Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.     

Genome Sequence of the Lager Brewing Yeast, an Interspecies Hybrid

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.Key words: Saccharomyces pastorianus, beer, genome, interspecies hybrid, larger yeast     

Gene Responses to Oxygen Availability in Kluyveromyces lactis: an Insight on the Evolution of the Oxygen-Responding System in Yeast

The whole-genome duplication (WGD) may provide a basis for the emergence of the very characteristic life style of Saccharomyces cerevisiae—its fermentation-oriented physiology and its capacity of growing in anaerobiosis. Indeed, we found an over-representation of oxygen-responding genes in the ohnologs of S. cerevisiae. Many of these duplicated genes are present as aerobic/hypoxic(anaerobic) pairs and form a specialized system responding to changing oxygen availability. HYP2/ANB1 and COX5A/COX5B are such gene pairs, and their unique orthologs in the ‘non-WGD’ Kluyveromyces lactis genome behaved like the aerobic versions of S. cerevisiae. ROX1 encodes a major oxygen-responding regulator in S. cerevisiae. The synteny, structural features and molecular function of putative KlROX1 were shown to be different from that of ROX1. The transition from the K. lactis-type ROX1 to the S. cerevisiae-type ROX1 could link up with the development of anaerobes in the yeast evolution. Bioinformatics and stochastic analyses of the Rox1p-binding site (YYYATTGTTCTC) in the upstream sequences of the S. cerevisiae Rox1p-mediated genes and of the K. lactis orthologs also indicated that K. lactis lacks the specific gene system responding to oxygen limiting environment, which is present in the ‘post-WGD’ genome of S. cerevisiae. These data suggested that the oxygen-responding system was born for the specialized physiology of S. cerevisiae.     

Convergent adaptation of Saccharomyces uvarum to sulfite,an antimicrobial preservative widely used in human-driven fermentations

Different species can find convergent solutions to adapt their genome to the same evolutionary constraints, although functional convergence promoted by chromosomal rearrangements in different species has not previously been found. In this work, we discovered that two domesticated yeast species, Saccharomyces cerevisiae, and Saccharomyces uvarum, acquired chromosomal rearrangements to convergently adapt to the presence of sulfite in fermentation environments. We found two new heterologous chromosomal translocations in fermentative strains of S. uvarum at the SSU1 locus, involved in sulfite resistance, an antimicrobial additive widely used in food production. These are convergent events that share similarities with other SSU1 locus chromosomal translocations previously described in domesticated S. cerevisiae strains. In S. uvarum, the newly described VII^XVI and XI^XVI chromosomal translocations generate an overexpression of the SSU1 gene and confer increased sulfite resistance. This study highlights the relevance of chromosomal rearrangements to promote the adaptation of yeast to anthropic environments.     

CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2

Background

Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation) and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD) event (ohnologs) versus small-scale duplications (SSD) to determine if there exist any differences in their patterns of sequence evolution.

Results

For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like) in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression.

Conclusions

Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.     

Evolution of ecological dominance of yeast species in high‐sugar environments

In budding yeasts, fermentation in the presence of oxygen evolved around the time of a whole genome duplication (WGD) and is thought to confer dominance in high‐sugar environments because ethanol is toxic to many species. Although there are many fermentative yeast species, only Saccharomyces cerevisiae consistently dominates wine fermentations. In this study, we use coculture experiments and intrinsic growth rate assays to examine the relative fitness of non‐WGD and WGD yeast species across environments to assess when S. cerevisiae’s ability to dominate high‐sugar environments arose. We show that S. cerevisiae dominates nearly all other non‐WGD and WGD species except for its sibling species S. paradoxus in both grape juice and a high‐sugar rich medium. Of the species we tested, S. cerevisiae and S. paradoxus have evolved the highest ethanol tolerance and intrinsic growth rate in grape juice. However, the ability of S. cerevisiae and S. paradoxus to dominate certain species depends on the temperature and the type of high‐sugar environment. Our results indicate that dominance of high‐sugar environments evolved much more recently than the WGD, most likely just prior to or during the differentiation of Saccharomyces species, and that evolution of multiple traits contributes to S. cerevisiae's ability to dominate wine fermentations.     

Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5′ portion derives from S. cerevisiae and the 3′ portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not require repeated backcrossing to one of the parental species.     

