Loss of Heterozygosity Drives Adaptation in Hybrid Yeast

1Hybridization is now recognized as a common phenomenon across the tree of life. Historically however, the detection of hybrids has been difficult, and its incidence may be under-reported for both plants and animals, and almost certainly for certain eukaryotes like insects and fungi. Its importance as an evolutionary force has thus been maligned, as hybrids appeared both rare and typically at a reduced fitness. In addition to potential postreproductive barriers, the hybrid is theorized to be ill-adapted to its environment and will also suffer minority cytotype disadvantage, because other hybrids are uncommon and backcrosses to parental species may be unfit. However, hybrids can have a variety of advantages over their parents, including heterozygote advantage, extreme phenotypic traits, and reproductive isolation (usually resulting from polyploidy), and can thus facilitate adaptation to novel or stressful conditions, invade unoccupied ecological niches, and even increase biodiversity.

2Some hybridization events lead to new hybrid species, whereas most result in introgression from hybrid backcrosses to the more abundant parental species. Hybridization introduces genetic variation into a population at orders of magnitude greater than what mutation alone can achieve, in a sense operating as a “multi-locus macro-mutation”. Therefore, hybridization via introgression, polyploidy, or homoploid hybrid speciation may offer a rapid strategy for adaptation to changing environmental conditions. For example, in Darwin’s finches, adaptive introgression supplied the morphological variation that allowed the species to survive following an El Niño event, and in ancient humans, introgression allowed adaptation to high altitudes, among other traits. The most iconic example comes from the hybrid sunflower species Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus, from the parents Helianthus annuus and Helianthus petiolaris. These three hybrid species are locally adapted to extreme desert, salt marsh, and dune habitats, respectively, and show traits such as increased drought or salt tolerance relative to their parents.

3Agriculture and industry use both intra- and interspecific hybrids as a tool to increase yield or robustness, introduce resistance to pests, and create novel phenotype or flavor profiles. For example, plant breeders have crossed domesticated species to wild species to introduce resistance to a variety of pathogens in wheat, potato, and canola, and almost all maize grown in the United States is grown from intraspecific hybrid seeds, which has increased yield and provided improved resistance to biotic and abiotic factors. Vintners and brewers have created interspecific hybrids to select for traits such as lower acetic acid concentration, and many incidental fungal hybrids have been discovered in brewing and industry, including Pichia sorbitophila, and various hybrids across the Saccharomyces clade, most notably the lager-brewing yeast, Saccharomyces pastorianus. It is presumed that the severe selection pressures exerted during industrial processes have selected for interspecific hybrid genomes that may be more able to cope with the extreme environments.

4At the genomic level, hybridization induces chromosome loss/aneuploidy, chromosomal rearrangements, gene loss, changes in gene expression, changes in epigenetic modifications, transposable element mobilization, and large-scale loss of heterozygosity (LOH), in which the allele of one species is lost and the allele of the other species is retained and may even be duplicated via gene conversion or break-induced replication. These extensive changes can result in a chimeric, stabilized hybrid, although the period of time for genome stabilization to occur can range dramatically. It is unknown whether there are structural and functional biases in the ways in which genes/alleles are lost or modified. Both drift and selection influence the resolution of the hybrid genome, but their contributions are difficult to untangle.

5Researchers have long been exploring the genetics of hybrid traits in the lab, particularly in agricultural crops, although this is often slowed by infertility and reduced viability in many interspecific hybrids. The Saccharomyces genus, which includes the budding yeast Saccharomyces cerevisiae, lends itself particularly well to experimental study. Many hybrids of this genus have been discovered in brewing, industrial, and natural environments; indeed, the genus itself is speculated to have been founded by the product of an ancient hybridization event. Viable interspecific hybrids can be created de novo in the lab, and their ability to grow mitotically means that the catastrophic postzygotic barriers to speciation that generally doom other obligate sexually reproducing hybrids can be avoided. This experimental system allows us to observe evolution in real time in the laboratory environment, and the genetic and genomic tools available in this model genus facilitate characterization of the connection between genotype and phenotype, including fitness.

