Hybrid adaptation is hampered by Haldane’s sieve

Bautista, Carla; Gagnon-Arsenault, Isabelle; Utrobina, Mariia; Fijarczyk, Anna; Bendixsen, Devin P.; Stelkens, Rike; Landry, Christian R.

doi:10.1038/s41467-024-54105-4

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Published: 28 November 2024

Hybrid adaptation is hampered by Haldane’s sieve

Nature Communications volume 15, Article number: 10319 (2024) Cite this article

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Abstract

Hybrids between species exhibit plastic genomic architectures that could foster or slow down their adaptation. When challenged to evolve in an environment containing a UV mimetic drug, yeast hybrids have reduced adaptation rates compared to parents. We find that hybrids and their parents converge onto similar molecular mechanisms of adaptation by mutations in pleiotropic transcription factors, but at a different pace. After 100 generations, mutations in these genes tend to be homozygous in the parents but heterozygous in the hybrids. We hypothesize that a lower rate of loss of heterozygosity (LOH) in hybrids could limit fitness gain. Using genome editing, we first demonstrate that mutations display incomplete dominance, requiring homozygosity to show full impact and to entirely circumvent Haldane’s sieve, which favors the fixation of dominant mutations. Second, tracking mutations in earlier generations confirmed a different rate of LOH in hybrids. Together, these findings show that Haldane’s sieve slows down adaptation in hybrids, revealing an intrinsic constraint of hybrid genomic architecture that can limit the role of hybridization in adaptive evolution.

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Introduction

Hybridization rapidly generates novel genotypes that can also lead to new and sometimes extreme phenotypes^1,2,3,4,5. As a result, hybrids may thrive and often outcompete their parents^6,7,8. Empirical and theoretical work has shown that hybridization can promote rapid evolution^{2,3,9,10,11,12,13,14,15}, including during evolutionary rescue, species diversification, and adaptive radiations^{2,11,12,16,17,18}. The generation of adaptive diversity through hybridization has long been successfully employed in biotechnology for fermentation^19,20,21 and in agriculture for crop improvement^22,23,24.

The combination of divergent genomes in the same organism can also lead to genomic instability^25,26. From microorganisms^27,28 to multicellular eukaryotes such as plants²⁹ and vertebrates³⁰, genomic instability has been frequently observed in hybrids. While the causes of instability are not always clear, evidence points to the alteration of the molecular pathways and components responsible for genome stability themselves. For instance, cell cycle checkpoints and DNA repair pathways observed to be altered in hybrids^31,32 can lead to inaccurate chromosome segregation^33,34, resulting in changes in ploidy^35,36,37,38, aneuploidies^39,40,41, and elevated mutation rates^42,43,44,45.

In spite of these potential negative consequences, genomic instability can also paradoxically enhance F1 hybrid traits. One example is the restoration of meiotic recombination through genome homogenization, which contributes to rescuing hybrid fertility⁴⁶. In this scenario, recurrent loss of heterozygosity (LOH) facilitates crossing over by providing segments of sequence similarity, thereby promoting proper chromosome segregation. Similarly, whole-genome duplication can also restore fertility in interspecific hybrids⁴⁷. However, the enhancement of these hybrid traits is typically observed under stable laboratory conditions. Some environmental conditions and stresses could further enhance genome instability, so what appears as a feature that enhances adaptability in benign conditions could become a liability in extreme ones. Motivated by this question, in a previous study⁴⁸, we produced F1 hybrids between the budding yeasts Saccharomyces cerevisiae and Saccharomyces paradoxus to measure their adaptive potential in an environment containing a drug that increases genomic instability by mimicking UV radiation^49,50. These two species diverged 5–10 million years ago^{51,52,53,54,55}, live in similar ecological niches in nature⁵⁶, and carry signs of introgression in their mitochondrial and nuclear genomes with adaptive potential in some cases^{46,57,58,59,60}. Replicated experimental evolution across 100 generations revealed that hybrids showed smaller fitness gains than their parents in conditions mimicking UV radiation⁴⁸.

Here, we investigate the genomic and genetic bases of differential adaptation rates in hybrids by testing two non-mutually exclusive hypotheses: (1) Hybrid adaptation to UV radiation is hampered by genomic instability, and (2) Hybrids do not have access to the same adaptive mutations as the parental species. We use Whole Genome Sequencing (WGS) of 270 ancestral and evolved clones of hybrid and parental genotypic backgrounds to: (1) Investigate major genomic changes in copy number, such as ploidy changes, aneuploidies, or LOH, to determine if they are more prevalent in hybrid genomes, and (2) Identify potentially differential de novo mutations in hybrids compared to parents. We functionally validate seven putative adaptive mutations with site-directed mutagenesis in both S. cerevisiae and S. paradoxus haploid backgrounds and by CRISPR-Cas9 genome editing. We examine if there are differences in the population dynamics of adaptive mutations between hybrids and parents by tracking genotype frequency over time through the historical resurrection of frozen stocks. We find that neither of our two starting hypotheses is supported. Rather, adaptation to UV mimetic conditions often proceeds through two types of mutations, nucleotide substitution and LOH, the second occurring more slowly in the hybrids, and thus slowing down their rate of adaptation.

Results

Adaptive dynamics of hybrids and parents in UV mimetic conditions

We previously evolved 90 populations (30 F1 hybrid replicates and 30 replicates of each of the parental S. cerevisiae and S. paradoxus genotypic backgrounds) for 100 generations in the presence of a DNA damaging agent, the UV mimetic drug 4-nitroquinoline 1-oxide (4-NQO), and in a control condition (rich medium) (Fig. 1a)⁴⁸. We found that hybrids achieved a lower rate of adaptation compared to the parental genotypic backgrounds, with a lower average gain of fitness over the course of evolution (Fig. 1b).

**Fig. 1: Hybrids show a reduced adaptation rate in UV mimetic conditions.**

To compare the number and type of genetic changes between evolved hybrid and parental populations, we measured DNA content by flow cytometry and sequenced the genomes of 269 isolated clones (refer to the Methods section for detailed information on the sample size) derived from the evolved populations and their ancestors (Fig. 1a and Supplementary Data 1) (genome-wide mean read depth of 100X, Supplementary Fig. 1). In parallel, we measured the growth of these isolated clones in control and UV mimetic conditions (Supplementary Fig. 2). We confirmed our previous finding that evolved hybrids showed significantly lower growth rate than parental replicated populations when evolved under UV mimetic conditions (Supplementary Fig. 2, all p < 0.001). The growth rates of isolated clones from populations and that of their populations of origin were strongly correlated (Fig. 1c: rs = 0.75, p < 0.0001), supporting that the growth of isolated clones is representative of the general fitness dynamics observed in the previous evolution experiment⁴⁸. From now on, we will refer to lines when discussing observations on these isolated clones derived from their populations of origin, and we use growth rate as a proxy for fitness. While we acknowledge that growth rate measurements do not directly capture all fitness components, our findings demonstrate an improvement in growth rate, which provides evidence of selection acting on this trait.

