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. 2022 Jun 17;11:e79114. doi: 10.7554/eLife.79114

Figure 1. Genome-wide comparison between the two C. depauperatus strains and phylogenetic placement of C. depauperatus.

(A) Circos plot comparing the genome assemblies of C. depauperatus CBS7841 and CBS7855. The two assemblies are overall syntenic, except for five inversions (labelled ‘a’ to ‘e’; see Figure 1—figure supplement 2 for details). Other genomic features are depicted in different tracks for each chromosome, as shown in the key. (B) Genome-based phylogeny recovers C. depauperatus as a sister species to the human pathogenic Cryptococcus clade. The tree was inferred by maximum likelihood using a concatenation-based approach on a data matrix composed of protein alignments of 4074 single-copy genes shared across selected strains of seven Cryptococcus species and an outgroup (Kwoniella mangrovensis). Log-likelihood of the tree: lnL = –16948764.2158. A coalescence-based tree topology inference obtained by ASTRAL was completely congruent with the concatenation-based phylogeny (see Figure 1—figure supplement 5). The reliability of each internal branch was evaluated by 1000 replicates of the Shimodaira–Hasegawa approximate likelihood ratio test (SH-aLRT) and ultrafast bootstrap (UFboot) in the concatenation-based tree, and local posterior probability (LPP) in the coalescence-based tree. Branch lengths are given in number of substitutions per site (scale bar). For each branch of the tree, three additional measures of genealogical concordance are shown: the gene concordance factor (gCF), the site concordance factor (sCF), and quartet support for the main topology (q1)(see ‘Materials and methods’ for details). Scanning electron microscopy images illustrating sexual reproductive structures (basidia with spore chains) of C. neoformans (H99 × KN99; top), C. depauperatus CBS7841 (middle) and C. amylolentus (CBS6039 × CBS6273; bottom). Scale bars = 5μm.

Figure 1.

Figure 1—figure supplement 1. Statistics of the genome assemblies of CBS7841 and CBS7855 and electrophoretic karyotyping.

Figure 1—figure supplement 1.

(A) Genome sequencing data generated, final genome assembly statistics, and (B) genome completeness assessment upon aligning C. depauperatus gene sets to 4284 Benchmarking Universal Single-Copy Orthologs (BUSCOs) included in the tremellomycetes_odb10 dataset. (C) Electrophoretic karyotypes (clamped homogeneous electrical field [CHEF]) of CBS7841 and CBS7855. Chromosomes of Saccharomyces cerevisiae (Scer) and Hansenula wingei (Hwin) served as size markers. For CBS7841, all contigs (except the largest one that includes the rDNA array, which runs at a higher size range) could be assigned to a band in the gel. For CBS7855, we found two discrepancies: (i)a band (marked with a question mark) that could not be assigned to any of the assembled contigs, and (ii) the fact that contigs 3 and 4 seem to be unaccounted for in the gel. This might suggest inherent chromosome instability in this strain (e.g., a subpopulation of cells undergoing chromosomal rearrangements/breaks/fusions), which will be the subject of future studies.
Figure 1—figure supplement 1—source data 1. Raw images of clamped homogeneous electrical field (CHEF) gels.
Figure 1—figure supplement 2. Inversions between the two C. depauperatus isolates.

Figure 1—figure supplement 2.

(A) Synteny maps of C. depauperatus CBS7841 and CBS7855 highlighting two large and three small inversions detected between the two strains, with each inversion containing more than two genes (labeled ‘a’ to ‘e’ and denoted by a teal blue bar). (B) Except for inversion ‘a’, all the others (inversion ‘b’ is shown as an example) are associated with duplicated sequences at the borders (highlighted in yellow). Gray or pink bars/lines connect similar regions between the two strains (BLASTN hits>200bp) in the same or inverted orientation, respectively. An ‘r’ next to the chromosome number (e.g., Chr 6r in CBS7855) indicates that the orientation is reversed relative to the orientation in the assembly.
Figure 1—figure supplement 3. Centromere length comparison between CBS7841 and CBS7855.

Figure 1—figure supplement 3.

(A) Box plots depicting the predicted centromere length. Each dot represents one centromere, and the horizontal red lines depict mean centromere lengths in the corresponding strain. Shaded boxes represent the interquartile ranges, and upper or lower whiskers show the largest or smallest observations, respectively. The centromere length differences observed between the two strains are not statistically significant (p=0.1036; Wilcoxon test). (B) Bar graph depicting the length difference between centromeres of homologous chromosomes of the two strains. (C) List of open reading frames (ORFs) flanking the candidate centromeric regions, genomic coordinates of the predicted centromeres and their exact length. Centromere length was defined as the distance between centromere-flanking ORFs.
Figure 1—figure supplement 4. Genome-wide divergence (k, with Jukes–Cantor correction) of C. depauperatus CBS7855 relative to CBS7841 genome reference.

Figure 1—figure supplement 4.

This analysis shows no evidence of introgression (which would appear as regions with zero or nearly zero divergence) between the two strains (y-axis values represent percentages; x-axis values represent mega base pairs).Centromeres, rDNA, and repeat content are depicted as indicated in the key. Each data point represents the average divergence using a non-overlapping sliding window of 5000 sites. A gray line shows the mean divergence for that chromosome.
Figure 1—figure supplement 5. Tree topology inferred from the coalescence-based approach.

Figure 1—figure supplement 5.

(A) Tree topology derived from the coalescence approach is fully congruent with the maximum likelihood (ML) tree derived from a concatenation-based approach. Numerical values on the branches represent quartet support for the main topology (q1).All branches received 1.0 local posterior probability (LPP). (B) Examination of support among individual gene trees for alternative hypotheses of conflicting branches in the species phylogeny. Green bars and topology (T1)reflect the relationships inferred using a concatenation-based approach on the full data matrix, which also corresponds to the best quartet support, while orange and blue bars and topologies (T2 and T3)correspond to the two alternative hypotheses supported by the two alternative resolutions of each quartet. Dashed horizontal lines mark expectation for a hard polytomy. Cn: C. neoformans; Cde: C. deneoformans; Cg: C. gattii; Cd: C. depauperatus; Ca: C. amylolentus; Cw: C. wingfieldii; Cf: C. floricola.