Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe
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Evaluation Summary:
Meiotic drivers are selfish elements that distort segregation to be over-represented in offspring of heterozygotes. Multiple meiotic drive elements are known in the yeast Schizosaccharomyces pombe, which can seem puzzling as this fungus has long been thought to undergo moslty same-clone mating because of its mating-type switching system. This manuscript reports theoretical and experimental analyses suggesting that the outcrossing rate can be high enough in this species to explain the spread of multiple meiotic drive elements. The findings support the emerging view that homothallic fungi can undergo quite high rates of outcrossing, which is also in agreement with evolutionary considerations on the evolution of mating types. This study can thus be of high relevance for scientists studying meiotic drivers and/or mating systems and their evolution.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Meiotic drivers are genetic elements that break Mendel’s law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe , a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.
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Author Response:
Reviewer #1:
This manuscript reports theoretical and experimental analyses of a meiotic drive element in the yeast Schizosaccharomyces pombe, to understand whether the outcrossing rate is high enough in this species, long thought to undergo mostly same-clone mating, to explain the spread of multiple meiotic drive elements. The topic is of general interest, the experiments and analyses are clever and sound, and provide interesting answers. The experiments indeed show that the outcrossing rate in the laboratory varies among natural isolates and density conditions, and can be substantial ; the theoretical model shows that the estimated outcrossing rates do allow meiotic drive to spread.
However, the outcrossing rates measured in the laboratory may be really different from those in nature and population genomic data …
Author Response:
Reviewer #1:
This manuscript reports theoretical and experimental analyses of a meiotic drive element in the yeast Schizosaccharomyces pombe, to understand whether the outcrossing rate is high enough in this species, long thought to undergo mostly same-clone mating, to explain the spread of multiple meiotic drive elements. The topic is of general interest, the experiments and analyses are clever and sound, and provide interesting answers. The experiments indeed show that the outcrossing rate in the laboratory varies among natural isolates and density conditions, and can be substantial ; the theoretical model shows that the estimated outcrossing rates do allow meiotic drive to spread.
However, the outcrossing rates measured in the laboratory may be really different from those in nature and population genomic data are available that could allow estimate actual outcrossing rates in natural populations.
We fully agree that rates measured in the laboratory may be different than those in nature, especially given our observations on outcrossing rates varying under different cell densities. We now note that explicitly in the introduction and the discussion to make that point more clear.
We disagree, however, that the population genomic data are sufficient to estimate actual outcrossing rates in nature for S. pombe. Our position stems from empirical analyses of what happens when S. pombe isolates outcross. Traditional population genetics models assume Mendelian allele transmission and a baseline recombination rate. These assumptions are strongly violated in S. pombe, so applying traditional population genetics models to S. pombe to determine outcrossing rate is problematic (Hu et al., 2017; Nuckolls et al., 2017; Zanders et al., 2014). The causes of these violations are so complex that amending existing models to adequately predict outcrossing would be a major undertaking in population genetics that is well beyond the scope of this work. We added to the introduction and to the discussion to illustrate the complexity of the situation and explain why we did not provide estimates.
Indeed, outcrossing rates depend mostly on where and when in nature dispersal and clonal multiplication occur, while laboratory experiments typically use high densities of clonemates on plates. Overall this study brings support to the idea that homothallic fungi probably do not undergo mostly same-clone mating in nature, in contrast to the most accepted view in the fungal literature, but in agreement with evolutionary considerations ; the study and the findings would thus benefit from being placed in the right evolutionary context (doi: 10.1111/j.1420-9101.2012.02495.x ; 10.1111/j.1469-185X.2010.00153.x ; 10.1128/EC.00440-07 ; 10.1038/hdy.2014.37 ; 10.1111/nph.17039).
We have added citations and statements clarifying that homothallic fungi, and specifically, S. pombe can outcross to the introduction. We also extensively revised the introduction to better contextualize our study within the current assumptions about S. pombe biology and its capacity to harbor selfish genes.
The terms selfing and outcrossing as used in the manuscript does not correspond to the diploid selfing and outcrossing that occur in plants and animals, and the term can thus be misleading.
To address this confusion, we now more explicitly define the terms we used in the paper in the introduction. We also have added a supplemental figure to help illustrate S. pombe’s mating process and the terms we use (Figure 1-figure supplement 1).
