Figure 3. Maximum likelihood phylogenetic tree using whole-genome sequences. A concatenated alignment of 2,422 single-copy orthologs was used to construct the ML tree. P. tannophilus was used as an outgroup. Branch lengths denote amino acid substitutions per site.
To validate species discrimination, we employed the Average Nucleotide Identity (ANI) analysis across Nakazawaea genomes. Our examination revealed an average ANI value of 72.4% between N. atacamensis and the remaining genomes (Table S4 ). Consequently, this finding supports the classification of N. atacamensis as a novel species, consistent with the established yeast species delineation criteria (Lachance et al. , 2020). ANI serves as a robust parameter for demarcating species boundaries in yeasts using genome sequence data. Specifically, ANI values below 95%, which are indicative of distinct bacterial species, have been found also to be a good guideline for a group of well-defined yeast species (Lachanceet al. , 2020).
Notably, the genome size of N. atacamensis is comparable to that of other Nakazawaea species, such as N. ishiwadae GDMCC 60786. Gene prediction and component analysis of the N. atacamensis genome using the GeneMark tool resulted in the identification of 5,394 predicted genes. Among these genes, 5,116 protein-coding genes (95%) were annotated with InterProScan (Table S5 ). To facilitate the reconstruction of the molecular network from the predicted proteins, we employed KofamKOALA and assigned KEGG Orthologs (KOs). We identified 2,782 genes involved in 385 pathways (Table S6 ). Most of the predicted genes are associated with metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and biosynthesis of cofactors (Figure 4A ). Given that N. atacamensis is a fermenting yeast, we specifically focused on carbon metabolism. Our analysis revealed the presence of 68 genes encoding enzymes involved in various carbon source metabolism pathways (Figure 4B , Table S6 ). These pathways include glycolysis/gluconeogenesis, pyruvate metabolism, the citrate cycle (TCA), and the pentose phosphate pathway, all of which are critical for sugar fermentation through the central carbon metabolism. Additionally, other pathways, such as glycogen biosynthesis and degradation, nucleotide sugar biosynthesis, and UDP-N-acetyl-D-glucosamine biosynthesis, may also be present in N. atacamensis .