Novakomyces olei sp. nov., the First Member of a Novel Taphrinomycotina Lineage

Abstract

Taphrinomycotina is the smallest subphylum of the phylum Ascomycota. It is an assemblage of distantly related early diverging lineages of the phylum, comprising organisms with divergent morphology and ecology; however, phylogenomic analyses support its monophyly. In this study, we report the isolation of a yeast strain, which could not be assigned to any of the currently recognised five classes of Taphrinomycotina. The strain of the novel budding species was recovered from extra virgin olive oil and characterised phenotypically by standard methods. The ultrastructure of the cell wall was investigated by transmission electron microscopy. Comparisons of barcoding DNA sequences indicated that the investigated strain is not closely related to any known organism. Tentative phylogenetic placement was achieved by maximum-likelihood analysis of the D1/D2 domain of the nuclear LSU rRNA gene. The genome of the investigated strain was sequenced, assembled, and annotated. Phylogenomic analyses placed it next to the fission Schizosaccharomyces species. To accommodate the novel species, Novakomyces olei, a novel genus Novakomyces, a novel family Novakomycetaceae, a novel order Novakomycetales, and a novel class Novakomycetes is proposed as well. Functional analysis of genes missing in N. olei in comparison to Schizosaccharomyces pombe revealed that they are biased towards biosynthesis of complex organic molecules, regulation of mRNA, and the electron transport chain. Correlating the genome content and physiology among species of Taphrinomycotina revealed some discordance between pheno- and genotype. N. olei produced ascospores in axenic culture preceded by conjugation between two cells. We confirmed that N. olei is a primary homothallic species lacking genes for different mating types.

Keywords: functional gene analysis; genome sequence; novel class; novel yeast species; olive oil; phylogenomics.

Figure 1

Phylogenetic placement of Novakomyces olei sp. nov. in the subphylum Taphrinomycotina using maximum-likelihood analysis and the Tamura–Nei model [73]. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Analysis was performed based on the sequences of the LSU rRNA gene D1/D2 domain. Bootstrap percentages (1000 replicates) exceeding 50% are given at branch nodes. Bar, 5% nucleotide sequence divergence. Ustilago maydis, Filobasidiella neoformans, and Rhodosporidium toruloides were used as the designated outgroup species. Taphrinomycotina is highlighted by the blue background.

Figure 2

The phylogenetic relationships of Taphrinomycotina and related fungi based on concatenated alignment of 110 orthologous, single-copy BUSCO gene amino acid data matrix. The maximum likelihood (ML) phylogeny was reconstructed under the LG + GAMMA substitution model using RAxML as determined by IQ-TREE as the best model for a given dataset. Branch support values are indicated only for the nodes with <95% bootstrap support. Scale bar, number of amino acid substitutions per site. Cryptococcus neoformans and Ustilago maydis were the designated outgroup species in the analysis.

Figure 3

Significantly overrepresented slim gene ontology (GO) terms for biological processes of genes absent in Novakomyces olei sp. nov. compared to the reference gene set of Schizosaccharomyces pombe as its close relative. The sizes of the rectangles are proportional to the percentage of missing genes in a GO category.

Figure 4

Novakomyces olei NCAIM Y.02187^T. TEM micrograph of a section of a budding cell (5% malt extract, 3 days, 25 °C). Bud scar is indicated by arrows. Bar, 1000 nm. The image was taken by Bence Rácz.

Figure 5

Ascosporulating culture of Novakomyces olei NCAIM Y.02187^T. One two-spored ascus is shown in each panel. In panel (A), the remnant of an ascus formed by the conjugation of the mother cell and its bud is indicated by arrow. Bar, 10 μm for both panels (A,B).

Figure 6

Correlation between assimilation profiles and gene content of Novakomyces olei and related species of Taphrinomycotina. Assimilation responses are represented by +, positive; −, negative; v, variable; s, slow; n, not determined (from Kurtzman et al. [82] and this study). Numbered boxes with blue colour intensity indicate the gene copy number determined by tBLASTn analyses. The maximum likelihood (ML) phylogeny was reconstructed under LG + GAMMA substitution model using RAxML. The scale bar presents amino acids substitutions per site.