lutzii</i>) in previous studies [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004606#pntd.0004606.ref014" target="_blank">14</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004606#pntd.0004606.ref031" target="_blank">31</a>].</p

Collection areas of environmental samples (soil, aerosol and animals) in Brazil.

<p>The evaluated states (RO, MG and GO) are highlighted in different colors and the municipalities of sampling in different traces indicated with arrows.</p

Fungal structures (400X) visualized by FISH and TSA-FISH techniques for aerosol samples and controls.

<p><b>A:</b> aerosol samples from Goiás with DAPI. <b>B:</b> aerosol samples from Goiás with <i>P</i>. <i>brasiliensis</i> probe. <b>C:</b> aerosol sample from Rondônia, with DAPI. <b>D:</b> aerosol samples from Rondônia with <i>P</i>. <i>brasiliensis</i> probes. <b>E:</b> aerosol sample from Goiás with DAPI. <b>F:</b> aerosol sample from Goiás with <i>P</i>. <i>lutzii</i> probe. <b>G and I:</b> <i>Histoplasma capsulatum</i> with DAPI. <b>H:</b> <i>Histoplasma capsulatum</i> with <i>P</i>. <i>brasiliensis</i> probe (specificity control). <b>J:</b> <i>Histoplasma capsulatum</i> with <i>P</i>. <i>lutzii</i> probe (specificity control). <b>K:</b> isolate Pb01 (<i>P</i>. <i>lutzii</i>) with <i>P</i>. <i>lutzii</i> probe (positive control). <b>L:</b> isolate T16B1 (<i>P</i>. <i>brasiliensis</i>) with <i>P</i>. <i>brasiliensis</i> probe. The probe for <i>P</i>. <i>brasiliensis</i> is conjugated with Horseradish Peroxidase/HRP and <i>P</i>. <i>lutzii</i> probe is labeled with Texas Red/TXR, and genetic material is labeled with DAPI.</p

Georeferenced sites for the field work (soil, aerosols and armadillo collection).

<p>Georeferenced sites for the field work (soil, aerosols and armadillo collection).</p

The ITS region with the location of oligo probes designed for the detection and differentiation of <i>P</i>. <i>brasiliensis</i> and <i>P</i>. <i>lutzii</i>, labeled with HRP and Texas Red, respectively.

<p>The ITS region with the location of oligo probes designed for the detection and differentiation of <i>P</i>. <i>brasiliensis</i> and <i>P</i>. <i>lutzii</i>, labeled with HRP and Texas Red, respectively.</p

Armadillos (<i>Dasypus novemcinctus</i>), and their respective organ fragments, evaluated in the Brazilian states of Goiás, Minas Gerais and Rondônia.

<p>Armadillos (<i>Dasypus novemcinctus</i>), and their respective organ fragments, evaluated in the Brazilian states of Goiás, Minas Gerais and Rondônia.</p

Conidia production by isolates belonging to the four species of the <i>Paracoccidioides</i> genus.

<p>− 0.</p><p>+ up to 5.</p><p>++ from 5 to 20.</p><p>+++ from 20 to 50.</p><p>++++ >50 conidia per microscopic field.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037694#pone-0037694-t003" target="_blank">Table 3</a>, for the <i>ARF</i> gene), followed by the species found in this haplotype (S1, PS2, PS3 or <i>P. lutzii</i>).Marine incursion events, which occurred while <i>Lacazia</i> and <i>Paracoccidioides</i> genera diverged, are indicated with blue arrows along the time scale axis.</p