Major genomic changes in copy number reveal increased genomic instability in UV mimetic conditions

The majority of lines remained diploid during evolution. Ploidy changes were generally not more frequent under UV mimetic conditions (Supplementary Figs. 3 and 4). Likewise, we did not observe a larger number of whole-genome level ploidy changes in hybrids in UV mimetic compared to control conditions, suggesting that ploidy changes do not account for the observed reduction in hybrid adaptation rate (Supplementary Fig. 3). Conversely, evolution under UV mimetic conditions did result in a larger number of lines with aneuploidies in hybrid and both parental genotypic backgrounds when compared to control conditions (Fig. 2a). While only the increase in aneuploidy rate observed in S. paradoxus was statistically significant (Supplementary Fig. 5) compared to control conditions, the presence of aneuploidies predominantly in UV mimetic-treated populations suggests that UV mimetic exposure may compromise genome stability across all genotypes. However, the number of aneuploidies did not correlate with the increases in growth rate observed in any group (Supplementary Fig. 6, rs = − 0.18, p > 0.05; rs = − 0.22, p > 0.05 and, rs = − 0.27, p > 0.05; for S. cerevisiae, S. paradoxus, and hybrid respectively). Hybrids also did not show a larger number of aneuploidies compared to the parents, making it unlikely to explain their reduced adaptability (Fig. 2a).

**Fig. 2: Genomic changes accumulating during experimental evolution in parents and hybrids.**

The analysis of the relative read depth revealed a particular type of alteration, for instance, in chromosomes XII and XV (Fig. 2a). Intriguingly, gains and losses occurred simultaneously in the homologous chromosomes of S. cerevisiae and S. paradoxus, i.e., when a hybrid lost a portion of the S. cerevisiae chromosome, it simultaneously gained a portion of the homologous S. paradoxus one. This prompted us to map the chromosome relative read depth to determine if there were patterns in the distribution of species-specific chromosome gains and losses. The observed changes affected large regions of chromosomes (Fig. 2b). These patterns can be caused by the reciprocal exchange of segments of homologous chromosomes in diploids during mitosis, resulting in LOH (Fig. 2c)^61,62. We identified LOH as regions where there were simultaneous increases and decreases in relative read depth in homologous chromosomes along a size threshold of 20 kb (Supplementary Fig. 7). We detected two types of LOH, interstitial LOHs (i-LOHs), which often originate from gene conversion involving short exchanges, and terminal LOHs (t-LOHs), typically resulting from mitotic crossovers that encompass larger regions^61,63. We identified some large i-LOHs (for example, in line 17 on chromosome XIV, Supplementary Fig. 7) but we focused on t-LOHs, which were more frequent.

The number of t-LOH events in hybrid genomes was significantly positively correlated with chromosome size (Supplementary Fig. 8a, rs = 0.81, p < 0.05). The pattern of t-LOHs showed similarities across hybrid lines, with specific regions enriched on chromosomes XII and XV (Fig. 2d). These regions have been previously identified to be susceptible to t-LOHs⁶⁴. Certain t-LOHs were found to cluster around positions enriched with repetitive loci, such as the rDNA locus on chromosome XII^61,65 or the gene STE4, another common t-LOH target on chromosome XV⁶⁶. We found a much larger number of hybrid lines with t-LOHs when evolved under UV mimetic conditions (96% of the lines) compared to control conditions (8% of the lines) (Fig. 2e, p < 0.0001). Only one line evolved under UV mimetic conditions did not show any t-LOH (4%). This suggests that either DNA damage triggers t-LOH, thus enhancing its occurrence rate, or that t-LOH is particularly advantageous under these conditions and selected for. Advantageous LOHs have been associated with fitness increase in both the brewery and natural settings, generally under stressful environmental conditions^64,67. However, LOHs in themselves may not cause variation in the rates of adaptation among hybrid lines. We indeed did not find a significant correlation between the increase in growth rate (%) and the number of t-LOH events (Supplementary Fig. 8b, rs = 0.33, p > 0.05).

In summary, although we cannot examine t-LOHs in parental genomes due to their overall homozygosity, we found that aneuploidy frequencies are similar across parental species and hybrids, suggesting that overall genome instability is not specifically increased in hybrids. There is, therefore, no support for our initial hypothesis that increased genomic instability hampers adaptation in hybrids.

Parents and hybrids have parallel access to adaptive de novo mutations in the same genes

Lower rates of adaptation to UV mimetic conditions may also be explained by hybrids not having access to the same adaptive mutations as the parental lines, as per our alternative hypothesis. For instance, some mutations could have strong background-dependent effects. We investigated whether hybrids show single nucleotide polymorphisms (SNPs) in similar genes or gene functions, focusing on missense variants, as their potential impacts are easier to interpret, and data is more robust in this category. We found a median of 20 missense variants per line in S. cerevisiae, 12 in S. paradoxus, and 42 in the hybrids (Supplementary Fig. 9), with a comparable trend in control conditions. A gene ontology (GO) analysis revealed differences in terms of the biological functions affected by mutations but also some similarities (Supplementary Fig. 10). Enrichments are not significant, but interestingly, the most enriched GO terms for S. cerevisiae included double-strand break repair via sister chromatid exchange (GO:1990414), potentially aiding in coping with 4-NQO-induced DNA breaks, and the regulation of cell differentiation (GO:0045595). For S. paradoxus, the most enriched terms included ER-associated misfolded protein catabolic processes (GO:0071712) and endocytic vesicles (GO:0030139), potentially disrupting the invagination of extracellular substances, such as the 4-NQO drug (UV mimetic conditions). In the hybrid, the most enriched terms comprised trehalose metabolic process (GO:0005991) and ABC-type transporter activity (GO:0140359), comprising efflux pumps involved in expelling xenobiotic compounds, such as 4-NQO.

We found that mutations occurred in parallel in two specific genes across all three genotypic backgrounds (Fig. 3a), suggesting that the same molecular changes provide strong selective advantages in both hybrids and parents. The most frequent parallel changes involved the pleiotropic drug response genes PDR1 and YRR1 (Fig. 3a). PDR1 and YRR1 encode zinc finger transcription factors regulating multidrug and stress responses^68,69,70. Among their targets is PDR5, a well-characterized yeast efflux pump that actively transports toxic compounds out of the cell^71,72. Three S. cerevisiae lines, eight S. paradoxus lines, and eight hybrid lines had non-synonymous mutations in PDR1, while each genotypic background had at least four lines with YRR1 mutations (Fig. 3a). This is a surprising level of parallelism, given that drug resistance mutations have been shown to sometimes be genotype-specific⁷³ and that S. cerevisiae and S. paradoxus diverged 10 million years ago.