Reviewer #2:
The authors combine cytological, genetic, mathematical, and experimental evolution techniques to connect variation in mating behavior with variation in the population dynamics of meiotic drive in the yeast Schizosaccharomyces pombe. First, the authors use cytological and genetic methods to document variation across strains of pombe in their (i) propensity to inbreed, (ii) efficiency of mating, (iii) rate of mate type switching, and (iv) variability of ascus morphology. These results will be of major standalone interest to the yeast community, and will likely find experimental use in many settings. Then the authors use population genetic modeling to study the theoretical implications of this variation in mating behavior for the spread of meiotic drivers (which have recently been shown to be pervasive in pombe). Finally, the authors use cytological techniques to track the spread of introduced drivers in experimental populations of pombe, and show that the drivers follow frequency trajectories that agree well with predictions from the theoretical analysis. These results will be of major interest to geneticists working on meiotic drive, as well as workers in the currently burgeoning field of synthetic gene drives for population control.
The analysis is carefully done, and I am confident in the results as presented (with one minor exception detailed below). My only major suggestion for improvement concerns the scope of the population genetic modeling. As it stands, this modeling is primarily used to generate predicted frequency trajectories of meiotic drivers against which the trajectories observed in the evolution experiments can be compared. The fact that the experimental and theoretical trajectories match well is impressive, and very promising for the future of pombe as an experimental system in meiotic drive research. However, substantively, as the authors recognize, this agreement tells us mainly that the population genetic model that they use to generate the predicted trajectories takes into account all relevant parameters and is well calibrated. Thus, from the population genetic modeling and evolution experiments, we get only an indirect picture of how variation in mating behavior has actually impacted the natural spread of drivers in this species.
I believe that the population genetic modeling, with minor modifications, could in fact be used to make more direct predictions about the natural history of drive in pombe. For example, should strains with less inbreeding harbor more fixed drivers? And in strains with more inbreeding, should drivers---because they have very long fixation times---be more likely to be observed as polymorphisms? Such questions are, I believe, well within reach of the authors' population genetic modeling.
We agree that this is a very interesting line of inquiry. Populations with more than one driver operating have not yet been considered by population genetic modeling. Given that it has become apparent that genomes housing multiple drivers are not rare, this is also a very important question to address. Your comment emboldened us to attempt to tackle this problem, which we previously considered beyond our reach. Thank you for this push!
An additional unpublished caveat to reconstructing the natural history of drive in S. pombe is that our lab and Li-Lin Du’s lab have found that wtf meiotic drivers are quite ancient. Fission yeast lineages have been harboring multiple meiotic drivers for over 100 million years and some species have even more drivers than S. pombe! Because of this, we are interested in exploring how multiple drivers are maintained for long periods in addition to addressing how multiple drivers can arise within a lineage.
We are a long way away from accomplishing our goals on this project and consider this ongoing work to be beyond the scope of this paper.
A minor concern: To track the spread of a driver introduced in their experimental populations, the authors linked the driving allele to one fluorescent marker (GFP/mCherry) and the non-driving allele to the other (mCherry/GFP), and compared the spread of the one marker relative to the other. To use their model to generate expected frequency trajectories for these experiments, the authors needed to measure, in controlled settings, the intrinsic fitness costs of GFP vs mCherry; they estimate that GFP is relatively costly in sexual (but not vegetative) reproduction. However, their estimates of the relative fitness cost of GFP are based on frequency trajectories across just 6 generations, and assume additive dominance, so that the fitness cost to a GFP homozygote is twice that to a heterozygote. It is unclear how statistically noisy the estimation procedure is given the small number of generations used, and whether it is justified to assume additive dominance (which is especially relevant since the dominance of fitness costs is known to be a critical factor in determining the frequency dynamics of meiotic drive).
Thank you for this comment. We did use only 6 generations of data for these calculations, but we pooled data from 4 distinct control experiments each of which had 3 independent replicate populations. After 6 generations, fluorescent marker loss becomes a bigger factor in our results and the populations behave less predictability.
We did not, however, have a good justification for using additive dominance. Because of this, we reran the maximum likelihood and allowed both the fitness cost and dominance to vary. We found that the parameters that best fit our data was a fitness cost of 0.234 and a dominance of 0.083. This fitness cost is similar to our previous value, but this revealed it was incorrect for us to assume additive dominance. We have since updated the paper and the Figures 4 and 5 to reflect the use of these values.
Reviewer #3:
Weaknesses
Even though the experiments find some important parameters for meiotic-driver spread in fission yeast, the results are not sufficient to explain the apparent "success of meiotic drivers in this species". The links that the authors suggest between mating type switching efficiency, the amount of outcrossing, the speed of invasion of the driver and the cost associated with the driver cannot explain the success of drivers. Furthermore, the causality of the different factors is not explained.