6Previous work in our lab group has utilized experimental evolution to investigate adaptive events in haploid and homozygous diploid S. cerevisiae. To investigate genome evolution post hybridization, we utilize an interspecific hybrid, S. cerevisiae × Saccharomyces uvarum, and its parentals: a homozygous diploid S. uvarum and an intraspecific hybrid S. cerevisiae GRF167 × S. cerevisiae S288C. This allows us to understand the impact of varying levels of heterozygosity on adaptation and genome evolution, ranging from none (S. uvarum and previous S. cerevisiae experiments), to intraspecific heterozygosity (S. cerevisiae GRF167 × S. cerevisiae S288C), to the most extreme case of interspecific hybrids. Saccharomycesuvarum is one of the most distantly related species of S. cerevisiae in the Saccharomyces clade, separated by 20 My and 20% sequence divergence at coding sites. Despite this extensive divergence, S. cerevisiae and S. uvarum are largely syntenic and create hybrids, though less than 1% of spores are viable. The two species differ in their stress tolerances, for example, S. cerevisiae being more thermotolerant, S. uvarum being cryotolerant. Previous evolution experiments using lab-derived hybrids have revealed novel and/or transgressive phenotypes for ammonium limitation, ethanol tolerance, and growth on xylose. Notably, revealed several LOH events and a repeatable nonreciprocal translocation that produces a gene fusion at the high-affinity ammonium permease MEP2 after selection in ammonium limitation, offering insight into potential mutational events in the adaptation and/or stabilization of S. cerevisiae × S. uvarum hybrids.

7Here, we evolved these hybrids and diploids in replicate in three nutrient-limited conditions for hundreds of generations. Using whole genome sequencing, we found whole chromosome aneuploidy, genome rearrangements, copy number variants, de novo point mutations, and LOH. We sought to determine how initial heterozygosity affects adaptation to novel conditions and explore whether neutral or selective forces are influencing the resolution of the hybrid genome over time. In particular, we investigated a reoccurring LOH event observed in both intra- and interspecific hybrids and found support for the hypothesis that LOH at this locus is due to selection.

8An interspecific hybrid was created by crossing S. cerevisiae and S. uvarum (strains in supplementary table S1, Supplementary Material online) and evolved in continuous culture in the chemostat. In parallel, homozygous diploid S. uvarum and heterozygous diploid S. cerevisiae (GRF167 × S288C) were also evolved. Each strain was grown in two or more replicate independent cultures under three different nutrient limitations—glucose, phosphate, and sulfate—for 85–557 generations (median 158) at 30 °C, except for S. uvarum, which was unable to achieve steady state in all conditions at 30 °C and so was evolved at 25 °C. The population sizes were largely similar across strains, species, and conditions.

9Evolved clones were isolated from each population and subsequently competed individually against the appropriate green fluorescent protein (GFP)-tagged ancestor to gauge relative fitness. As expected, evolved hybrid and parental clones generally exhibit higher fitness than their unevolved ancestor, with typical relative fitness gains between 20% and 30% (tables 1 and 2). To explore whether these fitness gains are general or condition specific, we additionally competed each hybrid clone in the two nutrient-limited conditions in which the clone was not evolved. Results are variable, with some clones having negative or neutral fitness in the alternate conditions, suggesting condition-specific adaptation, and some clones experiencing fitness gains in multiple conditions, suggesting more general growth benefits (table 1). Only one clone exhibited fitness gains in all three nutrient environments, and no clones have a greater fitness gain in an alternate condition than the condition it was evolved in, signifying that clones are largely specifically adapted to the particular condition in which they were evolved.

10To identify mutations in the evolved hybrids, we generated whole genome sequencing data for 16 clones from the end points of the evolution experiments (table 1). We thus captured data from a range of nutrient limitations (phosphate: 6; glucose: 3; sulfate: 7) and generations (100–285, median: 154). Each clone had an average of 2.4 point mutations, a number of which have been previously identified in prior S. cerevisiae evolution experiments. For example, a nonsynonymous mutation in the S. cerevisiae allele of the glucose-sensing gene SNF3 has been identified in glucose-limited experiments in S. cerevisiae. To our knowledge, 20/27 coding point mutations are unique to these experiments.

11In evolved parentals, we again sequenced one clone from the end point of each population. In total, we sequenced 16 clones, 6 from each of the 3 nutrients (2 S. uvarum diploids and 4 S. cerevisiae diploids), except in glucose limitation in which only 2 S. cerevisiae populations were sampled. The generations ranged from 234 to 557 (median: 477) in S. uvarum, with an average of 2.83 mutations per clone, and from 127 to 190 (median: 166.5) in S. cerevisiae, with an average of 0.9 point mutations per clone (table 2). This discrepancy in point mutations between S. cerevisiae and S. uvarum may be explained by differences in generation number.

