Biology subjects

Fay, J. C.

Publications and source records attributed to Fay, J. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Deconstruction of Saccharomyces hybrids reveals multiple genomic regions required for thermotolerance

The genetic basis of phenotypic differences between species is fundamental to our view of evolution. In contrast to variation within species, phenotypic differences between species can result from a long-term sequential process whereby prior steps and multiple changes at a single locus can play important roles. However, investigating the genetic basis of phenotypes that have evolved over long time periods is limited by reproductive barriers between species. In this study, we use hybrid genetic analysis to examine reproductively isolated Saccharomyces species that have diverged in their thermal growth limits over the last 15 million years. By selectively eliminating S. cerevisiae chromosomes in hybrids with S. uvarum or S. kudriavzevii, we show that most chromosomes cause a substantial loss of thermotolerance. Reciprocal loss of chromosome 5 (Chr 5) confirms that the hybrid's thermotolerance depends on the S. cerevisiae chromosome and shows that it is partially inhibited by the S. uvarum chromosome. Using CRISPR-Cas9 to induce targeted loss of heterozygosity, we find that loss of either the left or right arm of Chr 5 results in loss of thermotolerance. Fine-mapping, complementation and reciprocal hemizygosity analysis identify MMS21, which encodes an essential subunit of the Smc5-Smc6 complex, as responsible for loss of thermotolerance associated with the left arm of Chr 5. Our findings suggest that divergence in thermotolerance over long time-periods is caused by multiple loci across the genome, with each playing a critical role in enabling growth at high temperatures.

evolutionary biology↗

bioRxiv2026

Functional diversification across yeast lineages of the nucleus-vacuole junction forming protein Nvj1

Nucleus-vacuole junctions (NVJs) in Saccharomyces cerevisiae serve as an inter-organellar hub for multiple cell processes, including lipid transport and biosynthesis, an intra-nuclear quality control mechanism, and piecemeal microautophagy of the nucleus. NVJs are formed by complexes between Vac8 in the vacuole membrane and Nvj1 in the nuclear envelope. Nvj1 also links the inner and outer nuclear membranes across the perinuclear lumen. Orthologs of Nvj1 had previously only been found in yeasts of the order Saccharomycetales, raising the possibility that NVJs are restricted to this clade. Using homology and synteny, we discovered scores of novel Nvj1 orthologs across 200 million years of evolution within the subphylum Saccharomycotina. Not all orthologs mediate NVJ formation when expressed in S. cerevisiae, and some lack a sequence motif necessary for this function. Furthermore, the sequence motif required for binding the oxysterol binding protein Osh1 and an associated novel sequence motif, both found only in the order Saccharomycetales, were independently lost in three different lineages. This collection suggests specific opportunities to explore evolutionary and functional adaptations of Nvj1s in ecologically and physiologically diverse yeasts.

cell biology↗

bioRxiv2025

Colonization of vineyards by non-Saccharomyces yeast species without evolution of copper and sulfite resistance

Humans have generated ecological and environmental disturbances, such as vineyards, across the globe. Disturbed environments create widespread and repeated selective pressures that can drive colonization and local adaptation in microbial species. We investigated the distribution of fermentative yeast species in vineyards compared to nearby arboreal habitats and measured their resistance to two commonly used vineyard antimicrobials, copper and sulfite. We analyzed 4,101 strains, representing 70 species, collected from grapevine- and oak-associated substrates at 17 vineyard and 20 non-vineyard sites in the USA and Slovenia. Species frequency varied with geography and substrate, but the majority of species commonly present in vineyards were also found in non-vineyard arboreal environments, representing a potential source for vineyard colonization and exploitation of sugar from grapes. Species varied in both copper and sulfite resistance, but only Saccharomyces cerevisiae showed elevated resistance in vineyard compared to non-vineyard samples. Our results indicate that S. cerevisiae has uniquely taken advantage of vineyard environments through adaptations that appear either unnecessary or inaccessible to other yeast species present in vineyards.