We agree we do not offer a complete explanation of wtf genes in S. pombe, but we claim our work ‘helps explain,’ which we feel is a well-supported claim. Or revised introduction, we hope, better contextualizes our study. The current understanding of S. pombe is that the species inbreeds. This has been hard to understand because one wouldn’t expect the species with the most known meiotic drivers to be an inbreeding species. Our data shows that S. pombe mating phenotypes in the lab are highly variable and include considerable outcrossing. We also show how the range of parameters we observe are consistent with the spread of meiotic drivers under specific conditions.
That outcrossing increases the speed of invasion is true (see also Durand et al 1997 PMID:9093861), but the argument that 'reduced levels of mating type switching could lead to less inbreeding' is not supported. There are two problems with this statement. First, it is not clear to me if this is theoretically true. If switching occurs infrequently but consistently, the chances of a cell to be positioned to another cell of the opposite mating type either from self or opposite type will probably not be that different. Only in a narrow range of cell density will this probably play a role, however, this should be properly modelled in a structural environment or tested experimentally.
Thank you for pointing this out. Our modified text makes the support for the argument that switching rates can affect inbreeding more clear. In short, the experiments we present in Figure 1-figure supplement 1 show that inbreeding is increased within an h90 population at low density. The simplest explanation of this observation is that the cells plated at low density have fewer opportunities to mate outside their clonal lineage, so instead they more frequently rely on intra-lineage mating following switching. Cells at higher density have the opportunity to mate outside their lineage, even before a switching event has enabled mating within a lineage.
Extending that logic, if switching happens less frequently within a clonal lineage, cells within that lineage will need to undergo more generations of mitosis before a switching event enables intra-lineage mating. Those extra divisions will make it more likely that cells of a given lineage will encounter cells of the opposite mating type from a distinct lineage. This would lead to more outcrossing.
We acknowledge that this considers that mating occurs on a surface and that the cell populations are stationary (unmixed) immediately prior to mating. These are the conditions used for all analyses in this work. These conditions likely do not recapitulate all matings that occur in nature, but we argue they are feasible in nature.
A comparison between heterothallic and homothallic strains is - contrary to what the authors argue in line 138 - not appropriate for this test, as the first cannot reproduce by selfing. Using strains that have intermediate amounts of mating type switching (e.g. using h90 Sp strains mutant in the switching pathway; Maki et al. PMID:29852001) could give more insight in this. Reduced switching will lead to reduced spore production, because fewer of the cells will be located next to a cell of the opposite mating type (as shown in Nieuwenhuis et al. 2018 PMID:29691402 and by the authors in Fig. 1-S3), but this does not have to affect outcrossing efficiency. This also becomes apparent from the data presented in Figs 1D and 1E, which do not seem correlated.
Thank you for this comment. We have modified the text to highlight our (previously poorly-expressed) intent of using these heterothallic controls. Briefly, these heterothallic cell mixtures model a randomly mating population in that they have an equal mix of h+ and h- cells of both colors, but cells cannot mate within a clonal lineage. This should necessarily lead to random mating in our assays. This is what we observe, which we interpret as support that our assays work as intended. We then later use this same control to model the effect of random mating in our experimental evolution analyses.
Second, the authors have not measured mating-type switching, but used the amount of mother daughter matings as a proxy for mating type switching. This method introduces a bias towards the correlation switching and selfing, because the latter is used as a proxy for the first. Fluorescent proteins under control of a mating-type specific promotor is an established method (e.g. Jakočiūnas et al 2013 PMCID:PMC6420890, Vještica et al 2021 PMID:33406066), which will give direct observations of the mating type. The observation that the shmoo length is associated with outcrossing is very interesting, and - without changing switching frequency - appears to affect outcrossing.
We acknowledge that we did not assay mating type switching directly in Sk. To our knowledge, no one has ever reported an experiment that assayed mating type switching directly in S. pombe. Lineage tracing paired with mating assays, like those presented in Figure 2B, were done to establish the currently accepted mating-type switching model in S. pombe.
We agree that this is unsatisfying and that factors other than mating type switching could affect the behavior of cells in this assay. We did not, however, exclusively rely on sibling cell matings as support of our argument in support of a reduced rate of mating-type switching in Sk. The foundation of our hypothesis is the discovery of Singh and Klar (2002) that there are fewer switching-inducing DSBs in Sk. We also observed that Sk cells divided more times than Sp prior to mating at low cell density, suggesting they required more divisions to have mating competent cells within a clonal lineage. Still, because we were unable to measure switching directly, we explicitly state that less switching in that strain is an unproven model consistent with the available data.