12With the limited number of samples we have from hybrid and parental clones, it is difficult to draw any conclusions regarding unique point mutations in hybrids. Furthermore, we have not tested the fitness of these mutations to prove they are adaptive. However, one class of mutations that may be of particular interest in hybrids are genomic mutations that may interact with the mitochondria, as previous work has shown that nuclear–mitochondria interactions can underlie hybrid incompatibility. Other studies have found that only the S. cerevisiae mitochondria are retained in S. cerevisiae × S. uvarum hybrids, and we recapitulate these findings, potentially setting the stage for conflicting interactions between the S. uvarum nuclear genome and the foreign mitochondria. We observe several mitochondria-related mutations in hybrids in both S. cerevisiae and S. uvarum alleles. For example, one point mutation, a nonsynonymous mutation in the S. cerevisiae allele of the mitochondrial ribosomal protein gene MHR1, was seen in two separate clones independently evolved in phosphate limitation. This gene may be of particular interest as it was discovered in a previous screen as being haploproficient (increased fitness of 19%) in hybrids in which the S. cerevisiae allele is missing and the S. uvarum allele is retained (Lancaster S, Dunham MJ, unpublished data), suggesting that this mutation may alter or disable the S. cerevisiae protein in some way. Another example involves the gene IRC3, a helicase responsible for the maintenance of the mitochondrial genome, which has a nonsynonymous mutation in the S. uvarum allele in clone Gh3 and is deleted in clone Gh2, potentially suggesting that the S. uvarum allele is deleterious in the hybrid background. Although our sample size is small, 4/27 point mutations in hybrids are related to mitochondria function compared with 0/26 in parentals and may represent interesting targets for further exploration.

13Yeasts in both natural and artificial environments are known to frequently experience changes in copy number, ranging from single genes to whole chromosomes. This holds true in our evolution experiments: We observe copy number changes across all genetic backgrounds (fig. 1; supplementary figs. S1–S3, Supplementary Material online). Clones were compared with array comparative genomic hybridization of populations to confirm that clones are representative of populations (see Materials and Methods). The evolved hybrid clones displayed an average of 1.5 copy number variants (CNVs) per clone (fig. 1, table 1; Supplementary fig. S3, Supplementary Material online), as defined by the number of segmental or whole chromosome amplifications/deletions (though it is likely that some of these CNVs were created in the same mutational event). The evolved S. cerevisiae clones had an average of 1.5 CNV per clone and the evolved S. uvarum clones had an average of 1 CNV per clone (table 2; supplementary figs. S1 and S2, Supplementary Material online). The most common event across nutrient limitations in the interspecific hybrids was an amplification of the S. cerevisiae copy of chromosome IV, which occurred in four independent hybrid clones (three in phosphate limitation, one in glucose limitation; supplementary fig. S3, Supplementary Material online). Several other characteristic rearrangements occurred in the evolved S. cerevisiae clones, including the amplification of the left arm of chromosome 14 accompanied by segmental monosomy of the right arm of chromosome 14, an event seen previously in other evolved populations. All copy number events in S. cerevisiae had break points at repetitive elements known as Ty elements, except those located on chrII, which may be mediated by another mechanism. In contrast, copy number variants in the hybrid were rarely facilitated by repetitive elements, perhaps in part because S. uvarum has no full length Ty elements; however, why S. cerevisiae Ty elements and long terminal repeat (LTR) sequences from either genome were not utilized remains unknown.

