Fig 1.
The GAL pathway and the distribution of galactose metabolism, GAL genes, and preferred codon usage across the Saccharomycotina.
(A) The 3 enzymes of the GAL pathway metabolize galactose into glucose-1-phosphate, which can then enter glycolysis after being converted into glucose-6-phosphate. (B) Various features of galactose metabolism plotted on a phylogeny of the budding yeast subphylum Saccharomycotina; the 12 major clades of the subphylum are color coded. The presence and codon optimization (measured by estAI) of the 3 GAL genes are represented in the inner 3 rings. Clades with 3 or more species without a complete GAL pathway were condensed and are shown as triangles; for the full tree, see S1 Fig. The GAL clusters in the Dipodascaceae/Trichomonascaceae, Pichiaceae, and Phaffomycetaceae were recently found to have likely originated from horizontal gene transfer events from the CUG-Ser1 clade [72]. We did not identify any GAL genes from species in the CUG-Ser2 clade or the family Saccharomycodaceae. In every other major clade examined, we identified complete and clustered occurrences of the GAL pathway (filled-in blue squares and circles, respectively.) High codon optimization (darker colors) in the GAL pathway is not restricted to any one major clade. The ability to metabolize galactose (filled-in green triangle) was assessed either experimentally in this study or taken from the literature. In some instances, where only literature data were available, there were conflicting or variable reports of galactose metabolism (5 species; empty green triangles). The majority of species in the Saccharomycotina have also been shown to have genome-wide selection on codon usage (denoted by the yellow stars) [59]. Species names and ecological information can be found in S1 Fig and Table A S1 Data.
Fig 2.
Codon optimization in the GAL pathway is positively and significantly correlated with growth rate on galactose.
Phylogenetically independent contrasts (PIC) analyses of galactose growth (y axis) versus GAL gene optimal codon usage (x axis) in species that grow on galactose containing medium. Each point represents an independent contrast calculated between 2 species for either optimal codon usage or growth. A plot of the uncorrected data can be found in S11 Fig. There is a significant and positive correlation between the PIC values for codon optimization and galactose growth in GAL1 (A), GAL10 (B), and GAL7 (C). The best fit and strongest correlation is between growth on galactose and optimization in GAL7 (C). The analyses included 94 species with a growth rate on galactose greater than 0, a complete GAL cluster, and evidence of genome-wide translational selection on codon usage. One species, Metschnikowia matae var. matae, was removed as an obvious outlier based on residual analysis. Underlying data can be found in Tables B–D S4 Data.
Fig 3.
Codon optimization in the GAL pathway is correlated with specific ecological niches in 2 different major clades of budding yeasts.
p-values less than 0.01 are indicated with ** and less than 0.05 with *. (A) In the CUG-Ser1 clade, species associated with a human niche or human and insect niches (13 species) have significantly higher codon usage optimization values in all GAL genes (p-values of 0.022, 0.028, and 0.006 for GAL1, GAL10, and GAL7, respectively) when compared to species that are associated with insect niches but not human niches (44 species). Only 11 species were not associated with either human or insect niches. (B) In the Saccharomycetaceae, species associated with only dairy niches (5 species) have significantly higher codon usage optimization values in all of the GAL genes (p-values of 0.010, 0.002, and 0.014 for GAL1, GAL10, and GAL7, respectively) versus species associated with only alcohol niches (14 species). A total of 9 species are associated with both dairy and alcohol niches. Underlying data can be found in Tables E and F S4 Data.
Fig 4.
Closely related Kluyveromyces species exhibit differential codon optimization in the GAL pathway associated with isolation from dairy environments.
All 4 Kluyveromyces species were shown experimentally to metabolize galactose. (A) Species phylogeny of 4 closely related Kluyveromyces species. K. marxianus and K. lactis are both associated with dairy niches and have high codon optimization values in their GAL pathway genes. In contrast, K. aestuarii is associated with marine mud, and K. dobzhanskii is associated with flies. (B) The genome-wide distribution of codon optimization (stAI) values for the 4 Kluyveromyces species included in this study. The 50th and 75th percentiles are shown with black dashed lines. In the 2 species associated with dairy niches, the codon optimization for all 3 GAL genes falls in the top 25th percentile. In the 2 species not associated with dairy, the GAL genes fall below the top 25th percentile. Genes encoding ribosomal proteins are well established to rank among the most highly optimized genes within a genome. (C) The distribution of terminal synonymous (dS) branch lengths (in unrooted gene trees) calculated for the 651 BUSCO genes. All 3 GAL genes fall within the interquartile range for K. aestuarii and K. marxianus. In K. dobzhanskii, all 3 GAL genes lie above the 70th percentile with GAL1 in the upper quartile. In K. lactis, GAL7 falls below the interquartile range, while GAL10 lies above. The 50th and 75th percentiles are shown with black dashed lines. Underlying data can be found in Tables G–P S4 Data.
Table 1.
KEGG Orthology (KO) annotated genes that have a codon optimization correlated with GAL1, GAL10, or GAL7.
This table contains the significant (multiple test corrected p-value <0.001***) results with the greatest absolute slope for each GAL gene and 3 additional results that are significant in both GAL7 and GAL10. Gene names for S. cerevisiae are listed where available.
Fig 5.
A model for ecological codon adaptation in the GALactose metabolism pathway.
The ancestral species in environment A maintains the GALactose metabolism pathway at an intermediate codon optimization. Upon introduction to environment B, which contains abundant galactose, there is increased demand for the GALactose metabolism enzymes to take advantage of this energy source. In this new environment, substitutions that increase codon optimization of the GAL genes will be selectively advantageous. Codon optimization will continue to increase under translational selection until it is no longer a barrier to expression or optimal flux through the pathway has been achieved.