There are mating-type reporters in the papers mentioned, but the reporters were not used for lineage tracing of mating type switching in the papers cited or any other papers we could find. Lineage tracing is required to assay switching frequency as a clonal population founded by a cell with reduced switching frequency will produce a population with a balanced number of h+ and h- cells after a limited number of divisions, as long as the two types of switches (h+ to h- and vice versa) occur at the same rate.
Similar to the reviewer, we were frustrated with our inability to assay switching rate directly. We previously attempted to use the markers described in Jakočiūnas et al 2013 for lineage tracing in Sp (lab isolate) cells. In agreement with the published work, we saw in initial snapshots of the cell population a roughly equal number of yellow (h-) and blue (h+) cells. When we imaged cells over time, however, they did not behave as expected. Most strikingly, all mating events were not between yellow and blue cells, as would be expected if they were absolute markers of h+ and h- cells. Instead, many of the matings were between two blue cells. This could be due to fluorophore carryover and/or delayed accumulation of the new fluorophore after switching. In addition, the fluorophore switching pattern we observed generally did not follow the expectation that 1 out of 4 cells derived from a single progenitor should have a different mating type than the other three cells. These observations were sufficient to convince us that the markers were not suitable, under our experimental conditions, for lineage tracing to assay switching patterns. We have now included a figure documenting our attempts to use this assay as other readers may also be curious why we relied on indirect measures.
Finally, the authors argue that meiotic drivers are evolving rapidly, can invade fast and that this can occur even when selfing is prevalent. The model seems to contract this. Let's start with the claim that novel drivers can invade in a population. Novel alleles arise at a frequency of 1/N (N = population size, bottom left corner Fig 3A, not at 5% as used in the analyses) and as drive is as strong as the inverse of the population size the fitness difference is initially extremely low giving plenty of time for drift (when driver is neutral) or selection (when driver is deleterious) to remove the novel allele.
We have added to the analysis in figure 3A to show that under our model (lacking drift) drivers can invade a population that is not exclusively inbreeding with any initial frequency greater than zero (Figure3-figure supplement 1). We have also extended our analysis to include simulations of drift (Figure 3-figure supplement 1). Note that in our models, drive is never neutral as it kills ~half the meiotic products (i.e. the progeny) made by heterozygotes.
In addition, we note that the frequency of the driver at the time of mating and meiosis is the essential parameter. A local population of S. pombe could be founded by relatively few individuals. If a mutation generating a novel driver occurs during the clonal expansion of this population, it could rise to relatively high frequency within that population before the cells starve and mate. This effect of clonal expansion is the reason microbiologists must do fluctuation analyses to assay mutation rates: there are jackpot cultures (analogous to local populations, founded by a limited number of individuals) with a high frequency of mutants and others with few to no mutations.
In order for drivers to increase to levels that will give 'rapid wtf gene evolution' (line 112) a prolonged level of mostly drift is probably necessary. It is difficult to make statements about the speed of wtf evolution in the fission yeast system, without having a better description of the variation of the paralogs and their ages in fission yeast. The speed of wtf evolution is not clear, as shown in earlier findings from this group that shows very old wtf loci; Eickbush et al. 2019. Comparing wtf evolution relative to neutrally evolving loci might give more insight in wtf evolution speed. Especially when drive is costly (as suggested by the authors, though not shown or quantified) the time to substantial frequencies is large. It could also be possible that drive itself is beneficial (e.g. resources from the killed spores made available to the killers or through released local competitive pressure), which will lead to increased fitness though combined drive and increased viability, even at low frequencies.
Eickbush et al 2019 demonstrates rapid evolution of the wtf gene family. We did find in that study that most wtf loci are shared between different isolates of S. pombe. The critical point is, however, that the wtf genes that are at a given locus are generally dramatically different due to rapid evolution. Even when two strains share a driver at the same locus, they generally have distinct sequences and are thus expected to be mutually killing. We have explained this situation more clearly in the revised discussion.
Because of this rapid evolution and the functional consequences of this variation, which we have demonstrated in cited studies, very subtle changes in wtf gene sequence leads to the birth of a novel driver. Therefore, generation of wtf driver heterozygosity does not require mating between significantly diverged previously isolated populations- a single point mutation can generate a novel driver that self-selects via drive.