evolutionary biology↗

bioRxiv2024

Pervasive divergence in protein thermostability is mediated by both structural changes and cellular environments

Temperature is a universal environmental constraint and organisms have evolved diverse mechanisms of thermotolerance. A central feature of thermophiles relative to mesophiles is a universal shift in protein stability, implying that it is a major constituent of thermotolerance. However, organisms have also evolved extensive buffering systems, such as those that disaggregate and refold denatured proteins and enable survival of heat shock. Here, we show that both cellular and protein structural changes contribute to divergence in protein thermostability between two closely related Saccharomyces species that differ by 8{degrees}C in their thermotolerance. Using thermal proteomic profiling we find that 85% of S. cerevisiae proteins are more stable than their S. uvarum homologs and there is an average shift of 1.6{degrees}C in temperature induced protein aggregation. In an interspecific hybrid of the two species, S. cerevisiae proteins retain their thermostability, while the thermostability of their S. uvarum homologs is enhanced, indicating that cellular context contributes to protein stability differences. By purifying orthologous proteins we show that amino acid substitutions underlie melting temperature differences for two proteins, Guk1 and Aha1. Amino acid substitutions are also computationally predicted to contribute to stability differences for most of the proteome. Our results imply that coordinated changes in protein thermostability impose a significant constraint on the time scales over which thermotolerance can evolve.

evolutionary biology↗

bioRxiv2024

The distribution of beneficial mutational effects between two sister yeast species poorly explains natural outcomes of vineyard adaptation

Domesticated strains of Saccharomyces cerevisiae have adapted to resist copper and sulfite, two chemical stressors commonly used in winemaking. S. paradoxus, has not adapted to these chemicals despite being consistently present in sympatry with S. cerevisiae in vineyards. This contrast represents a case of apparent evolutionary constraints favoring greater adaptive capacity in S. cerevisiae. In this study, we used a comparative mutagenesis approach to test whether S. paradoxus is mutationally constrained with respect to acquiring greater copper and sulfite resistance. For both species, we assayed the rate, effect size, and pleiotropic costs of resistance mutations and sequenced a subset of 150 mutants isolated from our screen. We found that the distributions of mutational effects displayed by the two species were very similar and poorly explained the natural pattern. We also found that chromosome VIII aneuploidy and loss of function mutations in PMA1 confer copper resistance in both species, whereas loss of function mutations in REG1 were only a viable route to copper resistance in S. cerevisiae. We also observed a single de novo duplication of the CUP1 gene in S. paradoxus but none in S. cerevisiae. For sulfite, loss of function mutations in RTS1 and KSP1 confer resistance in both species, but mutations in RTS1 have larger average effects in S. paradoxus. Our results show that even when the distributions of mutational effects are largely similar, species can differ in the adaptive paths available to them. They also demonstrate that assays of the distribution of mutational effects may lack predictive insight concerning adaptive outcomes.

evolutionary biology↗

bioRxiv2024

Sodium azide mutagenesis induces a unique pattern of mutations

The nature and effect of mutations are of fundamental importance to the evolutionary process. The generation of mutations with mutagens has also played important roles in genetics. Applications of mutagens include dissecting the genetic basis of trait variation, inducing desirable traits in crops, and understanding the nature of genetic load. Previous studies of sodium azide-induced mutations have reported single nucleotide variants (SNVs) found in individual genes. To characterize the nature of mutations induced by sodium azide, we analyze whole-genome sequencing (WGS) of 11 barley lines derived from sodium azide mutagenesis, where all lines were selected for diminution of plant fitness owing to induced mutations. We contrast observed mutagen-induced variants with those found in standing variation in WGS of 13 barley landraces. Here, we report indels that are two orders of magnitude more abundant than expected based on nominal mutation rates. We found induced SNVs are very specific, with C[->]T changes occurring in a context followed by another C on the same strand (or the reverse complement). The codons most affected by the mutagen include the sodium azide-specific CC motif (or the reverse complement), resulting in a handful of amino acid changes and relatively few stop codons. The specific nature of induced mutations suggests that mutagens could be chosen based on experimental goals. Sodium azide would not be ideal for gene knockouts but will create many missense mutations with more subtle effects on protein function.

