Phylogram showing the genetic relationships between the studied <i>Cryptococcus gattii</i> isolates.

<p>Dendrogram based on the maximum likelihood analysis of the concatenated seven ISHAM consensus MLST loci (8) using the program MEGA version 5.05 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105076#pone.0105076-Tamura1" target="_blank">[51]</a>. Bootstrap values higher than 50 are shown above the branches. Source, country, major molecular type, sequence type (ST), mating type and virulence of the strains in the <i>Galleria</i> model are indicated. Colour of the labels indicates clinical (black), veterinary (red) and environmental (green) strains; * mating type a, all others are mating type alpha; two letter country abbreviation (AR = Argentina, AU = Australia, BR = Brazil, CA = Canada, CO = Colombia, ES = Spain, IN = India, IT = Italy, NZ = New Zealand, PG = Papua New Guinea, TH = Thailand, US = USA, and ZA = South Africa); crosses represent more virulent (+++), similarly virulent (++), and less virulent strains (+), compared with the highly virulent VGIIa Vancouver Island outbreak strain CDCR265. Dash represents avirulent strains.</p

<i>Cryptococcus gattii</i> strains studied, including general information.

<p>Clin: clinical; Env: environmental; Vet: veterinary.</p

Microscopic photographs of the most virulent strain of each <i>Cryptococcus gattii</i> molecular type and CDCR265.

<p>Strains were stained with India ink before and after infection of <i>Galleria mellonella</i> larvae.</p

Gel view of the MSP of the obtained mass spectra of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> strains according to their major molecular type.

<p>Gel view of the MSP of the obtained mass spectra of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> strains according to their major molecular type.</p

Growth rate of <i>Cryptococcus gattii</i> strains with different level of virulence at 37°C.

<p>Crosses represent more virulent (+++), similarly virulent (++), and less virulent strains (+), compared with the highly virulent VGIIa Vancouver Island outbreak strain CDCR265. Dash represents avirulent strains.</p

Melanin production of <i>Cryptococcus gattii</i> strains.

<p>Strains are grouped by major molecular type and distributed in the X-axis from more to less virulence. Line colour of the bars represents more virulent (red), similarly virulent (blue), and less virulent strains (black), compared with the highly virulent VGIIa Vancouver Island outbreak strain CDCR265. Grey bars represent avirulent strains. Both the quantification of the laccase activity and the ability of the strains to produce melanin in Niger seed agar are represented.</p

Survival curve of <i>Galleria mellonella</i> larvae inoculated with <i>Cryptococcus gattii</i>.

<p>C: clinical; E: environmental; V: veterinary. Larvae inoculated with strains more virulent (continuous line) and as virulent as the highly virulent VGIIa Vancouver Island outbreak strain CDCR265 (dashed line), as well as larvae inoculated with PBS or not inoculated (dotted line), are represented.</p

Scores obtained for the identification of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> isolates, using the original un-supplemented MALDI Biotyper BDAL MSP library (Bruker).

*<p>2.300–3.000: highly probable species identification; 2.000–2.299: secure genus identification, probable species identification; 1.700–1.999: probable genus identification.</p

Capsule size, cell size and capsule percentage distribution of <i>Cryptococcus gattii</i> strains after larvae inoculation.

<p>Strains are grouped by major molecular type and distributed in the X-axis from more to less virulence. Symbol colour represents more virulent (red), similarly virulent (blue), and less virulent strains (black), compared with the highly virulent VGIIa Vancouver Island outbreak strain CDCR265. Empty rhombuses represent avirulent strains.</p

Values comparing survival curves and median survival times of <i>Galleria mellonella</i> larvae after being infected with the highly virulent strain of <i>Cryptococcus gattii</i> CDCR265 (VGIIa) and other strains of the molecular types VGI, VGII, VGIII and VGIV.

<p>Clin: clinical; Env: environmental; Vet: veterinary.</p><p>*In comparison with the highly virulent strain CDCR265 (++).</p>#<p>Reference strains of the highly and low virulent major sub-genotypes VGIIa (CDRC265) and VGIIb (CDRC272), respectively, of the Vancouver Island outbreak (11, 37).</p><p>NA: not applicable.</p><p>ND: no determined.</p