We have directly measured the fitness cost of Sk wtf4 heterozygosity when expressed in Sp, precisely the scenario assayed in this paper (Nuckolls et al 2017). We use that fitness cost in our modeling studies. In addition, the observed changes in our experimental evolution analyses were quite close to expected trajectories. This provides additional support that the parameters we used in these analyses were appropriate for our experimental conditions. We do, however, acknowledge that there are other ecological conditions under which the fitness costs of drivers could differ.
Minor comments
The loss of mCherry alleles due to reversion of ura+ occurs more rapidly than that in GFP. It is likely that this variable change in reversion affects the observed change in frequency. This should be corrected for in the raw data.
We agree that the loss of the fluorescent alleles makes our data noisy, but we do not have precise measurements of these rates. We judged this phenotype did not affect the conclusions in this paper, so we did not invest in correcting this limitation of our system.
Inbreeding is a term generally used in population genetics, where it refers the the amount of mating between related individuals. Even though it is fundamentally correct, a more appropriate term would be haploid selfing or intra-clonal mating, as mating in these strains and experiments is actually between clones. Inbreeding in this context is confusing to people who are not familiar with facultatively sexual species.
We have provided additional guidance and explanation to avoid confusion with our use of terms.
The effect of inbreeding on driver alleles has been studied theoretically before, showing qualitatively similar results (e.g. see Durand et al 1997 PMID:9093861; Martinossi-Allibert et al. 2021 PMID:33764512, Ament-Velásquez).
Reference to driver systems in other fungal species (Neurospora and Podospora) that are highly selfing is completely missing (Svedberg et al. 2018, 2021; Vogan et al 2019, 2020; Martinossi-Allibert et al. 2021)
Thank you for pointing out these omissions, we have added citations.
There seems to be quite some variation between the different replicate experiments (Fig 1E vs Fig 2-S3 for example).
We agree, but were satisfied that the data support our claims.
Line 76: This paragraph is a bit misleading and internally contradicting. The data from Farlow et al. does not take into consideration the recent hybridisation of diverged populations as shown in Tusso et al. 2018 and thus overestimates the time between outcrossing events. The estimate that 20-60 outcrossing events (underestimate due to homogenization and potential meitotic drive) occurred in the last 500 years suggests a higher number than 1 per 800,000. Citing this number is obsolete.
We removed the mention of the out of date Farlow reference.
line 730: The inbreeding coefficient in Sun et al. 2017 (probability of IBD which is between 0 and 1) is different from the one used in Hartl & Clark 2007 between -1 and 1.
Thank you, we have corrected this.
The speculations on the 4913bp insertion and its effect on mating type switching is not substantiated. Variation around the mating type is rampant (see for example Beach 1986 and Nieuwenhuis et al. 2018) and the authors even show that is likely is not the case that this element affects switching in FY29033. The insertion is an interesting observation, but just that.
We decided to keep this in the paper because if we were interested in pursuing a potential cause of changed mating phenotypes, we would likely start with testing the transposon, even though the phenotype of FY29033 argues against the hypothesis. Genetic context frequently affects phenotypes and Sk and FY29033 are different strains. Although we do not plan on following this up, we wanted to present the ideas to others who may be interested in pursuing these phenotypes further.
