Median-joining haplotype network (A) of environmental <i>C. laurentii</i> isolates based on concatenated nucleotide sequences of the 5′ end of 18S-SSU, D1/D2 of 28S-LSU, and ITS regions.

<p>The tree represents 103 <i>Cryptococcus</i> spp. isolates from Brazil, Botswana, Canada, Japan, India, and the United States. The seven <i>C. laurentii</i> and three <i>C. flavescens</i> haplotypes are clearly distinguished. The Botswana ancestral haplotype (H4) of <i>C. laurentii</i> is presented and highlighted in yellow. Each circle represents a unique haplotype (H), and the circumference is proportional to haplotype frequency (H1: 44 isolates; H2: 1; H3: 18; H4: 2; H5: 8; H6: 2; H7: 1; H8: 1; H9: 8; H10: 2; H11: 3; H12: 2; H13: 10; H14: 1; outgroup <i>C. albidus</i> CBS 142). Yellow dots represents the number of mutation sites, excluding gaps, between the haplotypes. Black dots (median vectors) are hypothetical missing intermediates. Minimum spanning trees (B) using the goeBURST algorithm confirm the haplotype relationships among <i>C. laurentii</i> isolates determined by median-joining network analysis. The size of the circle corresponds to the number of isolates within that haplotype, and the numbers between haplotypes represent the genetic distance of each haplotype, excluding the gaps. Minimum spanning trees as described in B modified to show the distribution of haplotypes according to the country of origin (C) or environmental source (D).</p

Phylogenetic analysis of 100 environmental <i>Cryptococcus</i> spp. isolates generated by the neighbor-joining, UPGMA, and maximum likelihood methods using partial nucleotide sequences of the (A) 5′end of 18S SSU-rDNA and (B) D1/D2 region of 28S LSU-rDNA.

<p>Numbers at each branch indicate bootstrap values>50% based on 1,000 replicates (NJ/UPGMA/ML). The analysis involved 103 and 105nucleotide sequences for the 18S-SSU and 28S-LSU respectively. ^T: Type strain.</p

Differential interference contrast (A) and India Ink staining (B) of <i>C. aspenensis</i> sp. nov. DS573^T (CBS 13867) cells after 3 days at 25°C in YPD broth. Scale bar of 20 µm is shown.

<p>Differential interference contrast (A) and India Ink staining (B) of <i>C. aspenensis</i> sp. nov. DS573^T (CBS 13867) cells after 3 days at 25°C in YPD broth. Scale bar of 20 µm is shown.</p

Intraspecific and interspecific pairwise distance of the three ribosomal regions of the environmental <i>Cryptococcus</i> spp. calculated by the Kimura 2-parameter model revealed higher variability of the ITS region compared with the 18S-SSU and 28S-LSU regions.

<p>Intraspecific and interspecific pairwise distance of the three ribosomal regions of the environmental <i>Cryptococcus</i> spp. calculated by the Kimura 2-parameter model revealed higher variability of the ITS region compared with the 18S-SSU and 28S-LSU regions.</p

PCR conditions and primers used for the amplification of the ribosomal loci.

<p>PCR conditions and primers used for the amplification of the ribosomal loci.</p

DNA polymorphisms in the ribosomal loci of the 75 <i>C. laurentii</i> environmental isolates.

<p><b><i>S</i></b>: number of polymorphic sites. <b><i>π</i></b>: nucleotide diversity. <b><i>k</i></b>: average number of nucleotide differences per sequence. <b><i>h</i></b>: number of haplotypes. <b><i>H_d</i></b>: haplotype diversity. <b><i>D</i></b><b>, </b><b><i>F_D</i></b><b>, </b><b><i>F_F</i></b><b> and </b><b><i>Fs</i></b>: Tajima's D, Fu and Li's D*, Fu and Li's F* and Fu's Fs, respectively.</p>a<p>: p value<0.05.</p><p>DNA polymorphisms in the ribosomal loci of the 75 <i>C. laurentii</i> environmental isolates.</p

Phylogenetic analysis of 100 environmental <i>Cryptococcus</i> spp. isolates generated by the neighbor-joining, UPGMA, and maximum likelihood methods using partial nucleotide sequences of the (A) internal transcribed spacer (ITS) and (B) concatenated sequences of the three ribosomal regions.

<p>Numbers at each branch indicate bootstrap values>50% based on 1,000 replicates (NJ/UPGMA/ML). The analysis involved 105 and 103 nucleotide sequences for ITS and concatenated sequences respectively. ^T: Type strain.</p

Isolate, species, source, and GenBank accession numbers of <i>Cryptococcus</i> spp. environmental isolates.

<p>NA: Not applicable.</p>T<p>: Type strain. Hap: Haplotype number.</p>#<p>: <i>C. aspenensis</i> sp. nov. (CBS 13867).</p><p>Mopane trees (<i>Colophospermum mopane</i>). Douglas fir (<i>Pseudotsuga menziesii</i>). Norway spruce (<i>Picea abies</i>). Trembling aspen (<i>Populus tremuloides</i>). CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. 18S-SSU: Small subunit rDNA. 28S-LSU: Large subunit rDNA. ITS: Internal transcribed spacer region.</p><p>Isolate, species, source, and GenBank accession numbers of <i>Cryptococcus</i> spp. environmental isolates.</p