Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species

Abstract

Background: Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.

Methods: In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.

Results: BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.

Conclusions: Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

Figure 1. Phylogenetic analysis of 100 environmental Cryptococcus 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.

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.

Figure 2. Phylogenetic analysis of 100 environmental Cryptococcus 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.

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.

Figure 3. Intraspecific and interspecific pairwise distance of the three ribosomal regions of the environmental Cryptococcus spp. calculated by the Kimura 2-parameter model revealed higher variability of the ITS region compared with the 18S-SSU and 28S-LSU regions.

Figure 4. Median-joining haplotype network (A) of environmental C. laurentii isolates based on concatenated nucleotide sequences of the 5′ end of 18S-SSU, D1/D2 of 28S-LSU, and ITS regions.

The tree represents 103 Cryptococcus spp. isolates from Brazil, Botswana, Canada, Japan, India, and the United States. The seven C. laurentii and three C. flavescens haplotypes are clearly distinguished. The Botswana ancestral haplotype (H4) of C. laurentii 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 C. albidus 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 C. laurentii 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).

Figure 5. Species tree of the C. laurentii species complex resulting from coalescent analyses of the concatenated data set.

The speciation of C. aspenensis from C. laurentii and C. rajasthanensis took place 37.9 million years ago. The C. laurentii haplotype (H4) from Botswana was the first haplotype to be differentiated (6.8 million years ago). Numbers at branches represent the Bayesian posterior support values while the bold numbers represent the nodes ages (in millions of years).

Figure 6. Differential interference contrast (A) and India Ink staining (B) of C. aspenensis sp. nov. DS573^T (CBS 13867) cells after 3 days at 25°C in YPD broth. Scale bar of 20 µm is shown.