Fig. 3: PDR1 shows parallel adaptive changes among genotypic backgrounds. — **Fig. 3: *PDR1* shows parallel adaptive changes among genotypic backgrounds.**

From here on, we will focus on the analysis of PDR1 since it was the most often mutated gene. Non-synonymous mutations in PDR1 were not randomly located along the gene but instead occurred in particular clusters that overlapped among all three genotypic backgrounds (Supplementary Fig. 11) when mapped on the protein tertiary structure (Fig. 3b). We found identical or similar substitutions as those from previous studies on S. cerevisiae exposed to different drugs, ethanol, and antifungals^{74,75,76,77,78} (Fig. 3b). Localization on the protein structure revealed the presence of a cluster also found in other species, for instance, in the pathogen Nakaseomyces glabratus (Supplementary Fig. 12a, b), for which antifungal resistance often arises from mutations in PDR1^{79,80,81,82,83,84,85}. Parallelism even occurred at the level of amino acid changes across genotypic backgrounds (Fig. 3c). For instance, a mutation at amino acid 308/307 (corresponding to the respective S. cerevisiae and S. paradoxus positions) independently occurred up to four times. This specific amino acid change has also been observed in other studies⁷⁵, conferring resistance to the same UV mimetic drug we employed (4-NQO)⁷⁴.

The identification of shared adaptation hotspots and shared amino acid changes suggests a common adaptive landscape across genotypic backgrounds, indicating that specific regions within PDR1 and YRR1 harbor similar potential for adaptive mutations to occur in both hybrid and parents.

Fitness increases with the copy number of PDR1 adaptive alleles

The occurrence of mutations in PDR1 across all three (hybrid and two parental) genotypic backgrounds raises an intriguing question: Why do these mutations not confer similar adaptive benefits to hybrids as they do to the parental species? We observed that some lines carrying PDR1 mutations showed particularly high increases in growth rate (%) under UV mimetic conditions within their own genotype (Fig. 3c - S. cerevisiae lines: 2, 25 and 27; S. paradoxus lines: 29, 22, 28, 27, 9 and 24; hybrid lines: 30, 27, 13, 17 and 28). A closer analysis revealed that all these lines share a common characteristic, namely the presence of multiple mutated copies of PDR1 (Fig. 3c), with the exception of two lines (line 17 of hybrid and line 9 of S. paradoxus), which are discussed in detail below. Although mutations are expected to initially be heterozygous, we observed a diversity of genotypes at the PDR1 locus. While some lines have undergone LOH that made the mutations homozygous, others show changes in ploidy, resulting in an increased number of chromosomes harboring PDR1 mutations (Fig. 3c). The regions of LOH (t-LOH) found in the hybrids on chromosome VII, which result in the homozygosity of PDR1 mutations (lines 13 and 30 in Supplementary Fig. 7), confer an adaptive advantage as homozygous lines have higher growth rates than heterozygous ones (see the comparison among lines 30 and 13 versus lines 24, 9 and 2 in Fig. 3c). An exception occurred in hybrid line 17, where heterozygosity leads to increased growth rates (%) comparable to that of homozygous lines, potentially attributed to additional mutations in genes like PDR18, which codes for a xenobiotic transporter⁸⁶. Another exception is observed in line 9 of S. paradoxus (Fig. 3c). While being heterozygous for the PDR1 mutation, this line’s high improvement in growth rate is likely due to an additional mutation in the YRR1 transcription factor, resulting in simultaneous mutations of both transcription factors, potentially increasing their activity.

Although not exhaustively investigated here, our observations in YRR1 also suggest that LOHs confer a selective advantage at this locus. Mutations in the hybrid lines consistently coincide with LOH around a specific region of chromosome XV (hybrid lines 5, 6, 8, and 15, in Supplementary Fig. 7). This suggests that mutations in this gene were followed by LOH, rendering the mutation homozygous and potentially conferring a fitness advantage. We could not compare the potential benefits of homozygous vs. heterozygous YRR1 mutations as all hybrids were homozygous for this gene. But given that YRR1 encodes a transcriptional regulator, like PDR1, we suspect that the adaptive mutations displayed incomplete dominance and their full adaptive impact is only achieved following LOH. We expand on this below for PDR1.

Fitness increased as a function of the number of PDR1 mutated copies in all three genotypic backgrounds (Fig. 3d, e top panel rs = 0.55, p < 0.05). This effect was particularly strong in hybrids (Fig. 3e bottom panel, rs = 0.79, p < 0.05). Among S. paradoxus and hybrid lines, the two with the largest observed increase in growth rate within their respective genotypic contexts (134% in S. paradoxus line 24 and 123% in hybrid line 28) each carried three copies of the PDR1 mutant alleles (Fig. 3c, d). Hybrid line 28 showed a complex pattern in which a whole-chromosome LOH was combined with an increase in ploidy (Fig. 3c*, Supplementary Figs. 13 and 7).

Taken together, our findings demonstrate an association between genomic changes leading to the amplification of mutated PDR1 copies and increases in growth rate (%) in UV mimetic conditions. Hybrids with PDR1 mutations do show improvements in growth rate (%) but to a smaller extent than both parental lines. This advantage could derive from the fact that parental genomes achieve higher levels of homozygosity for these mutations (Fig. 3c): The LOH frequency in hybrids containing PDR1 mutations was 37.5% (3/8 hybrid lines had a homozygous PDR1 mutation), whereas it was 100% across the parental S. cerevisiae lines (3/3) and 62.5% across S. paradoxus lines (5/8), making PDR1 mutations more visible to selection in parental lines. Thus, the limiting factor may not be the rate and type of mutation but their limited allelic amplification in hybrids (Fig. 4a).

Fig. 4: PDR1 mutations show additive phenotypes such that allele copy number correlates with gains in growth. — **Fig. 4: *PDR1* mutations show additive phenotypes such that allele copy number correlates with gains in growth.**

The challenge of attaining homozygosity accounts for the reduced adaptive potential of hybrids

The likelihood and rate of a mutation becoming fixed in a population is shaped by the architecture of the trait under selection, including allelic dominance, and the strength of selection. Therefore, not only does an adaptive mutation need to occur, but it also needs to be in a proper genotype for the individuals to fully benefit from it⁸⁷. Dominant mutations are more likely to become fixed than recessive ones, a principle referred to as Haldane’s sieve^88,89,90. This poses a problem to non-obligate sexual species and in systems that only rarely reproduce sexually, like many unicellular microbes such as fungi. In asexual populations, recessive or incompletely dominant beneficial mutations can bypass Haldane’s sieve by achieving homozygosity through LOH⁹¹, although completely recessive alleles would be invisible to selection and could thus be lost before an LOH occurs. The rate of LOH is not uniform among genotypes, and it has been shown to be lower in heterozygous ones⁹². In hybrids, which are highly heterozygous, LOH rates are even more limited (Fig. 4a)^41,44. Once a beneficial mutation occurs in the parental genomes, high rates of mitotic recombination can rapidly lead to LOH at the site where the mutation occurred and make a mutation homozygous. This would occur at a lower rate in hybrids, slowing down the rate of adaptation.

In order to test if Haldane’s sieve slows down adaptation in hybrids, we need to assess these conditions: (1) The initial PDR1 mutation is recessive or incompletely dominant, requiring homozygosity to unmask its full fitness benefits. We thus predict that homozygous PDR1 mutants will display higher fitness than heterozygous PDR1 mutants; (2) LOH occurs at a slower pace in hybrid genomes^41,44,92.

