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 .