14Frequently in nutrient-limited evolution experiments, copy number variants involve amplification of the nutrient-specific transporter, and indeed, we also observed amplification of these transporters in many of the clones. In sulfate limitation, the S. cerevisiae allele of the high-affinity sulfate transporter gene SUL1 is amplified in 7/7 hybrid clones and 4/4 S. cerevisiae clones (fig. 1, tables 1 and 2; supplementary figs. S1 and S3, Supplementary Material online). Interestingly, SUL2 is the preferred sulfate transporter in S. uvarum and was not observed to be amplified in the evolved hybrids (supplementary fig. S2 and table S2, Supplementary Material online). In glucose limitation, previous S. cerevisiae evolution experiments found frequent amplification of the high-affinity glucose transporter genes HXT6/7. In our experiments, the S. uvarum alleles of the HXT6/7 transporters are amplified in 3/3 hybrid clones and both S. uvarum clones but are not amplified in evolved S. cerevisiae clones. This is suggestive that the S. uvarum HXT6/7 alleles confer a greater fitness advantage compared with S. cerevisiae; alternatively, the genomic context could be more permissive to amplification in S. uvarum (fig. 1, tables 1 and 2; supplementary figs. S1–S3, Supplementary Material online). Finally, in phosphate limitation, the S. cerevisiae copy of the high-affinity phosphate transporter gene PHO84 is amplified, and the S. uvarum allele is lost in 3/6 hybrid clones in an event known as LOH (figs. 1 and2, table 1; supplementary fig. 3, Supplementary Material online). Intriguingly, the evolved S. cerevisiae clones also display LOH and accompanied amplification favoring the allele derived from strain GRF167 over the S288C allele in 4/4 clones (fig. 2, table 2). All hybrid clones carry the “preferred” GRF167 S. cerevisiae allele, as this was the allele used to create the de novo hybrid.

15Selection on heterozygosity, as a LOH event could represent, is an underappreciated source of adaptation in microbial experimental evolution, as typical experiments evolve a haploid or homozygous diploid strain asexually and, as a result, have little variation to select upon. LOH is observed in natural and industrial hybrids, but here we document its occurrence in both intra- and interspecific hybrids in the laboratory as a result of short-term evolution (also see). LOH is observed across all nutrient conditions, with 13 independent LOH events detected in S. cerevisiae and 9 independent events documented in the hybrids (figs. 1 and2, tables 1 and 2; supplementary figs. S1 and S3, Supplementary Material online). It thus appears that this type of mutational event is both common and can occur over short evolutionary timescales.

16The LOH event can result in copy-neutral (where one allele is lost and the other allele is amplified) or non-neutral chromosomal segments (where one allele is lost, rendering the strain hemizygous at that locus) and can favor the retention of either allele. In S. cerevisiae, there is a bias in resolution where LOH events favor retaining the GRF167 allele over the S288C allele (10/13 events, P = 0.0169; table 2; supplementary fig. S4, Supplementary Material online). One unique case in clone Sc4 has a small approximately 5 kb LOH event on chrXV favoring GRF167, which switches to favoring S288C for the rest of the chromosome. The retention of S. cerevisiae is slightly more common in the hybrids (5/9 events, P = 1.0; table 1; supplementary fig. S3, Supplementary Material online), though not as drastic as the observed genome resolution in the hybrid S. pastorianus, where LOH favors S. cerevisiae over S. eubayanus. The size of the event ranges from approximately 25 kb to the whole chromosome level in the hybrids and from 5 kb to 540 kb in S. cerevisiae. Where LOH is accompanied by an amplification event, the LOH event always occurs first and does not share break points with the amplification event. Unlike many CNV events, most LOH events do not appear to be mediated by existing repetitive sequence such as a transposable element in the hybrid or S. cerevisiae and are most likely a product of break-induced replication or mitotic gene conversion, with break-induced replication as the favored method as all events extend to the telomere. The exceptions are in hybrid clones Ph4, Ph5, and Sh1, where there is a non-copy neutral loss of S. cerevisiae mediated by a Ty element or LTR, and S. cerevisiae clones Sc1 and Sc4, where there is a 6.5-kb deletion of the S288C allele flanked by two Ty elements.

17LOH events in hybrids could signify several ongoing processes in hybrid genome evolution: LOH regions may represent 1) loci with incompatibilities, 2) selection on existing variation, or 3) genetic drift eroding genomic segments. It is impossible to definitively rule out any of these hypotheses without further experimentation; however, there are several arguments disfavoring the incompatibility hypothesis. First, although our sample size is modest, failing to see repeated LOH events across nutrient conditions may indicate a lack of general incompatibility between species (although we cannot preclude condition-specific incompatibility,; ). This is consistent with previous studies in yeast, which suggest that classic Dobzhanksy–Muller incompatibilities are rare. Furthermore, LOH events observed in evolved S. cerevisiae suggest that this mutation type is not unique to interspecific hybrids. Instead, repeated events within a particular condition, such as the repeated LOH at PHO84 in phosphate limitation or the 6.5 kb segment on chrXIII in sulfate limitation, suggest that these events are beneficial and are indeed selection on one allele over the other.

