Table 1.
Characteristics of the studied Brazilian regions.
Fig 1.
Phylogenetic analysis of Brazilian Cryptococcus gattii VGII isolates inferred by maximum likelihood (ML), neighbour-joining (NJ), and maximum parsimony (MP) methods using the concatenated data set of the seven MLST genes.
All Brazilian Sequence types (81) from this study and 42 additional Sequence types representing all previously published VGII sequence types maintained in the MLST database (mlst.mycologylab.org) were included in the analysis. The tree with the highest log likelihood (-9194.7663) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 0.0500]). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 6.6983% sites). The tree is drawn to scale, with branch lengths measuring the number of substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 4,172 positions in the final dataset. Numbers at each branch indicate bootstrap values >50% based on 1,000 replicates by each of the three (ML/NJ/MP) algorithms which presented similar topologies. The taxa nomenclature includes the sequence type number (ST), Mating Type (a or alpha), country of isolation and for those isolates from Brazil the region of isolation [N = North, NE = Northeast, CW = Central-West, and SE = Southeast], and source (E = environmental; all others are clinical). All country abbreviations are designated according to the alpha-2 code of ISO 3166–1. AU: Australia, BR: Brazil, CA: Canada, CN: China, CO: Colombia, CD: Democratic Republic of the Congo, CH: Switzerland, DK: Denmark, DE: Germany, ES: Spain, FR: France, GF: French Guiana, GR: Greece, KR: Republic of Korea, SN: Senegal, TH: Thailand, US: United States of America, UY: Uruguay.
Fig 2.
Minimum spanning tree using the goeBURST algorithm.
(A) Minimum spanning tree using the goeBURST algorithm showing the high diversity identified among the 145 Brazilian Cryptococcus gattii VGII isolates and nine Brazilian sequence types (ST) according to the state where they were recovered. Dividing the country in two macro regions such as North (composing of the States of Amazonas, Roraima, Pará, Piauí, and Bahia) and South (States of Mato Grosso do Sul, São Paulo, and Rio de Janeiro) three main groups can be identified: 1) isolates mainly recovered from the South, representing all isolates derived from the ST301; 2) isolates mainly recovered from the North, representing those originated from the ST7 (VGIIb), and 3) the mixed group, which contains isolates derived from ST281. The main clonal complexes (CC) in each of these groups are: CC40, CC5, and CC20. The ancestors of the CC is highlighted by a black line. (B) Minimum spanning tree using the goeBURST algorithm of the isolates presented in (A), and their comparison with 42 STs identified in different countries previously published. All country abbreviations are designated according to the alpha-2 code of ISO 3166–1. In both figures each circle represents a unique ST, and the circumference is proportional to the number of isolates within each ST. Solid, grey and dashed branches represent at least one, two to three, and more than four or five differences, respectively. All STs are different VGII lineages, only the three PNW outbreak genotypes are labelled specifically as VGIIa (ST20), VGIIb (ST7), and VGIIc (ST6).
Fig 3.
Number of populations using in the STRUCTURE analysis calculated according to [35].
The results presenting in (A) show three populations in the Brazilian Cryptococcus gattii VGII and in (B) show four populations in the STs identified in different countries previously published and in Brazil.
Fig 4.
Population structure analysis inferred using multilocus sequence genotypes of Cryptococcus gattii isolates recovered from Brazil and using K = 3 (A), and comparison of these isolates with isolates recovered from different continents using K = 4 (B).
Clusters of individuals based on prior-defined populations are referred according to the Brazilian States and/or Continent. Each vertical line represents one of the isolates included and each color (red, dark blue, dark green, and yellow) represents the most likely ancestry of each isolate from one of the three populations (A) or four populations (B). Individuals with multiple colours have admixed genotypes from the prior-defined populations. One clone per region was included, thus Figure A contains 100 isolates while Figure B contain 125 isolates. The taxa nomenclature includes AM: Amazonas, PA: Pará, PI: Piauí, RR: Roraima, BA: Bahia, ES: Espírito Santo, RJ: Rio de Janeiro, MS: Mato Grosso do Sul, AS: Asia, AU: Australia, AF: Africa, SA: South America, NA: North America, EU: Europe.
Fig 5.
Linkage disequilibrium heat maps between polymorphic sites for all studied MLST loci (CAP59, GPD1, PLB1, SOD1, IGS1, LAC1 and URA5).
Pairwise D’ metricises are represented by heat colours (Colour Key). Recombination amongst Brazilian VGII strains has been shown, as evidenced amongst four sites (0.7%) in the locus CAP59 and six sites (0.8%) in the locus SOD1 (Fisher’s Exact Test P-value ≤ 0.05).
Table 2.
Characteristics of the MLST Loci studied in 145 Brazilian C. gattii VGII isolates.
Fig 6.
Split decomposition analysis using the Neighbor-net algorithm of the each of seven MLST genes evidencing the diversity and branching ambiguities attributable to recombination events in the CAP59 and SOD1.
The phi test result implemented in the software SplitsTree is presented next to each allele.