genomics↗

bioRxiv2023

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation in the ribosome. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8{Delta} mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8{Delta} suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8{Delta} suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1{Delta} mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1{Delta} temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation does not significantly inhibit GAAC activation. These results suggest that ribosomal protein mutations suppress the temperature sensitivity of S. pombe trm8{Delta} and tan1{Delta} mutants due to reduced ribosome concentrations, leading to both a reduced requirement for tRNA, and reduced ribosome collisions and GAAC activation. Results with S. cerevisiae trm8{Delta} trm4{Delta} mutants are consistent with this model, and fuel speculation that similar results will apply across eukaryotes.

molecular biology↗

bioRxiv2023

Divergence in the Saccharomyces species' heat shock response is indicative of their thermal tolerance

The Saccharomyces species have diverged in their thermal growth profile. Both S. cerevisiae and S. paradoxus grow at temperatures well above the maximum growth temperature of S. kudriavzevii and S. uvarum, but grow more poorly at lower temperatures. In response to thermal shifts, organisms activate a stress response that includes heat shock proteins involved in protein homeostasis and acquisition of thermal tolerance. To determine whether Saccharomyces species have diverged in their response to temperature we measured changes in gene expression in response to a 12{degrees}C increase or decrease in temperature for four Saccharomyces species and their six pairwise hybrids. To ensure coverage of subtelomeric gene families we sequenced, assembled and annotated a complete S. uvarum genome. All the strains exhibited a stronger response to heat than cold treatment. In response to heat, the cryophilic species showed a stronger response than the thermophilic species. The hybrids showed a mixture of parental stress responses depending on the time point. After the initial response, hybrids with a thermophilic parent were more similar to S. cerevisiae and S. paradoxus, and the S. cerevisiae x S. paradoxus hybrid showed the weakest heat shock response. Within the hybrids a small subset of temperature responsive genes showed species specific responses but most were also hybrid specific. Our results show that divergence in the heat shock response is indicative of a strains thermal tolerance, suggesting that cellular factors that signal heat stress or resolve heat induced changes are relevant to thermal divergence in the Saccharomyces species.

evolutionary biology↗

bioRxiv2023

Fluctuating selection facilitates the discovery of broadly effective but difficult to reach adaptive outcomes in yeast

Evolutionary compromises are thought to be common under fluctuating selection because the mutations that best enable adaptation to one environmental context can often be detrimental in others. Yet, prior experimental work has shown that generalists can sometimes perform as well as specialists in their own environments. Here we use a highly replicated evolutionary experiment (N = 448 asexual lineages of the brewers yeast) to show that even though fluctuation between two environmental conditions often induces evolutionary compromises (at least early on), it can also help reveal difficult to reach adaptive outcomes that ultimately improve performance in both environments. Specifically, we begin by showing that yeast adaptation to chemical stress can involve fitness tradeoffs with stress-free environments and that, accordingly, lineages that are repeatedly exposed to occasional stress tend to respond by trading performance for breadth of adaptation. We then show that on rare occasions, fluctuating selection leads to the evolution of no-cost generalists that can even outcompete constant selection specialists in their own environments. We propose that the discovery of these broader and more effective adaptive outcomes under fluctuating selection could be partially facilitated by changes in the adaptive landscape that result from having to deal with fitness tradeoffs across different environmental conditions. Overall, our findings indicate that reconciling the short- and long-term evolutionary consequences of fluctuating selection could significantly improve our understanding of the evolution of specialization and generalism.