18To test the hypothesis that LOH events provide a selective advantage, we used allele replacement, in which the allele of one species/strain is replaced with the allele of the other species/strain in an otherwise isogenic background. We tested this hypothesis using the most commonly seen LOH event, LOH at PHO84. Although the region extends from 25 kb to 234 kb in length in the hybrids and from 40 kb to 85 kb in S. cerevisiae and thus includes many genes, PHO84 was a prime candidate driving this event. PHO84 is one of only ten genes encompassed in the region extending from the telomere to the break point of the shortest LOH event and is included in every other LOH event on chromosome XIII (fig. 2). It is a high-affinity phosphate transporter responsible for inorganic phosphate uptake in high and low phosphate conditions, and previous work identified a point mutation in PHO84 (an alanine to valine substitution at the 5ʹ end of the gene), which increased fitness by 23% in phosphate-limited conditions. Finally, prior work with other nutrient transporters has shown amplification of nutrient transporters to be a key event in adapting to nutrient-limited conditions.

19We thus selected a region of approximately 2.5 kb encompassing the PHO84 ORF, its promoter, and 3ʹ-UTR. We created allele replacement strains using the two alleles of S. cerevisiae in a S. cerevisiae diploid background; the two alleles are 99.1% identical in this region (supplementary fig. S5, Supplementary Material online) and each strain is identical to the ancestral strain used in our evolution experiments except at the PHO84 locus. The S. cerevisiae ancestor carries one copy of GRF167 (preferred) and one copy of S288C (“un-preferred”), so named due to which allele was retained and amplified in the evolved clones. To measure any resultant changes in fitness, we competed each strain individually against a fluorescent ancestral strain and measured which strain overtook the culture. Relative fitness is thus defined as the growth advantage per generation. Two copies of the un-preferred allele decreased relative fitness by −5.31% (±1.86), whereas two copies of the preferred allele increased relative fitness by 9.93% (±0.27). This displays an overall difference in relative fitness of 15.24% between the un-preferred and preferred alleles. By comparing the fitness of these allele replacement strains with the evolved clones (table 2), the allele replacement does not fully recapitulate the fitness gain observed in the evolved clone. One explanation is that the additional mutations present in the evolved strains also contribute to their total fitness. Another explanation could be the increased copy number of the PHO84 region that we see in these evolved clones. To further explore this fitness difference, we cloned the GRF167 allele onto a low copy number plasmid and transformed the allele replacement strain carrying two preferred S. cerevisiae alleles to simulate increased copy number of PHO84 and saw only a minimal fitness increase of 1.76% (±1.06; note all fitness competitions involving plasmids were done in conjunction with empty plasmids to take into account any fitness effects from the plasmid itself). This supports the conclusion that relative fitness gains in the evolved clone are largely due to the loss of the S288C allele, and selection and amplification of the GRF167 allele, with little additional benefit from further amplification. It could also be the case that co-amplification of other genes in the segment is required to attain the full benefit, as previously observed by the contribution of BSD2 to the SUL1 amplicon in sulfate limitation.

20To understand the fitness effects of LOH and amplification in the hybrids, we generated hybrid strains with varying numbers of S. cerevisiae GRF167 PHO84 alleles. Unfortunately, we were unable to obtain a successful strain carrying the preferred S. cerevisiae allele in a S. uvarum background; however, we were able to generate a S. uvarum PHO84 knockout strain, thus creating a hybrid with one copy of S. cerevisiae PHO84. When combined with a low copy plasmid carrying the same allele, this strain effectively has two or more copies of S. cerevisiae PHO84 in a hybrid background. This hybrid strain can serve as a proxy for the LOH event observed in the evolution experiments and has a relative fitness gain of 25.57% (±2.88). The ancestral hybrid with the same plasmid (effectively 1 S. uvarum allele and 2 or more S. cerevisiae alleles) has a relative fitness gain of 12.53% (±1.31). The difference between these two hybrids suggests that while amplification is beneficial, the highest fitness is achieved with the loss of the S. uvarum allele (P = 0.0061).

21Together, these results support the conclusion that the S. cerevisiae GRF167 allele is preferred over the S288C allele and that S. cerevisiae alleles are preferred over the S. uvarum allele in the hybrid and, hence, that the LOH events seen in both intra- and interspecific hybrids are the product of selection.