We first validated the adaptiveness of specific PDR1 mutations. Site-directed mutagenesis was used to introduce seven candidate mutations (G280R, G280S, M308I, and G1042W for S. cerevisiae; G279R, G279S, and G281V for S. paradoxus) on a plasmid carrying either S. cerevisiae or S. paradoxus PDR1 gene. After introducing these plasmids into an S. cerevisiae strain (BY4741) deleted for PDR1 (pdr1Δ), we found that the mutations conferred significantly higher growth rates in the presence of the UV mimetic drug compared to the wild-type (WT) (Supplementary Fig. 14a). These mutations also conferred benefits in the parental backgrounds with slight variations in the extent of effects (Supplementary Fig. 15). To further confirm the effects derived from the transcription factor activity of PDR1, we measured expression of the downstream drug efflux pump Pdr5^71,72. We fused Pdr5 to a Green Fluorescent Protein (mEGFP) and measured its expression in the same strain (BY4741) containing PDR1 mutations on a plasmid. The Pdr5-mEGFP strain exhibited higher fluorescence levels when carrying a plasmid containing specific PDR1 mutations, compared to wild-type (WT) (Supplementary Fig. 14b). Consistent with this observation, we found that the same PDR1 mutations also lead to resistance to several drugs other than 4-NQO (Supplementary Fig. 12c).

To assess the dominance of PDR1 mutations in a reference genomic background, we generated homozygous and heterozygous diploid mutants using CRISPR-Cas9 genome editing and by mating S. cerevisiae lab strains BY4741 and BY4742. We focused on the M308I mutation because it showed the most significant increase in growth rate (%) and activation of the Pdr5 efflux pump (Supplementary Fig. 14). We found that in the homozygous state, this mutation conferred a higher fitness advantage than in the heterozygous state (Fig. 4b), confirming its incomplete dominance. The hemizygote also showed a higher growth rate than the heterozygote, suggesting that the mutated allele confers higher benefits in the absence of the WT allele. We tested this assumption in S. paradoxus, specifically in line 27, which contained the same mutation in PDR1 (M307I) (Fig. 3c). We observed a similar pattern of incomplete dominance when comparing the various genotypes (see Supplementary Fig. 16). Since we lacked an evolved S. cerevisiae line with this specific mutation (Fig. 3c), we crossed haploid strains of the constructed PDR1 mutant (M308I) in the BY4741/2 genetic backgrounds and of the line 27 of S. paradoxus to generate both homozygous and heterozygous hybrids for the PDR1 locus. The pattern remained consistent among the hybrids, where homozygous mutants exhibited enhanced growth rates compared to their heterozygous counterparts (Supplementary Fig. 16). Taken together, our findings confirm the first key assumption that (1) the initial PDR1 mutation exhibits incomplete dominance and requires homozygosity to fully contribute to adaptation.

We archived populations regularly over the course of experimental evolution⁴⁸, so we could revive the frozen stocks and isolate some clones to determine the timing of appearance of the various PDR1 mutations. We revived ~ 350 clones from nine hybrid and parental populations that harbor homozygous PDR1 mutations at the end of the experimental evolution (based on WGS data) and sequenced the PDR1 locus using amplicon Sanger sequencing. We identified three types of zygosity at the PDR1 locus at intermediate time points (Fig. 4c): heterozygous, homozygous WT, and homozygous mutant. As expected for de novo mutations in diploids, mutations were consistently first detected as heterozygous (Fig. 4d), but in some cases, in the parental populations, the LOH was so rapid that we also detected homozygous mutants (Fig. 4c left).

This trend aligns with the increase in growth rate (%) recorded during experimental evolution in their populations of origin but also when analyzing specific isolated clones⁴⁸ (Supplementary Figs. 17 and 18, respectively). Mutations also became homozygous later (Fig. 4d), but with the important difference that the parents become homozygous at a higher frequency than the hybrids. This pattern persisted across all experimental populations: Homozygous genotypes appeared quickly after the initial PDR1 mutations occurred, and spread rapidly in the parental populations, whereas in the hybrids, even by generation 95, a high proportion of heterozygotes were still observed (Fig. 4d, see Supplementary Fig. 17 for detailed analysis). Supporting this trend, we saw in some of the parents (population 2 and 25 of S. cerevisiae, population 27 and 29 of S. paradoxus) that the emergence of homozygous and heterozygous mutations coincided, indicating that the mechanism of adaptation through LOH can operate quickly (Fig. 4c, d). The relative frequency of each mutation in each population through time further shows that the proportion of homozygous mutants was much lower in hybrids compared to parental species (Fig. 4d). To verify that the low homozygote frequency in hybrids was not due to reduced homozygote fitness in the specific hybrid genotypic background, we compared hybrid growth rates across generations and populations and confirmed that homozygotes were fitter than heterozygotes and homozygotes WT (Fig. 4e and Supplementary Fig. 18 for growth rate measurements across generations). These findings validate our final assumption that (2) LOH occurs at a slower pace in hybrids.

Discussion

Hybridization is a recurring phenomenon in nature that has captured the interest of scientists for decades^{1,9,14,93,94,95,96} in fundamental fields but also in applied research such as in agriculture and medical microbiology^{23,24,27,97,98}. The adaptive and non-adaptive roles of hybridization have been studied extensively^{8,46,99,100,101,102,103,104,105,106,107}. However, investigations on the negative consequences have mostly focused on reproductive isolation^108,109 and less on the potential reduction in adaptive rates of hybrids. We previously evolved hybrids of S. cerevisiae and S. paradoxus species during 100 generations under stress that mimics UV radiation and observed a reduced adaptive potential of hybrids⁴⁸. We used this system to investigate what could reduce the hybrid rate of adaptation.

We first observed that hybrid genomes exhibit similar rates of aneuploidy compared to parental species. Indeed, our findings reveal markedly lower levels of aneuploidy compared to prior investigations conducted in contexts such as experimental evolution under selective pressures¹¹⁰, industrial or natural environments²⁶. This notable variation in aneuploidy prevalence among hybrids hints at the potential influence of environment-dependent genetic effects, as demonstrated in a previous study using the same set of strains⁴¹.

Our results also reveal that hybrid and parental species have access to the same adaptive changes in key genes. In principle, adaptation can, therefore, occur through the same mechanisms and at the same rate. We examined the cases of mutations that impacted a transcriptional factor involved in drug resistance because they displayed strong parallelism. The mutations displayed incomplete dominance, thus partially avoiding Haldane’s sieve - the bias against the establishment of recessive beneficial mutations. However, achieving homozygosity through LOH allowed them to fully circumvent this filter^88,89,90. Experiments involving the evolution of heterozygous yeast populations have also shown that LOH frequently unmasks beneficial recessive alleles, which can confer significant fitness advantages^{91,111,112,113}. Because LOH depends on recombination and recombination depends on sequence identity^41,44,92, LOH occurs at a slower rate in hybrids, ultimately contributing to slowing down hybrid adaptation. This contrasts with de novo mutations, which accumulate in yeast hybrid genotypes at rates that are similar to those observed in the parental species¹¹⁴. Similarly, the absence of a higher frequency of aneuploidy in hybrids cannot compensate for the delayed onset of LOH in increasing mutated gene copy numbers. While this reduced rate of LOH represents one potential mechanism influencing hybrid adaptation, it is likely not the sole driver. Additional exploration may uncover alternative genetic interactions contributing to the lower adaptation of hybrid lines.