evolutionary biology↗

bioRxiv2022

Using colony size to measure fitness in Saccharomyces cerevisiae

Competitive fitness assays in liquid culture have been a mainstay for characterizing experimental evolution of microbial populations. Growth of microbial strains has also been extensively characterized by colony size and could serve as a useful alternative if translated to per generation measurements of relative fitness. To examine fitness based on colony size, we established a relationship between cell number and colony size for strains of Saccharomyces cerevisiae robotically pinned onto solid agar plates in a high-density format. This was used to measure growth rates and estimate relative fitness differences between evolved strains and their ancestors. After controlling for edge effects through both normalization and agar-trimming, we found that fitness based on colony size is as sensitive as competitive fitness assays grown in liquid medium. While fitnesses determined from liquid and solid mediums were not equivalent, our results demonstrate that colony size provides a sensitive means of measuring fitness that is particularly well suited to measurements across many environments.

evolutionary biology↗

bioRxiv2022

Macroevolutionary diversity of traits and genomes in the model yeast genus Saccharomyces

Species is the fundamental unit to quantify biodiversity. In recent years, the model yeast Saccharomyces cerevisiae has seen an increased number of studies related to its geographical distribution, population structure, and phenotypic diversity. However, seven additional species from the same genus have been less thoroughly studied, which has limited our understanding of the macroevolutionary leading to the diversification of this genus over the last 20 million years. Here, we report the geographies, hosts, substrates, and phylogenetic relationships for approximately 1,800 Saccharomyces strains, covering the complete genus with unprecedented breadth and depth. We generated and analyzed complete genome sequences of 163 strains and phenotyped 128 phylogenetically diverse strains. This dataset provides insights about genetic and phenotypic diversity within and between species and populations, quantifies reticulation and incomplete lineage sorting, and demonstrates how gene flow and selection have affected traits, such as galactose metabolism. These findings elevate the genus Saccharomyces as a model to understand biodiversity and evolution in microbial eukaryotes.

evolutionary biology↗

bioRxiv2021

Cis-regulatory variants affect gene expression dynamics in yeast

Evolution of cis-regulatory sequences depends on how they effect gene expression and motivates both the identification and prediction of cis-regulatory variants responsible for expression differences within and between species. While much progress has been made in relating cis-regulatory variants to expression levels, the timing of gene activation and repression may also be important to the evolution of cis-regulatory sequences. We investigated allele-specific expression (ASE) dynamics within and between Saccharomyces species during the diauxic shift and found appreciable cis-acting variation in gene expression dynamics. Within species ASE is associated with intergenic variants, but ASE dynamics are more strongly associated with insertions and deletions than ASE levels. To refine these associations we used a high-throughput reporter assay to test promoter regions and individual variants. Within the subset of regions that recapitulated endogenous expression we identified and characterized cis-regulatory variants that affect expression dynamics. Between species, chimeric promoter regions generate novel patterns and indicate constraints on the evolution of gene expression dynamics. We conclude that changes in cis-regulatory sequences can tune gene expression dynamics and that the interplay between expression dynamics and other aspects expression are relevant to the evolution of cis-regulatory sequences.

evolutionary biology↗

bioRxiv2020

The structure and diversity of strain level variation in vaginal bacteria

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core-genomes for six species of vaginal bacteria: G. vaginalis, L. crispatus, L. iners, L. jensenii, L. gasseri, and A. vaginae. Despite inhabiting the same environment, strain diversity and structure varies across species. All species except L. iners are characterized by multiple distinct groups of strains. Even so, strain diversity is lower in the Lactobacillus species, consistent with a more recent colonization of the human vaginal microbiome. Both strain diversity and the frequency of multi-strain samples is related to species-level diversity of the microbiome in which they occur, suggesting similar ecological factors influencing diversity within the vaginal niche. We conclude that the structure of strain level variation provides both the motivation and means of testing whether strain level differences contribute to the function and health consequences of the vaginal microbiome. Data SummaryAll vaginal metagenomic sequence data generated for this project can be found on the Sequence Read Archive under BioProject PRJNA639592.