The Sir2-Sum1 Complex Represses Transcription Using Both Promoter-Specific and Long-Range Mechanisms to Regulate Cell Identity and Sexual Cycle in the Yeast Kluyveromyces lactis

Deacetylases of the Sir2 family regulate lifespan and response to stress. We have examined the evolutionary history of Sir2 and Hst1, which arose by gene duplication in budding yeast and which participate in distinct mechanisms of gene repression. In Saccharomyces cerevisiae, Sir2 interacts with the SIR complex to generate long-range silenced chromatin at the cryptic mating-type loci, HMLα and HMR a. Hst1 interacts with the SUM1 complex to repress sporulation genes through a promoter-specific mechanism. We examined the functions of the non-duplicated Sir2 and its partners, Sir4 and Sum1, in the yeast Kluyveromyces lactis, a species that diverged from Saccharomyces prior to the duplication of Sir2 and Hst1. KlSir2 interacts with both KlSir4 and KlSum1 and represses the same sets of target genes as ScSir2 and ScHst1, indicating that Sir2 and Hst1 subfunctionalized after duplication. However, the KlSir4-KlSir2 and KlSum1-KlSir2 complexes do not function as the analogous complexes do in S. cerevisiae. KlSir4 contributes to an extended repressive chromatin only at HMLα and not at HMR a. In contrast, the role of KlSum1 is broader. It employs both long-range and promoter-specific mechanisms to repress cryptic mating-type loci, cell-type–specific genes, and sporulation genes and represents an important regulator of cell identity and the sexual cycle. This study reveals that a single repressive complex can act through two distinct mechanisms to regulate gene expression and illustrates how mechanisms by which regulatory proteins act can change over evolutionary time.     

Formation of Complex and Unstable Chromosomal Translocations in Yeast

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom''s-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers.     

Karyotype evolution of the Asterids insights from the first genome sequences of the family Cornaceae

Cornaceae is a core representative family in Cornales, the earliest branching lineage in the Asterids on the life tree of angiosperms. This family includes the only genus Cornus, a group of ~55 species. These species occur widely in Northern Hemisphere and have been used as resources for horticultural ornaments, medicinal and industrial manufacturing. However, no any genome sequences are available for this family. Here, we reported a chromosome­level genome for Cornus controversa. This was generated using high-fidelity plus Hi–C sequencing, and totally ~771.80 Mb assembled sequences and 39,886 protein-coding genes were obtained. We provided evidence for a whole-genome duplication event (WGD) unique to C. controversa. The evolutionary features of this genome indicated that the expanded and unique genes might have contributed to response to stress, stimulus and defense. By using chromosome-level syntenic blocks shared between eight living genomes, we found high degrees of genomic diversification from the ancestral core-eudicot genome to the present-day genomes, suggesting an important role of WGD in genomic plasticity that leads to speciation and diversification. These results provide foundational insights on the evolutionary history of Cornaceae, as well as on the Asterids diversification.     

Loss/retention and evolution of NBS-encoding genes upon whole genome triplication of Brassica rapa

A genome triplication took place in the ancestor of Brassiceae species after the split of the Arabidopsis lineage. The postfragmentation and shuffling of the genome turned the ancestral hexaploid back to diploids and caused the radiation of Brassiceae species. The course of speciation was accompanied by the loss of duplicate genes and also influenced the evolution of retained genes. Of all the genes, those encoding NBS domains are typical R genes that confer resistance to invading pathogens. In this study, using the genome of Arabidopsis thaliana as a reference, we examined the loss/retention of orthologous NBS-encoding loci in the tripled Brassica rapa genome and discovered differential loss/retention frequencies. Further analysis indicated that loci of different retention ratios showed different evolutionary patterns. The loci of classesII and III (maintaining two and three syntenic loci, respectively, multi-loci) show sharper expansions by tandem duplications, have faster evolutionary rates and have more potential to be associated with novel gene functions. On the other hand, the loci that are retained at the minimal rate (keeping only one locus, class I, single locus) showed opposite patterns. Phylogenetic analysis indicated that recombination and translocation events were common among multi-loci in B. rapa, and differential evolutionary patterns between multi- and single-loci are likely the consequence of recombination. Investigations towards other gene families demonstrated different evolutionary characteristics between different gene families. The evolution of genes is more likely determined by the property of each gene family, and the whole genome triplication provided only a specific condition.     