T-LOH events frequently originate in conserved regions because they contain similar sequences between chromosome pairs, facilitating the initiation of recombination. In the case of PDR1, since it is located near the centromere, recombination might be initiated in centromeric sequences that are highly conserved among related budding yeast species^115,116. Other elements, such as highly repetitive retrotransposons, can also trigger the initiation of mitotic recombination¹¹⁷. The observed high frequency of YRR1 mutations associated with t-LOHs in all examined hybrid lines (4/4) suggests a potentially elevated rate of LOH for this gene. This could be attributed to increased sequence homology between recombination-triggering regions. For instance, the t-LOHs we observed involving YRR1 may have originated around a Ty1 long-terminal repeat (LTR) (YORCdelta11). It is thus possible that not all loci, where adaptive mutations could occur, are limited to contributing to adaptation because of the low LOH rate in hybrids. Although not explored here, t-LOH in hybrids could also limit adaptation in other ways than simply occurring at a lower rate around the adaptive mutations. Since mitotic recombination often extends along the entire length of a chromosome arm^61,63 in heterozygous genomes⁶², an LOH that renders a beneficial mutation homozygous could also bring along other molecular changes or combination of changes that would negatively impact fitness⁶⁶. For example, Feri et al.¹¹⁸ demonstrated in Candida albicans the presence of several recessive lethal alleles that restrict LOH events. We also cannot disregard the importance of the hybrid background itself and the impact of interactions between divergent genomes, such as epistasis and hybrid incompatibilities^{119,120,121,122} which could modulate the fitness consequences of LOH.

Our findings contribute to the understanding of the genomic factors shaping asexual microbes. Such hybrids often evolve during domestication, for instance, many beer yeasts are among-species hybrids^123,124 and these hybrids are known to be largely sterile, i.e., to not have access to sexual reproduction¹²⁵. Even fungal pathogens evolve through recurrent hybridization events^{27,98,126,127,128,129,130}, acquire antifungal resistance and adapt to new hosts with de novo mutations and LOH^{131,132,133,134,135}. It has been shown for instance that to confer full resistance to antifungals, a mutation in a transcriptional regulator needed to be followed by an LOH in the fungal pathogen Candida albicans¹³⁶. Antimicrobial resistance has also been shown to depend on an LOH event in S. cerevisiae that is required again to render an LOF mutation homozygous^137,138. Understanding which conditions could slow down the rate of LOH, such as heterozygosity along the chromosome as we exemplify here, or the physical linkage to potentially deleterious mutations¹¹⁸, is therefore key to understanding evolution in an applied context such as antimicrobial resistance. Similarly, cancer cells reproduce somatically and usually evolve by LOH, as some of the mutations associated with tumor progression involve the loss of functional dominant alleles of tumor suppressors^139,140. The phenomenon we uncovered here, whereby some genotypes experience lower rates of LOH, thus has also consequences that extend beyond the study of hybrids.

Methods

Experimental crosses and previous experimental evolution

The S. cerevisiae and S. paradoxus strains used were described in Bautista et al.⁴⁸ and were derived from the natural strains LL13_054 and MSH-604 isolated in North American forests^13,47 (Supplementary Data 2). To prevent mating type switching in haploids, the HO locus was replaced with resistance cassettes (HPHNT1 for Hygromycin B resistance and NATMX4 for Nourseothricin resistance)¹⁴¹. A total of 90 experimental strains were constructed by crossing haploid strains (30 S. cerevisiae, 30 S. paradoxus, and 30 hybrids), so that each starting parental and hybrid diploid population is the result of an independent mating event as described in Bautista et al.⁴⁸. The 90 populations were evolved for 100 generations (Fig. 1a) as described in Bautista et al.⁴⁸. Briefly, we used as environments a non-DNA damaging growth condition we call control (YPD medium consists of 10 g/L yeast extract, 20 g/L tryptone, and 20 g/L glucose) and a DNA damaging growth condition, which was identical except for the addition of a UV mimetic molecule⁴⁹ 4-Nitroquinoline 1-oxide (4-NQO) (Sigma–Aldrich, cat. no. N8141, batch #WXBC3635V, Canada).

Growth assays on individual clones

Growth assays were conducted on individual clones, which were used for genome sequencing and isolated from the glycerol stocks from experimental evolution⁴⁸ (Supplementary Data 1). Ancestor strains (n = 90, once per clone) as well as the clones evolved in YPD (n = 90, once per clone) and in YPD + 4-NQO (n = 90, once per clone) were pre-cultured in 1 mL of YPD in 96 deep-well plates and incubated for 24 h at 25 °C. Subsequently, 20 µL of these pre-cultures were grown in 96-well flat-bottomed culture plates in 180 µL of medium (YPD or YPD + 4 µM of 4-NQO), resulting in an initial OD₅₉₅ of approximately 0.1. A transfer cycle was performed at 24 h after approximately ~ 5 generations in rich conditions. Each culture was diluted approximately 30-fold by transferring 6 µL of grown culture into 194 µL of fresh medium to initiate a new round of growth at an OD₅₉₅ starting at about 0.03 (this second cycle is used to calculate growth parameters, such as the growth rate). Incubation at 25 °C was performed directly in three temperature-controlled spectrophotometers (Infinite® 200 PRO, Tecan, Reading, UK) that read the OD₅₉₅ at intervals of 15 min throughout the two cycles performed. All samples were randomized across plates, temperature-controlled spectrophotometers, and days.

DNA extraction, library construction, and Whole Genome Sequencing (WGS)

We obtained whole-genome sequences of 270 individual clones derived from the 270 experimental populations⁴⁸ (Supplementary Data 1). We extracted genomic DNA from overnight YPD cultures derived from each clone according to the manufacturer’s instructions (MasterPure™ Yeast DNA Purification Kit, Biosearch Technologies - Lucigen, Wisconsin, USA) and purified on Axygen™ AxyPrep Magnetic PCR Clean-up SPRI beads (Axygen Inc, New York, USA). Five DNA pooled libraries were prepared using RIPTIDE™ High Throughput rapid DNA library prep in 96-well plate format (iGenomX, South San Francisco, USA)¹⁴². The quality of the libraries was verified using an Agilent BioAnalyzer 2100 electrophoresis system (Genomic Analysis Platform of the Institute of Integrative Biology and Systems of Université Laval, Québec, Canada). Pooled libraries were sequenced using paired-end 150 bp reads on different lanes of an Illumina NovaSeq 6000 (Illumina, San Diego, USA) at the Genome Quebec Innovation Center (Montréal, Canada). We excluded sequences of the S. cerevisiae line 16 evolved in UV mimetic due to poor performance in the construction of the library (resulting in a final sample size of n = 269).