microbiology↗

bioRxiv2019

High-throughput experimental evolution using barcoded strains of Saccharomyces cerevisiae

BackgroundExperimental evolution of microbes can be used to empirically address a wide range of questions about evolution and is increasingly employed to study complex phenomena ranging from genetic evolution to evolutionary rescue. Regardless of experimental aims, fitness assays are a central component of this type of research, and low-throughput often limits the scope and complexity of experimental evolution studies. We created an experimental evolution system in Saccharomyces cerevisiae that utilizes genetic barcoding to overcome this challenge. ResultsWe first confirm that barcode insertions do not alter fitness and that barcode sequencing can be used to efficiently detect fitness differences via pooled competition-based fitness assays. Next, we examine the effects of ploidy, chemical stress, and population bottleneck size on the evolutionary dynamics and fitness gains (adaptation) in a total of 76 experimentally evolving, asexual populations by conducting 1,216 fitness assays and analyzing 532 longitudinal-evolutionary samples collected from the evolving populations. In our analysis of these data we describe the strengths of this experimental evolution system and explore sources of error in our measurements of fitness and evolutionary dynamics. ConclusionsOur experimental treatments generated distinct fitness effects and evolutionary dynamics, respectively quantified via multiplexed fitness assays and barcode lineage tracking. These findings demonstrate the utility of this new resource for designing and improving high-throughput studies of experimental evolution. The approach described here provides a framework for future studies employing experimental designs that require high-throughput multiplexed fitness measurements.

evolutionary biology↗

bioRxiv2019

Multiple changes underlie allelic divergence of CUP2 between Saccharomyces species.

Under the model of micromutationism, phenotypic divergence between species is caused by accumulation of many small-effect changes. While mapping the causal changes to single nucleotide resolution could be difficult for diverged species, genetic dissection via chimeric constructs allows us to evaluate whether a large-effect gene is composed of many small-effect nucleotide changes. In a previously described non-complementation screen, we found allele difference of CUP2, a copper-binding transcription factor, underlie divergence in copper resistance between Saccharomyces cerevisiae and S. uvarum. Here, we tested whether the allele effect of CUP2 was caused by multiple nucleotide changes. By analyzing chimeric constructs containing four separate regions in the CUP2 gene, including its distal promoter, proximal promoter, DNA binding domain and transcriptional activation domain, we found that all four regions of the S. cerevisiae allele conferred copper resistance, with the proximal promoter showing the largest effect, and that both additive and epistatic effects are likely involved. These findings support a model of multiple changes underlying evolution and suggest an important role of both protein coding and cis-regulatory changes in evolution.