Fast Diploidization in Close Mesopolyploid Relatives of Arabidopsis

Mesopolyploid whole-genome duplication (WGD) was revealed in the ancestry of Australian Brassicaceae species with diploid-like chromosome numbers (n = 4 to 6). Multicolor comparative chromosome painting was used to reconstruct complete cytogenetic maps of the cryptic ancient polyploids. Cytogenetic analysis showed that the karyotype of the Australian Camelineae species descended from the eight ancestral chromosomes (n = 8) through allopolyploid WGD followed by the extensive reduction of chromosome number. Nuclear and maternal gene phylogenies corroborated the hybrid origin of the mesotetraploid ancestor and suggest that the hybridization event occurred ~6 to 9 million years ago. The four, five, and six fusion chromosome pairs of the analyzed close relatives of Arabidopsis thaliana represent complex mosaics of duplicated ancestral genomic blocks reshuffled by numerous chromosome rearrangements. Unequal reciprocal translocations with or without preceeding pericentric inversions and purported end-to-end chromosome fusions accompanied by inactivation and/or loss of centromeres are hypothesized to be the main pathways for the observed chromosome number reduction. Our results underline the significance of multiple rounds of WGD in the angiosperm genome evolution and demonstrate that chromosome number per se is not a reliable indicator of ploidy level.     

Pervasive and persistent redundancy among duplicated genes in yeast

The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (~100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.     

Screening of duplicated loci reveals hidden divergence patterns in a complex salmonid genome

A whole‐genome duplication (WGD) doubles the entire genomic content of a species and is thought to have catalysed adaptive radiation in some polyploid‐origin lineages. However, little is known about general consequences of a WGD because gene duplicates (i.e., paralogs) are commonly filtered in genomic studies; such filtering may remove substantial portions of the genome in data sets from polyploid‐origin species. We demonstrate a new method that enables genome‐wide scans for signatures of selection at both nonduplicated and duplicated loci by taking locus‐specific copy number into account. We apply this method to RAD sequence data from different ecotypes of a polyploid‐origin salmonid (Oncorhynchus nerka) and reveal signatures of divergent selection that would have been missed if duplicated loci were filtered. We also find conserved signatures of elevated divergence at pairs of homeologous chromosomes with residual tetrasomic inheritance, suggesting that joint evolution of some nondiverged gene duplicates may affect the adaptive potential of these genes. These findings illustrate that including duplicated loci in genomic analyses enables novel insights into the evolutionary consequences of WGDs and local segmental gene duplications.     

APT1, but Not APT2, Codes for a Functional Adenine Phosphoribosyltransferase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU intron in vivo

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiaeCBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiaeCBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron.     

A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae''s current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community.     

Three aldo–keto reductases of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote. The S. cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes. We have identified several open reading frames in the S. cerevisiae genome which show significant similarity to members of the aldo–keto reductase superfamily. The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways. Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined. Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined.     

Genome-Wide Reconstruction of Rediploidization Following Autopolyploidization across One Hundred Million Years of Salmonid Evolution

The long-term evolutionary impacts of whole-genome duplication (WGD) are strongly influenced by the ensuing rediploidization process. Following autopolyploidization, rediploidization involves a transition from tetraploid to diploid meiotic pairing, allowing duplicated genes (ohnologs) to diverge genetically and functionally. Our understanding of autopolyploid rediploidization has been informed by a WGD event ancestral to salmonid fishes, where large genomic regions are characterized by temporally delayed rediploidization, allowing lineage-specific ohnolog sequence divergence in the major salmonid clades. Here, we investigate the long-term outcomes of autopolyploid rediploidization at genome-wide resolution, exploiting a recent “explosion” of salmonid genome assemblies, including a new genome sequence for the huchen (Hucho hucho). We developed a genome alignment approach to capture duplicated regions across multiple species, allowing us to create 121,864 phylogenetic trees describing genome-wide ohnolog divergence across salmonid evolution. Using molecular clock analysis, we show that 61% of the ancestral salmonid genome experienced an initial “wave” of rediploidization in the late Cretaceous (85–106 Ma). This was followed by a period of relative genomic stasis lasting 17–39 My, where much of the genome remained tetraploid. A second rediploidization wave began in the early Eocene and proceeded alongside species diversification, generating predictable patterns of lineage-specific ohnolog divergence, scaling in complexity with the number of speciation events. Using gene set enrichment, gene expression, and codon-based selection analyses, we provide insights into potential functional outcomes of delayed rediploidization. This study enhances our understanding of delayed autopolyploid rediploidization and has broad implications for future studies of WGD events.