Flow cytometry analysis of ploidy

DNA content was measured by flow cytometry using the SYTOX™ green staining assay (Thermo Fisher, Waltham, USA)^41,47. Diploid strains of the S. cerevisiae isolate LL13_054 were used as the diploid control. Given the different genetic backgrounds used in our study, the inference of polyploidy was done by comparing strains and looking for outliers, as most strains aligned with each other and with the diploid control. Whole-genome ploidy changes were expected to result in a shift toward higher DNA content values, as shown in Supplementary Fig. 19. For the hybrid strains, WGS analysis enabled us to determine the specific ploidy state as triploid, due to the gain of an entire genome copy from one of the parental genotypic backgrounds (Supplementary Figs. 7 and 19). The 270 individual clones derived from the 270 experimental populations (Supplementary Data 1) from Bautista et al.⁴⁸ and used for WGS (n = 269) were thawed from glycerol stocks and grown on solid YPD omnitray plates (25 °C, 72 h). They were inoculated into 1 mL of YPD in 96 deepwell plates and incubated for 24 h at 25 °C. Cells were subsequently prepared for flow cytometry as in Gerstein et al.¹⁴³. They were fixed in 70% ethanol and kept frozen at −20 °C for further analysis. RNA was degraded using 0.25 mg mL⁻¹ RNase A during an overnight incubation at 37 °C. Cells were washed twice with sodium citrate (50 mM, pH 7) and stained with a SYTOX™ green concentration of 0.6 μM for 1 h at 25 °C in the dark. Cell concentration was adjusted in sodium citrate (50 mM, pH 7) to be less than 500 cells/μL. Five thousand cells from each of the 270 samples were analyzed in 96-well plates in a CytoFLEX Platform flow cytometer (Beckman Coulter, California, USA) at the Feldan Therapeutics facility (Québec, Canada). Cells were excited with the blue laser at 488 nm, and fluorescence was measured in a green fluorescence detection channel (525/40 nm). The distributions of the green fluorescence values were processed to find the two main density peaks, which correspond to the two cell populations in the G1 and G2 phases, respectively. Although two peaks (G1 and G2) are expected when lines are in the exponential phase, due to variations in growth, some lines may have reached the stationary phase (single peak in G0, which has the same DNA content as G1). Therefore, only those samples clearly exceeding the first peak of the diploid control were considered polyploids (Supplementary Fig. 19).

Quality assessment and read mapping of next-generation sequencing data

Raw reads from barcoded samples of the five libraries were demultiplexed using DemuxFastqs from fgbio tools¹⁴⁴ v1.5.0 (Supplementary Data 3). Reads were trimmed using Trimmomatic¹⁴⁵ v0.36 with parameters ILLUMINACLIP:Trimm_seqs.fa:6:20:10 and using Trimm_seqs.fa (Supplementary Data 4) as a list of adapter sequences used. To assess the quality of both pre- and post-trimming sequencing reads, we used FastQC v0.11.9¹⁴⁶ and MultiQC v1.11¹⁴⁷.

Reads from S. cerevisiae samples were mapped to the indexed reference genome of S. cerevisiae strain YPS128¹⁴⁸, which in our study is named LL13_054, and S. paradoxus samples were mapped to the S. paradoxus SpB (named MSH-604) genome^148,149. Reads from hybrid lines were mapped to a concatenated genome comprising the two respective parental genomes end to end. The BWA-MEM algorithm¹⁵⁰ v0.7.17 was used for mapping. Mapped reads were processed by genome-sorting algorithms using samtools v1.8¹⁵¹, and quality was assessed by mapping coverage with goleft v0.2.2¹⁵². The average mean read depth across samples was about 100X (Supplementary Fig. 1). We used Picard tools v2.26.11¹⁵³ for adding Read Groups with AddOrReplaceReadGroups, and we removed duplicate reads with MarkDuplicates with parameter REMOVE_DUPLICATES = true.

Analysis of sequencing read depth (aneuploidies and loss of heterozygosity)

Mean read depths over 1 kbp windows were obtained with BamStats04 from Jvarkit tools v2021.08.10¹⁵⁴ and make windows from bedtools tools v2.30.0¹⁵⁵. We first eliminated some sequences of the hybrid lines evolved in UV mimetic because the sequencing depth was low (lines 1 and 21) or because the content of one of the parental genomes was the majority (lines 10 and 25) (Supplementary Figs. 20–22). We removed these two lines (10 and 25) from the rest of the non-WGS analysis due to the uncertainty about their origin. We also verified the results of the Flow cytometry analysis of ploidy section and compared DNA content by measuring average read depth across genomes (Supplementary Fig. 19). We computed the median chromosome read depth and the median whole genome read depth for each line. In order to detect the number of gained or lost chromosomes, we divided each chromosome’s median read depth by the genome-wide median read depth. We standardized this value by the value of the corresponding ancestors to obtain the relative read depth (log2 fold change). Values with a chromosome median read depth higher than the genome-wide median represent gains in DNA content (gradient towards red in Fig. 2a, b, Supplementary Figs. 7 and 13a), and values with a median read depth lower than the genome-wide median represent losses in DNA content (gradient towards purple in Fig. 2a, b, Supplementary Figs. 7 and 13a) for each individual chromosome. We also computed the number of chromosomes with aneuploidies per line by considering an aneuploidy as a deviation (increase or decrease) of 30% with respect to the genome-wide median read depth as in Bendixsen et al.²⁶. The positional coverage mapping of hybrid genomes unveiled terminal regions with pronounced increases in read depth in one parental chromosome copy and concurrent decreases in the other copy (Supplementary Fig. 7), revealing the presence of reciprocal crossovers between chromosomes. These tracts that extend to the telomeres and usually measure between 50–100 kb correspond to terminal-loss of heterozygosity (t-LOH) regions^61,62,65. To quantitatively assess the number of t-LOH events in hybrid lines, we identified regions with simultaneous increases and decreases in read depth (deviation of 30% with respect to the genome-wide median read depth) in both chromosomal copies exceeding a size threshold of 20 kb. We excluded triploid strains from the t-LOH analyses due to the impossibility of detecting increases and decreases in read-depth. Therefore, the sample size for the evolved UV mimetic hybrids is n = 28 (excluding lines 10 and 25) for non-WGS analyses (such as in Flow cytometry analysis of ploidy or Growth assays on individual clones sections); n = 26 (excluding lines 1, 10, 21, and 25) when analyzing coverage patterns; n = 23 in UV mimetic conditions or n = 25 in control conditions when analyzing general LOH patterns (since we also exclude triploids which are displayed in Supplementary Fig. 19).