22In summary, we sought to understand forces underlying genome stabilization and evolution in interspecific and intraspecific hybrids as they adapt to novel environments. We evolved and sequenced clones from 16 hybrid populations and 16 parental populations to reveal a variety of mutational events conferring adaptation to 3 nutrient-limited conditions. Of particular note, we find LOH in both evolved intraspecific and interspecific hybrid clones in all nutrient environments, potentially signifying areas where selection has acted on preexisting variation present in the ancestral clone. We used an allele replacement strategy to test this hypothesis for a commonly repeated LOH event and show that selection is indeed driving the homogenization of the genome at this locus. Although other studies in natural, industrial, and lab-evolved isolates have observed LOH, we present the first empirical test of the causal evolutionary forces influencing these events. This work can begin to help us understand past hybridization events and subsequent genome resolution in hybrids in natural and artificial systems.

23Although our sample size is modest, we observe several interesting trends when comparing S. uvarum clones (homozygous), S. cerevisiae clones (intraspecific hybrid and previously published homozygous), and interspecific hybrid clones. First, theory and previous research predict the interspecific hybrid may experience more genome instability in the form of chromosomal rearrangements and CNV events. Instead, in our work, the interspecific hybrid and S. cerevisiae clones experience the same number of CNV events (both 1.5 CNVs/clone). However, the mechanism of CNV formation seems to differ between the hybrid and S. cerevisiae clones. Whereas S. cerevisiae CNV break points typically occur at transposable elements in our study, and previous studies, the interspecific hybrid CNVs do not utilize transposable elements, although they obviously share the same sequence background as the S. cerevisiae clones (albeit in one copy). Whether this difference is due to the absence of full-length transposable elements in S. uvarum is unknown, but this could potentially explain the lower number of CNV events in S. uvarum clones (1 CNV/clone) and presents an intriguing direction for future study.

24We now have many examples of predictable evolution in natural systems, and in laboratory experimental evolution, in which there often appears to be a limited number of high fitness pathways that strains follow when adapting to a particular condition. For example, it is well established that amplifications of nutrient transporters are drivers of adaptation in evolution in nutrient-limited conditions. Previous work in our group has particularly focused on the amplification of the high-affinity sulfate transporter gene SUL1 in sulfate-limited conditions, which occurs in almost every sulfate-limited evolution experiment and confers a fitness advantage of as much as 40% compared with the unevolved ancestor strain. The amplification of phosphate transporters has been markedly less common, and thus drivers of adaptation in this condition have been less clear. identified a whole chromosome amplification of chrXIII in one population. In a follow-up study, found whole or partial amplification of chrXIII in 3/8 populations. A genome-wide screen for segmental amplifications found a slight increase in fitness for a small telomeric segment of chromosome XIII, and a A49V point mutation in PHO84 was observed to increase fitness by 23%. However, screens by showed that although PHO84 is recurrently mutated in various experiments, it showed no benefit when amplified or deleted in phosphate-limited conditions. Finally, additional evolution experiments recapitulated the point mutation seen in ) in 24/32 populations and saw amplification of PHO84 in 8/32 populations (Miller A, Dunham MJ, unpublished data). It is important to note that all these experiments used a strain background derived from S288C or CEN.PK, both of which carry the same (un-preferred) PHO84 allele.

25In our work, we observed amplification of the S. cerevisiae GRF167 allele of PHO84 in 4/4 S. cerevisiae clones from 4 populations and 3/6 hybrid clones from 6 populations. This amplification was always preceded by the loss of the S288C allele in S. cerevisiae clones, and the LOH break points are never shared with the amplification break points. There is a 15% fitness difference between carrying two copies of the S288C allele of PHO84 compared with carrying two copies of the GRF167 allele of PHO84, and additional copies of the GRF167 allele do not provide substantial further fitness gains. The two alleles differ by several noncoding changes, and three nonsynonymous substitutions: a mutation from glutamic acid to aspartic acid (E229D), leucine to proline (L259P), and leucine to glutamine (L556Q), the latter two of which are considered “nonconservative” protein mutations due to changes in hydrophobicity and structure (supplementary fig. S5, Supplementary Material online). Intriguingly, the L259P mutation has actually been previously identified as being responsible for resistance to tetrachloroisophthalonitrile and partial resistance to pentachlorophenol in a QTL study of small-molecule drugs. Indeed, Perlstein et al. found proline at residue 259 to be conserved across fungal species, and even in orthologous human xenobiotic transporters, likely because proline-induced kinks in transmembrane spans have been shown to be essential to protein function. It thus appears that amplification of PHO84 has been less predictable, as the S288C allele does not confer a fitness advantage unless mutated, per. Together, these results imply that strain background can constrain adaptive pathways.