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Evaluation Summary:
Meiotic drivers are selfish elements that distort segregation to be over-represented in offspring of heterozygotes. Multiple meiotic drive elements are known in the yeast Schizosaccharomyces pombe, which can seem puzzling as this fungus has long been thought to undergo moslty same-clone mating because of its mating-type switching system. This manuscript reports theoretical and experimental analyses suggesting that the outcrossing rate can be high enough in this species to explain the spread of multiple meiotic drive elements. The findings support the emerging view that homothallic fungi can undergo quite high rates of outcrossing, which is also in agreement with evolutionary considerations on the evolution of mating types. This study can thus be of high relevance for scientists studying meiotic drivers and/or mating …
Evaluation Summary:
Meiotic drivers are selfish elements that distort segregation to be over-represented in offspring of heterozygotes. Multiple meiotic drive elements are known in the yeast Schizosaccharomyces pombe, which can seem puzzling as this fungus has long been thought to undergo moslty same-clone mating because of its mating-type switching system. This manuscript reports theoretical and experimental analyses suggesting that the outcrossing rate can be high enough in this species to explain the spread of multiple meiotic drive elements. The findings support the emerging view that homothallic fungi can undergo quite high rates of outcrossing, which is also in agreement with evolutionary considerations on the evolution of mating types. This study can thus be of high relevance for scientists studying meiotic drivers and/or mating systems and their evolution.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This manuscript reports theoretical and experimental analyses of a meiotic drive element in the yeast Schizosaccharomyces pombe, to understand whether the outcrossing rate is high enough in this species, long thought to undergo mostly same-clone mating, to explain the spread of multiple meiotic drive elements. The topic is of general interest, the experiments and analyses are clever and sound, and provide interesting answers. The experiments indeed show that the outcrossing rate in the laboratory varies among natural isolates and density conditions, and can be substantial ; the theoretical model shows that the estimated outcrossing rates do allow meiotic drive to spread. However, the outcrossing rates measured in the laboratory may be really different from those in nature and population genomic data are …
Reviewer #1 (Public Review):
This manuscript reports theoretical and experimental analyses of a meiotic drive element in the yeast Schizosaccharomyces pombe, to understand whether the outcrossing rate is high enough in this species, long thought to undergo mostly same-clone mating, to explain the spread of multiple meiotic drive elements. The topic is of general interest, the experiments and analyses are clever and sound, and provide interesting answers. The experiments indeed show that the outcrossing rate in the laboratory varies among natural isolates and density conditions, and can be substantial ; the theoretical model shows that the estimated outcrossing rates do allow meiotic drive to spread. However, the outcrossing rates measured in the laboratory may be really different from those in nature and population genomic data are available that could allow estimate actual outcrossing rates in natural populations. Indeed, outcrossing rates depend mostly on where and when in nature dispersal and clonal multiplication occur, while laboratory experiments typically use high densities of clonemates on plates. Overall this study brings support to the idea that homothallic fungi probably do not undergo mostly same-clone mating in nature, in contrast to the most accepted view in the fungal literature, but in agreement with evolutionary considerations ; the study and the findings would thus benefit from being placed in the right evolutionary context (doi: 10.1111/j.1420-9101.2012.02495.x ; 10.1111/j.1469-185X.2010.00153.x ; 10.1128/EC.00440-07 ; 10.1038/hdy.2014.37 ; 10.1111/nph.17039). The terms selfing and outcrossing as used in the manuscript does not correspond to the diploid selfing and outcrossing that occur in plants and animals, and the term can thus be misleading.
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Reviewer #2 (Public Review):
The authors combine cytological, genetic, mathematical, and experimental evolution techniques to connect variation in mating behavior with variation in the population dynamics of meiotic drive in the yeast Schizosaccharomyces pombe. First, the authors use cytological and genetic methods to document variation across strains of pombe in their (i) propensity to inbreed, (ii) efficiency of mating, (iii) rate of mate type switching, and (iv) variability of ascus morphology. These results will be of major standalone interest to the yeast community, and will likely find experimental use in many settings. Then the authors use population genetic modeling to study the theoretical implications of this variation in mating behavior for the spread of meiotic drivers (which have recently been shown to be pervasive in …
Reviewer #2 (Public Review):
The authors combine cytological, genetic, mathematical, and experimental evolution techniques to connect variation in mating behavior with variation in the population dynamics of meiotic drive in the yeast Schizosaccharomyces pombe. First, the authors use cytological and genetic methods to document variation across strains of pombe in their (i) propensity to inbreed, (ii) efficiency of mating, (iii) rate of mate type switching, and (iv) variability of ascus morphology. These results will be of major standalone interest to the yeast community, and will likely find experimental use in many settings. Then the authors use population genetic modeling to study the theoretical implications of this variation in mating behavior for the spread of meiotic drivers (which have recently been shown to be pervasive in pombe). Finally, the authors use cytological techniques to track the spread of introduced drivers in experimental populations of pombe, and show that the drivers follow frequency trajectories that agree well with predictions from the theoretical analysis. These results will be of major interest to geneticists working on meiotic drive, as well as workers in the currently burgeoning field of synthetic gene drives for population control.