Functional analysis of de novo mutations

We eliminated some sequences of the hybrid lines evolved in UV mimetic because the sequencing depth was low (lines 1 and 21) or because the content of one of the parental genomes was the majority (lines 10 and 25) (Supplementary Figs. 20– 22). SNP calling was performed with Haplotype Caller (gatk-v4.1.4.1)^156,157. Before generating the GVCFs (Genomic Variant Call Format files), we added an RG (read group) tag to individual BAM files. After SNP calling, genotyping of the GVCFs was performed with GenotypeGVCFs. For variant filtration, we applied standard hard filters with options: QUAL by depth (QD) < 2.0, mapping quality (MQ) < 40.0, Fisher’s exact tests of strand bias (FS) > 60.0, symmetric odds ratio test of strand bias (SOR) > 3.0, mapping quality rank sum test (MQRankSum) < − 12.5, rank sum test for site position within reads (ReadPosRankSum) < − 8.0, Genotype Quality (GQ) < 20, and Coverage (DP) < 3. We selected variants that passed the filters described above, and we excluded INDELS, focusing exclusively on substitutions (SNP) for subsequent analysis. We excluded the pre-existing genetic variation relative to the reference genome, by removing any variant that was already present in the ancestral strain for each evolved line (Supplementary Fig. 9). For annotation purposes, we used the S. cerevisiae genome assembly R64-1-1 (Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database, INSDC Assembly GCA_000146045.2, Sep 2011) for S. cerevisiae genomes. Subsequently, we generated maps annotations with Liftoff (v1.6.3)¹⁵⁸ of the S. paradoxus genome from the S. cerevisiae genome assembly R64-1-1 (Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database, INSDC Assembly GCA_000146045.2, Sep 2011). We finally generated a combined annotated genome for hybrid analysis. The variants were ultimately annotated using Ensembl Variant Effect Predictor (VEP) v110¹⁵⁹. We examined missense variants to perform Gene Ontology (GO) analysis using bioMart¹⁶⁰, simplifyEnrichment¹⁶¹, and clusterProfiler¹⁶².

Validation of the adaptiveness of mutations

We used ChimeraX v1.5¹⁶³ to visualize Pdr1 amino acid changes we found throughout the experimental evolution and other mutations found in the literature^{74,75,76,77,78} (Supplementary Data 5) on the AlphaFold 2^164,165 generated structure for Pdr1 (AF-P12383-F1, Supplementary Data 6). Subsequently, we analyzed the Nakaseomyces glabratus Pdr1 protein by superimposing structures and amino acid changes^79,82,83 across species (Supplementary Data 7; AF-B9VI40-F1, Supplementary Data 8). We next used a set of plasmids derived from the MoBy-ORF library, in which genes are controlled by their native promoter and terminator¹⁶⁶, to express the PDR1 sequences from either S. cerevisiae (BY4741) or S. paradoxus (MSH-604). Candidate mutations (G280R, G280S, M308I, and G1042W, being G1041W in LL13_054 strain for the S. cerevisiae sequence, G279R, G279S, and G281V for the S. paradoxus sequence) were inserted by site-directed mutagenesis. As controls, we used the plasmid without the PDR1 gene (Empty) or the plasmid containing the Wild-Type (WT) PDR1 sequence. We introduced these plasmids following a modified lithium acetate transformation protocol¹⁶⁷ in an S. cerevisiae lab strain BY4741, and in natural strains S. cerevisiae LL13_054 and S. paradoxus MSH-604 in both WT and pdr1Δ (previously constructed by replacing PDR1 locus with a NATMX4 module) backgrounds. Growth assays were performed following the same steps as described above in the Growth assays on individual clones section. We added extra conditions to the ones previously used (control and 4 µM of 4-NQO): 8 µM and 10 µM of 4-NQO (Supplementary Fig. 15). We also conducted an assay on the S. cerevisiae lab strain BY4741 pdr1Δ, containing the same mutations on the same plasmids, and exposed them to antifungal azoles. After adjusting cell density to an OD₅₉₅ of 1, we made three serial dilutions 1/5 in 200 μL of water (40 μL of cells in 160 μL of water). We spotted 5 μL of each dilution on YPD + 0.2% DMSO (control), YPD + 16 μg/mL Fluconazole (FLC), YPD + 2 μg/mL Itraconazole (ITR) or YPD + 0.5 μg/mL Voriconazole (VRC) and incubated at 30 °C for 48 h. We assessed the expression of the downstream Pdr5p drug efflux pump by fusing Pdr5 to a Green Fluorescent Protein (mEGFP) (Pdr5-mEGFP) in a BY4741 pdr1Δ lab strain expressing PDR1 mutants from the above-described pMoBY plasmids. For a comprehensive list of strains, refer to Supplementary Data 2, and for a comprehensive list of oligonucleotide sequences, refer to Supplementary Data 9.

Incomplete-dominance assay

To evaluate the dominance of the PDR1 mutations, we created S. cerevisiae diploids harboring either homozygous or heterozygous M308I substitutions using CRISPR-Cas9 genome editing and mating BY4741 and BY4742 haploids (Supplementary Data 2). We replaced the PDR1 locus with NATMX4 (in BY4741) or HPHNT1 (in BY4742) modules specifically targeted by two different gRNA (guide RNA) using a modified protocol from Ryan et al.¹⁶⁸. Yeast cells were transformed following a modified lithium acetate transformation protocol¹⁶⁷ with a pCAS-NAT or pCAS-HPH plasmid (Addgene plasmid 6084747 modified by Després et al.¹⁶⁹ and Lemieux et al.¹⁷⁰ using the same approach as in Dionne et al.¹⁷¹) expressing both the gRNA (guide RNA) of interest (targeting NATMX4 or HPHNT1), the Streptococcus pyogenes Cas9¹⁷² and a donor DNA sequence featuring 40 bp homology arms flanking the PDR1 DNA sequence. The donor DNA sequences were: PDR1 sequence (WT), PDR1 sequence with the desired mutation (M308I), or PDR1 sequence with a stop codon instead of the first methionine (M1Stop) (Oligonucleotide sequences can be found in Supplementary Data 9). Finally, we mated BY4741 and BY4742 haploids with the desired mutations to create the following diploid strains: WT homozygous (pdr1Δ::PDR1/pdr1Δ::PDR1), M308I homozygous (pdr1Δ::PDR1(M308I)/pdr1Δ::PDR1(M308I)), heterozygous (pdr1Δ::PDR1/pdr1Δ::PDR1(M308I) or pdr1Δ::PDR1(M308I)/pdr1Δ::PDR1) or hemizygous (pdr1Δ::PDR1(M308I)/pdr1Δ::M1Stop) (refer to Supplementary Data 2 for a comprehensive list of strains). We selected diploid strains using double selection (Synthetic complete (SC) medium -lysine -methionine which is used as a standard selection method for BY background). SC medium consists of 1.74 g/L yeast nitrogen base without ammonium sulfate, 20 g/L glucose, and 5 g/L ammonium sulfate. Growth assays were performed following the same steps as described above in the Growth assays on individual clones section.