26In hybrids, the amplification of the S. cerevisiae segment occurred in conjunction with the loss of the S. uvarum allele. Hybrid strains with the LOH had a 25.57% relative fitness gain, whereas hybrid strains with amplification of the S. cerevisiae PHO84 allele without the LOH had a 12.53% fitness gain. Thus, LOH confers an additional 13.04% fitness gain, showing that selection for LOH has a larger impact on fitness than amplification alone. Note that S. uvarum does have proline at residue 259, like the preferred GRF167 allele, and differs from both S. cerevisiae alleles at the other two coding substitutions (229N and 556K), but the amino acid and noncoding divergence is too high to speculate what substitutions are responsible for the selection of the GRF167 allele (supplementary fig. S6, Supplementary Material online). Why the loss of one allele is more beneficial remains unclear, as PHO84 is thought to function as a monomer, but it may be due to competition for cell wall space or negative interactions with other genes in the PHO pathway.

27The infusion of variation created by hybridization provides new templates for selection to act upon, which can be more important than either point mutations or copy number variants alone. Our work shows that outcrossing need not be common to have long-lasting effects on adaptation. This implication is particularly relevant in yeast, where outcrossing may occur quite rarely followed by thousands of asexual generations.

28The observation that LOH occurs in hybrid genomes is increasingly documented, although the reason(s) for this type of mutation has been unresolved. As most examples stem from allopolyploid events that occurred millions of years ago, understanding why LOH is important in hybrid genome evolution is difficult. Cancer cells are also known to experience LOH, sometimes involved in the inactivation of tumor suppressor genes, leaving only one copy of the gene that may be mutated or silenced. Data support the conclusion that LOH events are selected for during tumor development, as many LOH events involve specific chromosomal segments, although the underlying molecular and genetic reasons for selection is an open debate.

29Here, we experimentally demonstrate that LOH can occur in homoploid interspecific hybrids as well as in intraspecific hybrids. These events occur within a few hundred generations and are common mutations, more common on average in the intraspecific hybrid (1.3 events/clone) than the interspecific hybrid (0.56 events/clone). Interestingly, the LOH events do not share break points with the CNV events in S. cerevisiae; instead, they appear to occur independently and to precede any subsequent amplification (amplification occurs following 9/13 LOH events). Competition assays with the PHO84 locus provide support that LOH itself may be more beneficial than amplification or at least increase the selective benefit of amplification events. The observation that LOH in intraspecific hybrids occurs independently from copy number change provides different opportunities for adaptation to novel conditions.

30Other cases of LOH, like the copy number neutral events observed in Ph4, Ph5, and Sh4, all of which favor the retention of the S. uvarum allele (supplementary fig. S3, Supplementary Material online), may be due to hybrid incompatibility within a particular protein complex, other epistatic interactions, or neutral processes. We furthermore discover examples where one species allele appears to be preferred over the other without LOH, such as the repeated amplification of the S. uvarum high-affinity glucose transporters HXT6/7. When one species allele is amplified and the other is not amplified, one explanation is that the local sequence context can permit or deny amplification. In the case of HXT6/7, previous experiments in S. cerevisiae have shown that amplification of HXT6/7 is quite common, thus suggesting that when given a choice between this locus in S. cerevisiae or S. uvarum in the hybrid, the preferred allele is indeed S. uvarum, though more subtle differences in rate cannot yet be ruled out. A similar scenario is observed with S. cerevisiae SUL1. Together, our results show that the heterozygosity supplied by hybridization is an important contributor to adaptive routes explored by populations as they adapt to novel conditions.

31Although we cannot generalize our results from the PHO84 locus across the many other LOH events discovered in our hybrids and S. cerevisiae, in the future, we can use similar methodology to explore whether positive selection always drives LOH or whether other explanations such as incompatibility resolution contribute as well. Future experiments might also utilize a high throughput method to explore segmental LOH in hybrids at a genome-wide scale, similar to ongoing experiments at the gene level (Lancaster S, Dunham MJ, unpublished data). Although our sample size is moderate, this is a novel and necessary step in understanding forces underlying hybrid genome stabilization and highlighting an underappreciated mechanism of hybrid adaptation.