The analysis is carefully done, and I am confident in the results as presented (with one minor exception detailed below). My only major suggestion for improvement concerns the scope of the population genetic modeling. As it stands, this modeling is primarily used to generate predicted frequency trajectories of meiotic drivers against which the trajectories observed in the evolution experiments can be compared. The fact that the experimental and theoretical trajectories match well is impressive, and very promising for the future of pombe as an experimental system in meiotic drive research. However, substantively, as the authors recognize, this agreement tells us mainly that the population genetic model that they use to generate the predicted trajectories takes into account all relevant parameters and is well calibrated. Thus, from the population genetic modeling and evolution experiments, we get only an indirect picture of how variation in mating behavior has actually impacted the natural spread of drivers in this species. I believe that the population genetic modeling, with minor modifications, could in fact be used to make more direct predictions about the natural history of drive in pombe. For example, should strains with less inbreeding harbor more fixed drivers? And in strains with more inbreeding, should drivers---because they have very long fixation times---be more likely to be observed as polymorphisms? Such questions are, I believe, well within reach of the authors' population genetic modeling.
A minor concern: To track the spread of a driver introduced in their experimental populations, the authors linked the driving allele to one fluorescent marker (GFP/mCherry) and the non-driving allele to the other (mCherry/GFP), and compared the spread of the one marker relative to the other. To use their model to generate expected frequency trajectories for these experiments, the authors needed to measure, in controlled settings, the intrinsic fitness costs of GFP vs mCherry; they estimate that GFP is relatively costly in sexual (but not vegetative) reproduction. However, their estimates of the relative fitness cost of GFP are based on frequency trajectories across just 6 generations, and assume additive dominance, so that the fitness cost to a GFP homozygote is twice that to a heterozygote. It is unclear how statistically noisy the estimation procedure is given the small number of generations used, and whether it is justified to assume additive dominance (which is especially relevant since the dominance of fitness costs is known to be a critical factor in determining the frequency dynamics of meiotic drive).
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Reviewer #3 (Public Review):
Meiotic drivers act when the driver allele is in a heterozygous state, which is most likely when outcrossing is frequent. To explain the existence of drivers in the highly selfing fission yeast, the authors investigate the effect of selfing on spread of a driver allele. They show that outcrossing in fission yeast varies between different natural isolates, and show in a competition experiment that a meiotic driver increases in the population more rapidly under outcrossing than under selfing conditions. Additionally, they show that fitness costs associated with a driver will reduce the speed of invasion and the initial frequency required for invasion to be possible.
Strengths:
The research shows experimentally the change of allele frequency for drivers and how this spread differs between heterothallic …
Reviewer #3 (Public Review):
Meiotic drivers act when the driver allele is in a heterozygous state, which is most likely when outcrossing is frequent. To explain the existence of drivers in the highly selfing fission yeast, the authors investigate the effect of selfing on spread of a driver allele. They show that outcrossing in fission yeast varies between different natural isolates, and show in a competition experiment that a meiotic driver increases in the population more rapidly under outcrossing than under selfing conditions. Additionally, they show that fitness costs associated with a driver will reduce the speed of invasion and the initial frequency required for invasion to be possible.
Strengths:
The research shows experimentally the change of allele frequency for drivers and how this spread differs between heterothallic (obligatory outcrossing) and homothallic (haploid selfing capable) strains. The experiments are further supported by a simple model in which the invasion trajectories are well predicted. The addition of competitions where a driver is linked to a deleterious allele (GFP) shows the importance of the cost of drivers for their evolutionary dynamics which again fits with the predictions of the model.
The authors further show that the amount of haploid selfing, sporulation efficiency and probably mating type switching varies between natural isolates. These observations suggest that mating in nature is probably more messy than would be expected based on Leupold derived strains. They describe interesting observations in natural strains, such as shmoo-length variation, uncleaved (filamentous) cells that mate and potential mating interference.
These experiments are very promising and show that meiotic drive in the fission yeast system can be used as a tool to study meiotic drivers, being able to manipulate driver linked cost (and probably also benefits when linking the driver to an prototrophic marker), the ability to self- or outcross and possibly associate these markers to mating type alleles simulating "sex" specific drive.
Weaknesses:
Even though the experiments find some important parameters for meiotic-driver spread in fission yeast, the results are not sufficient to explain the apparent "success of meiotic drivers in this species". The links that the authors suggest between mating type switching efficiency, the amount of outcrossing, the speed of invasion of the driver and the cost associated with the driver cannot explain the success of drivers. Furthermore, the causality of the different factors is not explained.