evolutionary biology↗

bioRxiv2018

The fate of deleterious variants in a barley genomic prediction population

Targeted identification and purging of deleterious genetic variants has been proposed as a novel approach to animal and plant breeding. This strategy is motivated, in part, by the observation that demographic events and strong selection associated with cultivated species pose a \"cost of domestication.\" This includes an increase in the proportion of genetic variants where a mutation is likely to reduce fitness. Recent advances in DNA resequencing and sequence constraint-based approaches to predict the functional impact of a mutation permit the identification of putatively deleterious SNPs (dSNPs) on a genome-wide scale. Using exome capture resequencing of 21 barley 6-row spring breeding lines, we identify 3,855 dSNPs among 497,754 total SNPs. In order to polarize SNPs as ancestral versus derived, we generated whole genome resequencing data of Hordeum murinum ssp. glaucum as a phylogenetic outgroup. The dSNPs occur at higher density in portions of the genome with a higher recombination rate than in pericentromeric regions with lower recombination rate and gene density. Using 5,215 progeny from a genomic prediction experiment, we examine the fate of dSNPs over three breeding cycles. Average derived allele frequency is lower for dSNPs than any other class of variants. Adjusting for initial frequency, derived alleles at dSNPs reduce in frequency or are lost more often than other classes of SNPs. The highest yielding lines in the experiment, as chosen by standard genomic prediction approaches, carry fewer homozygous dSNPs than randomly sampled lines from the same progeny cycle. In the final cycle of the experiment, progeny selected by genomic prediction have a mean of 5.6% fewer homozygous dSNPs relative to randomly chosen progeny from the same cycle.\n\nAuthor SummaryThe nature of genetic variants underlying complex trait variation has been the source of debate in evolutionary biology. Here, we provide evidence that agronomically important phenotypes are influenced by rare, putatively deleterious variants. We use exome capture resequencing and a hypothesis-based test for codon conservation to predict deleterious SNPs (dSNPS) in the parents of a multi-parent barley breeding population. We also generated whole-genome resequencing data of Hordeum murinum, a phylogenetic outgroup to barley, to polarize dSNPs by ancestral versus derived state. dSNPs occur disproportionately in the gene-rich chromosome arms, rather than in the recombination-poor pericentromeric regions. They also decrease in frequency more often than other variants at the same initial frequency during recurrent selection for grain yield and disease resistance. Finally, we identify a region on chromosome 4H that strongly associated with agronomic phenotypes in which dSNPs appear to be hitchhiking with favorable variants. Our results show that targeted identification and removal of dSNPs from breeding programs is a viable strategy for crop improvement, and that standard genomic prediction approaches may already contain some information about unobserved segregating dSNPs.

genomics↗

bioRxiv2018

Mitochondria-encoded genes contribute to the evolution of heat and cold tolerance among Saccharomyces species

Over time, species evolve substantial phenotype differences. Yet, genetic analysis of these traits is limited by reproductive barriers to those phenotypes that distinguish closely related species. Here, we conduct a genome-wide non-complementation screen to identify genes that contribute to a major difference in thermal growth profile between two Saccharomyces species. S. cerevisiae is capable of growing at temperatures exceeding 40{degrees}C, whereas S. uvarum cannot grow above 33{degrees}C but outperforms S. cerevisiae at 4{degrees}C. The screen revealed only a single nuclear-encoded gene with a modest contribution to heat tolerance, but a large effect of the species mitochondrial DNA (mitotype). Furthermore, we found that, while the S. cerevisiae mitotype confers heat tolerance, the S. uvarum mitotype confers cold tolerance. Recombinant mitotypes indicate multiple genes contribute to thermal divergence. Mitochondrial allele replacements showed that divergence in the coding sequence of COX1 has a moderate effect on both heat and cold tolerance, but it does not explain the entire difference between the two mitochondrial genomes. Our results highlight a polygenic architecture for interspecific phenotypic divergence and point to the mitochondrial genome as an evolutionary hotspot for not only reproductive incompatibilities, but also thermal divergence in yeast.

evolutionary biology↗

bioRxiv2018

Mitochondrial DNA and temperature tolerance in lager yeasts

A growing body of research suggests that the mitochondrial genome (mtDNA) is important for temperature adaptation. In the yeast genus Saccharomyces, species have diverged in temperature tolerance, driving their use in high or low temperature fermentations. Here we experimentally test the role of mtDNA in temperature tolerance in synthetic and industrial hybrids (Saccharomyces cerevisiae x Saccharomyces eubayanus, or Saccharomyces pastorianus), which cold-brew lager beer. We find that the relative temperature tolerances of hybrids correspond to the parent donating mtDNA, allowing us to modulate lager strain temperature preferences. The strong influence of mitotype on the temperature tolerance of otherwise identical hybrid strains provides support for the mitochondrial climactic adaptation hypothesis in yeasts and demonstrates how mitotype has influenced the world's most commonly fermented beverage.\n\nOne Sentence SummaryMitochondrial genome origin affects the temperature tolerance of synthetic and industrial lager-brewing yeast hybrids.

genetics↗