Genomic factors shape carbon and nitrogen metabolic niche breadth across Saccharomycotina yeasts.

Organisms exhibit extensive variation in ecological niche breadth, from very narrow (specialists) to very broad (generalists). Two general paradigms have been proposed to explain this variation: (i) trade-offs between performance efficiency and breadth and (ii) the joint influence of extrinsic (environmental) and intrinsic (genomic) factors. We assembled genomic, metabolic, and ecological data from nearly all known species of the ancient fungal subphylum Saccharomycotina (1154 yeast strains from 1051 species), grown in 24 different environmental conditions, to examine niche breadth evolution. We found that large differences in the breadth of carbon utilization traits between yeasts stem from intrinsic differences in genes encoding specific metabolic pathways, but we found limited evidence for trade-offs. These comprehensive data argue that intrinsic factors shape niche breadth variation in microbes.

Genomic and ecological factors shaping specialism and generalism across an entire subphylum.

Organisms exhibit extensive variation in ecological niche breadth, from very narrow (specialists) to very broad (generalists). Paradigms proposed to explain this variation either invoke trade-offs between performance efficiency and breadth or underlying intrinsic or extrinsic factors. We assembled genomic (1,154 yeast strains from 1,049 species), metabolic (quantitative measures of growth of 843 species in 24 conditions), and ecological (environmental ontology of 1,088 species) data from nearly all known species of the ancient fungal subphylum Saccharomycotina to examine niche breadth evolution. We found large interspecific differences in carbon breadth stem from intrinsic differences in genes encoding specific metabolic pathways but no evidence of trade-offs and a limited role of extrinsic ecological factors. These comprehensive data argue that intrinsic factors driving microbial niche breadth variation.

Repeated horizontal gene transfer of GALactose metabolism genes violates Dollo's law of irreversible loss.

Dollo's law posits that evolutionary losses are irreversible, thereby narrowing the potential paths of evolutionary change. While phenotypic reversals to ancestral states have been observed, little is known about their underlying genetic causes. The genomes of budding yeasts have been shaped by extensive reductive evolution, such as reduced genome sizes and the losses of metabolic capabilities. However, the extent and mechanisms of trait reacquisition after gene loss in yeasts have not been thoroughly studied. Here, through phylogenomic analyses, we reconstructed the evolutionary history of the yeast galactose utilization pathway and observed widespread and repeated losses of the ability to utilize galactose, which occurred concurrently with the losses of GALactose (GAL) utilization genes. Unexpectedly, we detected multiple galactose-utilizing lineages that were deeply embedded within clades that underwent ancient losses of galactose utilization. We show that at least two, and possibly three, lineages reacquired the GAL pathway via yeast-to-yeast horizontal gene transfer. Our results show how trait reacquisition can occur tens of millions of years after an initial loss via horizontal gene transfer from distant relatives. These findings demonstrate that the losses of complex traits and even whole pathways are not always evolutionary dead-ends, highlighting how reversals to ancestral states can occur.

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts.

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.

Eukaryotic Acquisition of a Bacterial Operon.

Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.

Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum.

Budding yeasts (subphylum Saccharomycotina) are found in every biome and are as genetically diverse as plants or animals. To understand budding yeast evolution, we analyzed the genomes of 332 yeast species, including 220 newly sequenced ones, which represent nearly one-third of all known budding yeast diversity. Here, we establish a robust genus-level phylogeny comprising 12 major clades, infer the timescale of diversification from the Devonian period to the present, quantify horizontal gene transfer (HGT), and reconstruct the evolution of 45 metabolic traits and the metabolic toolkit of the budding yeast common ancestor (BYCA). We infer that BYCA was metabolically complex and chronicle the tempo and mode of genomic and phenotypic evolution across the subphylum, which is characterized by very low HGT levels and widespread losses of traits and the genes that control them. More generally, our results argue that reductive evolution is a major mode of evolutionary diversification.

Functional and evolutionary characterization of a secondary metabolite gene cluster in budding yeasts.

Secondary metabolites are key in how organisms from all domains of life interact with their environment and each other. The iron-binding molecule pulcherrimin was described a century ago, but the genes responsible for its production in budding yeasts have remained uncharacterized. Here, we used phylogenomic footprinting on 90 genomes across the budding yeast subphylum Saccharomycotina to identify the gene cluster associated with pulcherrimin production. Using targeted gene replacements in Kluyveromyces lactis, we characterized the four genes that make up the cluster, which likely encode two pulcherriminic acid biosynthesis enzymes, a pulcherrimin transporter, and a transcription factor involved in both biosynthesis and transport. The requirement of a functional putative transporter to utilize extracellular pulcherrimin-complexed iron demonstrates that pulcherriminic acid is a siderophore, a chelator that binds iron outside the cell for subsequent uptake. Surprisingly, we identified homologs of the putative transporter and transcription factor genes in multiple yeast genera that lacked the biosynthesis genes and could not make pulcherrimin, including the model yeast Saccharomyces cerevisiae We deleted these previously uncharacterized genes and showed they are also required for pulcherrimin utilization in S. cerevisiae, raising the possibility that other genes of unknown function are linked to secondary metabolism. Phylogenetic analyses of this gene cluster suggest that pulcherrimin biosynthesis and utilization were ancestral to budding yeasts, but the biosynthesis genes and, subsequently, the utilization genes, were lost in many lineages, mirroring other microbial public goods systems that lead to the rise of cheater organisms.