That outcrossing increases the speed of invasion is true (see also Durand et al 1997 PMID:9093861), but the argument that 'reduced levels of mating type switching could lead to less inbreeding' is not supported. There are two problems with this statement. First, it is not clear to me if this is theoretically true. If switching occurs infrequently but consistently, the chances of a cell to be positioned to another cell of the opposite mating type either from self or opposite type will probably not be that different. Only in a narrow range of cell density will this probably play a role, however, this should be properly modelled in a structural environment or tested experimentally. A comparison between heterothallic and homothallic strains is - contrary to what the authors argue in line 138 - not appropriate for this test, as the first cannot reproduce by selfing. Using strains that have intermediate amounts of mating type switching (e.g. using h90 Sp strains mutant in the switching pathway; Maki et al. PMID:29852001) could give more insight in this. Reduced switching will lead to reduced spore production, because fewer of the cells will be located next to a cell of the opposite mating type (as shown in Nieuwenhuis et al. 2018 PMID:29691402 and by the authors in Fig. 1-S3), but this does not have to affect outcrossing efficiency. This also becomes apparent from the data presented in Figs 1D and 1E, which do not seem correlated. Second, the authors have not measured mating-type switching, but used the amount of mother daughter matings as a proxy for mating type switching. This method introduces a bias towards the correlation switching and selfing, because the latter is used as a proxy for the first. Fluorescent proteins under control of a mating-type specific promotor is an established method (e.g. Jakočiūnas et al 2013 PMCID:PMC6420890, Vještica et al 2021 PMID:33406066), which will give direct observations of the mating type. The observation that the shmoo length is associated with outcrossing is very interesting, and - without changing switching frequency - appears to affect outcrossing.
Finally, the authors argue that meiotic drivers are evolving rapidly, can invade fast and that this can occur even when selfing is prevalent. The model seems to contract this. Let's start with the claim that novel drivers can invade in a population. Novel alleles arise at a frequency of 1/N (N = population size, bottom left corner Fig 3A, not at 5% as used in the analyses) and as drive is as strong as the inverse of the population size the fitness difference is initially extremely low giving plenty of time for drift (when driver is neutral) or selection (when driver is deleterious) to remove the novel allele. In order for drivers to increase to levels that will give 'rapid wtf gene evolution' (line 112) a prolonged level of mostly drift is probably necessary.
It is difficult to make statements about the speed of wtf evolution in the fission yeast system, without having a better description of the variation of the paralogs and their ages in fission yeast. The speed of wtf evolution is not clear, as shown in earlier findings from this group that shows very old wtf loci; Eickbush et al. 2019. Comparing wtf evolution relative to neutrally evolving loci might give more insight in wtf evolution speed. Especially when drive is costly (as suggested by the authors, though not shown or quantified) the time to substantial frequencies is large. It could also be possible that drive itself is beneficial (e.g. resources from the killed spores made available to the killers or through released local competitive pressure), which will lead to increased fitness though combined drive and increased viability, even at low frequencies.
# Minor comments
The loss of mCherry alleles due to reversion of ura+ occurs more rapidly than that in GFP. It is likely that this variable change in reversion affects the observed change in frequency. This should be corrected for in the raw data.
Inbreeding is a term generally used in population genetics, where it refers the the amount of mating between related individuals. Even though it is fundamentally correct, a more appropriate term would be haploid selfing or intra-clonal mating, as mating in these strains and experiments is actually between clones. Inbreeding in this context is confusing to people who are not familiar with facultatively sexual species.
The effect of inbreeding on driver alleles has been studied theoretically before, showing qualitatively similar results (e.g. see Durand et al 1997 PMID:9093861; Martinossi-Allibert et al. 2021 PMID:33764512, Ament-Velásquez).
Reference to driver systems in other fungal species (Neurospora and Podospora) that are highly selfing is completely missing (Svedberg et al. 2018, 2021; Vogan et al 2019, 2020; Martinossi-Allibert et al. 2021)
There seems to be quite some variation between the different replicate experiments (Fig 1E vs Fig 2-S3 for example).
Line 76: This paragraph is a bit misleading and internally contradicting. The data from Farlow et al. does not take into consideration the recent hybridisation of diverged populations as shown in Tusso et al. 2018 and thus overestimates the time between outcrossing events. The estimate that 20-60 outcrossing events (underestimate due to homogenization and potential meitotic drive) occurred in the last 500 years suggests a higher number than 1 per 800,000. Citing this number is obsolete.
line 730: The inbreeding coefficient in Sun et al. 2017 (probability of IBD which is between 0 and 1) is different from the one used in Hartl & Clark 2007 between -1 and 1.
The speculations on the 4913bp insertion and its effect on mating type switching is not substantiated. Variation around the mating type is rampant (see for example Beach 1986 and Nieuwenhuis et al. 2018) and the authors even show that is likely is not the case that this element affects switching in FY29033. The insertion is an interesting observation, but just that.
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