Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota)

Abstract

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

Keywords: Black yeast; Chaetothyriales; Comparative genomics; Ecology; Evolution; Herpotrichiellaceae; Phylogeny.

Fig. 1

Phylogenomic distribution of Chaetothyriales and related ascomycetes used for comparative genomics. The majority of species are placed in the families Cyphellophoraceae (C) and Herpotrichiellaceae (H). The main characteristics such as niche, isolation source (red boxes – anthropophilic pathogens, orange boxes, zoophilic pathogens and green boxes geophilic), anamorphs, teleomorphs and sexual locus organization are displayed for each compared species.

Fig. 2

Phylogenetic analysis of members of Chaetothyriales (Class Eurotiomycetes). The Maximum likelihood tree, based on 172 LSU sequences, was determined using MEGA v. 6 with Kimura 2-parameter model with default settings and statistical bootstrapping procedure involving 500 replicates. Bootstrap values above 70 % are shown at the nodes. Family boundaries are indicated with coloured blocks. The tree was rooted to Verrucula granulosaria AFTOL-ID 2304.

Fig. 2

Phylogenetic analysis of members of Chaetothyriales (Class Eurotiomycetes). The Maximum likelihood tree, based on 172 LSU sequences, was determined using MEGA v. 6 with Kimura 2-parameter model with default settings and statistical bootstrapping procedure involving 500 replicates. Bootstrap values above 70 % are shown at the nodes. Family boundaries are indicated with coloured blocks. The tree was rooted to Verrucula granulosaria AFTOL-ID 2304.

Fig. 3

Distribution of orthology classes in black yeasts and closely related fungi: core genes found in all genomes are shown in green, shared genes present in more than one but not all genomes in blue and genes that were unique to only one of the 28 analysed genomes in yellow.

Fig. 4

Genome-scale of chaetothyrialean phylogeny and divergence times. Calibration points are highlighted in blue and were used to infer the divergence times for Chaetothyriales (upper panel). The red node displays the divergence dates of Chaetothyriales and the red asterisk bolded area highlights a common era for both Cyphellophoraceae and Herpotrichiellaceae. The bottom scale presents the main geological and periods and eras.

Fig. 5

Dynamic evolution of protein families. (A) Phylogenetic tree showing the relationship between species and altered protein families. Pie diagrams and numbers at the nodes represent the abundance of contractions (red) expansion (blue) and No change (black) of 1771 protein families during evolution of black yeasts. (B) Heatmap showing expansion and contractions of protein families found in species belonging to the bantiana- and carrionii-clades, respectively. Domains are grouped by category similarity. All domains shown are significantly changed, and were identified using CAFE with cut-off of family p-values <0.05 and Viterbi p-values <0.01.

Fig. 6

Distribution of CYP p450 genes in Ascomycota. TOP 10 fungal species with highest CYP p450 numbers in ascomycetous genomes, based on search against the Fungal Cytochrome P450 Database (FCPD).

Fig. 7

Mating type locus structure of ancestral lineages C. apollinis and V. gallopava (top-right panel), the Cyphellophoraceae (bottom-left panel) and the salmonis-clade of Herpotrichiellaceae (top-left panel). Sexual loci for each fungal species are displayed in each respectively scaffold and the corresponding genes and accession numbers are displayed to each gene.

Fig. 8

Mating type locus structure of heterothallic species R. mackenziei (lower panel), E. dermatitidis and closely related homothallic Ca. coronata and Ca. epimyces (dermatitidis-clade upper panel). Mating type genes are represented in each corresponding assembled scaffold. Accession numbers are displayed to each gene.

Fig. 9

Mating type locus organization of heterothallic species from jeanselmei-clade, Herpotrichiellaceae. Mating type genes are represented in each corresponding assembled scaffold. Accession numbers are displayed to each gene.

Fig. 10

Mating type locus structure of heterothallic species from carrionii-clade, Herpotrichiellaceae. Mating type genes are represented in each corresponding assembled scaffold. Accession numbers are displayed to each gene.

Fig. 11

Mating type locus organization of heterothallic species from bantiana-clade, Herpotrichiellaceae. Mating type genes are represented in each corresponding assembled scaffold. Accession numbers are displayed to each gene.

Fig. 12

Distribution of heterokaryon (het) containing genes in 23 black yeast-like fungi and related Ascomycota. (A) Total counts of het containing genes (IPR010730 domain) for each species. (B) Pairwise similarity graphs generated by clustering analysis of 1439 het-containing proteins from Chaetothyriales and related species.