32The mutation events we observe in our experimentally evolved hybrids are in many ways quite representative of mutations observed in ancient hybrid genomes, suggesting that hybrid genome stabilization and adaptation can occur quite rapidly (within several hundred generations). Furthermore, our results illustrate that the infusion of variation introduced by hybridization at both the intra- and inter-species level can increase fitness by providing choices of alleles for selection to act upon, even when sexual reproduction is rare. This may be particularly important for leveraging existing variation for agricultural and industrial processes and as climate change potentially increases natural hybridization.

33A list of strains used in this study is included in supplementary table S1, Supplementary Material online. All interspecific hybrids were created by crossing a ura3 LYS2 haploid parent to a URA3 lys2 haploid parent of the other mating type, plating on media lacking both uracil and lysine, and selecting for prototrophs. The S. cerevisiae strain background, known as “GRF167”, is itself a cross between FL100 and the genomic type strain S288C (data not shown). GRF167 was chosen as a strain background for simultaneous work investigating transposable elements during experimental evolution, which will be addressed in a future study.

34Continuous cultures were established using media and conditions previously described. Detailed protocols and media recipes are available at http://dunham.gs.washington.edu/protocols.shtml (last accessed March 6, 2017). Samples were taken daily and measured for optical density at 600 nm and cell count; microscopy was performed to check for contamination; and archival glycerol stocks were made daily. An experiment was terminated when contamination, growth in tubing, or clumping appeared (number of generations at the end point for each population are presented in tables 1 and 2). Samples from each end point population were colony purified to yield two clones for further study.

35Populations from the end point of each evolution were analyzed for copy number changes using array comparative genomic hybridization following the protocol used in.

36DNA was extracted from overnight cultures using the Hoffman–Winston protocol and cleaned using the Clean & Concentrator kit (Zymo Research). Nextera libraries were prepared following the Nextera library kit protocol and sequenced using paired end 150 bp reads on the Illumina NextSeq 500 machine (sequencing coverage in supplementary table S2, Supplementary Material online). The reference genomes used were S. cerevisiae v3, S. uvarum, and a hybrid reference genome created by concatenating the two genomes. Sequence was aligned to the appropriate reference genome using bwa v0.6.2 and mutations were called using GATK and samtools 0.1.19. Mutations in evolved clones were filtered in comparison with the ancestor to obtain de novo mutations. All mutations were first visually inspected using Integrative Genomics Viewer. Subsequently, point mutations in the hybrids were confirmed with Sanger sequencing (supplementary table S3, Supplementary Material online). Copy number variants were visualized using DNAcopy for S. cerevisiae and S. uvarum. LOH events were called based on sequencing coverage in the hybrids and by identifying homozygous variant calls in S. cerevisiae. All break points were called by visual inspection of sequencing reads and are thus approximate.

37The pairwise competition experiments were performed in 20 ml chemostats. Each competitor strain was cultured individually until steady state was reached and then was mixed 50:50 with a GFP-tagged ancestor. Each competition was conducted in at least two biological replicates for approximately 15 generations after mixing. Samples were collected and analyzed twice daily. The proportion of GFP+ cells in the population was detected using a BD Accuri C6 flow cytometer (BD Biosciences). The data were plotted with ln [dark cells/GFP+ cells] versus generations. The relative fitness coefficient was determined from the slope of the linear region.

38Allele replacements for the PHO84 locus were done following the protocol of the Caudy lab with further modifications described here. The native locus was replaced with Kluyveromyces lactis URA3. The pho84Δ::URA3 strain was grown overnight in 5 ml of C-URA media, then inoculated in a flask of 100 ml yeast extract peptone dextrose (YPD) and grown to an optical density of 0.6–0.8. Cells were washed then aliquoted. 275 µl of transformation mix (35 µl 1 M lithium acetate, 240 µl of 50% 3500 polyethylene glycol), 10 µl of salmon sperm, and approximately 3 µg of polymerase chain reaction product were added to the cell pellet. It was incubated at 37 °C (S. uvarum) or 42 °C (S. cerevisiae) for 45 min, then plated to YPD. It was replica plated to 5-fluoroorotic acid the following day, and colonies were tested for the gain of the appropriate species allele. The GRF167 allele was cloned into the pIL37 plasmid using Gibson assembly. Correct assembly was verified by Sanger sequencing. All primers used can be found in supplementary table S3, Supplementary Material online.

39Supplementary data are available at Molecular Biology and Evolution online.