Incomplete-dominance assay on the three genotypic backgrounds

Since CRISPR-Cas9 editing was not feasible in the wild-type ancestral strains (S. paradoxus: MSH-604 or S. cerevisiae: LL13_054), we sporulated line 27 of Saccharomyces paradoxus (strain MSH-604) containing the M307I mutation (Fig. 3c) to obtain haploid cells for subsequent crosses with the BY4741 and BY4742 strains described above. For sporulation, diploid cells were grown overnight in 10 mL of selective medium, then transferred to fresh medium for a 3 h incubation with shaking at 30 °C. After centrifugation and washing with 20 mL of sterile water, the cells were resuspended in 10 mL of YEPA medium (10 g/L yeast extract, 20 g/L tryptone, and 20 g/L potassium acetate (CH₃CO₂K)) and incubated overnight at 30 °C with shaking. Subsequently, the cells were washed twice with 20 mL of sterile water and then resuspended in 25 mL of SPM medium (3 g/L potassium acetate (CH₃CO₂K) and 0.2 g/L raffinose) for 3–5 days to induce sporulation.

For yeast tetrad dissection, asci were incubated in 100 μL of zymolyase 20 T (200 μg/mL in water) for 20 min and then resuspended in 100 μL of sorbitol 1 M. Samples were spread onto Petri dishes containing YPD medium and dissected under a microscope before being incubated for 3 days at 30 °C. Finally, we mated BY4741, BY4742, S. paradoxus line 27 MATa and S. paradoxus line 27 MATα haploids with the desired mutations to create diploid strains: WT homozygous (pdr1Δ::PDR1 / pdr1Δ::PDR1), M308I/M307I homozygous (pdr1Δ::PDR1(M308I/M307I) / pdr1Δ::PDR1(M308I/M307I)) or heterozygous (pdr1Δ::PDR1 / pdr1Δ::PDR1(M308I/M307I) or pdr1Δ::PDR1(M308I/M307I) / pdr1Δ::PDR1) in the three backgrounds S. cerevisiae (diploid BY4743), S. paradoxus (MSH-604) and hybrid (refer to Supplementary Data 2 for a comprehensive list of strains). We then selected diploid strains using double selection (YPD + Nourseothricin + Hygromycin B) or by colony PCR screening¹⁷³ to identify diploid colonies when double selection was not feasible (when crossing BY4741/2 strains with S. paradoxus haploid strains MSH-604). Growth assays were performed following the same steps as described above in the Growth assays on individual clones section.

Allele frequency dynamics on PDR1 mutants

Whole population samples were archived regularly during the evolution experiment⁴⁸. From these samples, we isolated clones to estimate the timing of the appearance of the various PDR1 mutations and to track the dynamics between homozygous and heterozygous zygosities in the population. We revived approximately 350 clones from nine populations (Supplementary Data 1) and sequenced the PDR1 locus using amplicon Sanger sequencing (Oligonucleotide sequences can be found in Supplementary Data 10). We designed specific primers for each mutation (Oligonucleotide sequences can be found in Supplementary Data 9) and used standard colony PCR to amplify those regions. We performed an exhaustive zygosity analysis across time points (each time point represents five generations) to quantify the ratio of homozygous to heterozygous variants. We sequenced clones starting from the first detection of PDR1 mutations until the homozygous frequency exceeded 80%, or if this did not occur, we sequenced until time point 19 (corresponding to 95 generations). Growth assays in isolated hybrid clones were performed following the same steps as described above in the Growth assays on individual clones section.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1045261. The demultiplexing process details are available at Zenodo¹⁷⁴ (https://doi.org/10.5281/zenodo.10389557) and Supplementary Data 3. The processed data are available at Zenodo¹⁷⁴ (https://doi.org/10.5281/zenodo.10389557). PDB-formatted files of the AF2-generated models are Supplementary Data 6 and 8, and can also be found at Zenodo¹⁷⁴ (https://doi.org/10.5281/zenodo.10389557). Strains are available upon request.

Code availability

Data and scripts for figure generation can be found at Zenodo¹⁷⁴ (https://doi.org/10.5281/zenodo.10389557) and on GitHub (https://github.com/cbautistaro/Bautista2024_LOH_project_NatureCommunications). Data were analyzed using bash and R version 4.2.0.

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Acknowledgements

We thank Nadia Aubin-Horth for feedback on the manuscript, Alexandre Dubé, and Cynthia Gagné-Thivierge for assistance in the laboratory work, Angel Cisneros, Lorena Ament, Alexandre Andreas Rego, Damien-Biot Pelletier, Adarsh Jay, and David Bradley for help with data analysis and members of the Landry Lab for helpful discussions. We thank the “Centre d’expertise et de services Génome Québec” and Feldan Thérapeutiques Québec, especially Vanessa Théberge and David Guay, for their services in sequencing and flow cytometry analysis, respectively. This work was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to C.R.L. RGPIN-2020-04844. C.B. is supported by “la Caixa” Foundation (ID 100010434), under the agreement (LCF/BQ/AA16/11580051) and by the Fonds de recherche du Québec - Nature et technologies (FRQNT) (#274987) doctoral fellowships. M.U. was funded by the Human Frontier Science Program (HFSP) initiative “Science for Scientists” to support Ukrainian scientists. C.R.L. holds the Canada Research Chair in Cellular Systems and Synthetic Biology.

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Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, Canada
Carla Bautista, Isabelle Gagnon-Arsenault, Mariia Utrobina, Anna Fijarczyk & Christian R. Landry
Département de Biologie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada
Carla Bautista, Isabelle Gagnon-Arsenault, Mariia Utrobina, Anna Fijarczyk & Christian R. Landry
Regroupement québécois de recherche sur la fonction, la structure et l’ingénierie des protéines (PROTEO), Université Laval, Québec, Canada
Carla Bautista, Isabelle Gagnon-Arsenault, Anna Fijarczyk & Christian R. Landry
Centre de Recherche en Données Massives (CRDM), Université Laval, Québec, Canada
Carla Bautista, Isabelle Gagnon-Arsenault, Anna Fijarczyk & Christian R. Landry
Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec, Canada
Isabelle Gagnon-Arsenault & Christian R. Landry
National University of Kyiv-Mohyla Academy, Kyiv, Ukraine
Mariia Utrobina
Department of Zoology, Stockholm University, Stockholm, Sweden
Devin P. Bendixsen & Rike Stelkens

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Carla Bautista
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Isabelle Gagnon-Arsenault
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Mariia Utrobina
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Anna Fijarczyk
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Devin P. Bendixsen
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Christian R. Landry
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Contributions

C.B., I.G.A., and C.R.L. designed the research. CB performed the experiments collected and analyzed the data with the assistance of I.G.A., M.U., A.F., D.B., R.S., and C.RL. C.B. wrote the manuscript with the assistance of CRL. C.B., I.G.A., M.U., A.F., D.B., R.S., and C.R.L. edited the manuscript. C.R.L. was responsible for funding acquisition. All authors read and approved the final manuscript.

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Correspondence to Carla Bautista or Christian R. Landry.

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Bautista, C., Gagnon-Arsenault, I., Utrobina, M. et al. Hybrid adaptation is hampered by Haldane’s sieve. Nat Commun 15, 10319 (2024). https://doi.org/10.1038/s41467-024-54105-4

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Received: 20 December 2023
Accepted: 01 November 2024
Published: 28 November 2024
Version of record: 28 November 2024
DOI: https://doi.org/10.1038/s41467-024-54105-4

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