Joint inference of evolutionary transitions to selffertilization and demographic history using wholegenome sequences
Abstract
The evolution from outcrossing to selfing occurred recently across the eukaryote tree of life in plants, animals, fungi, and algae. Despite shortterm advantages, selfing is hypothetically an evolutionary deadend reproductive strategy. The tippy distribution on phylogenies suggests that most selfing species are of recent origin. However, dating such transitions is challenging yet central for testing this hypothesis. We build on previous theories to disentangle the differential effect of past changes in selfing rate or from that of population size on recombination probability along the genome. This allowed us to develop two methods using fullgenome polymorphisms to (1) test if a transition from outcrossing to selfing occurred and (2) infer its age. The teSMC and tsABC methods use a transition matrix summarizing the distribution of times to the most recent common ancestor along the genome to estimate changes in the ratio of population recombination and mutation rates overtime. First, we demonstrate that our methods distinguish between past changes in selfing rate and demographic history. Second, we assess the accuracy of our methods to infer transitions to selfing approximately up to 2.5N_{e} generations ago. Third, we demonstrate that our estimates are robust to the presence of purifying selection. Finally, as a proof of principle, we apply both methods to three Arabidopsis thaliana populations, revealing a transition to selfing approximately 600,000 years ago. Our methods pave the way for studying recent transitions to selffertilization and better accounting for variation in mating systems in demographic inferences.
Editor's evaluation
This manuscript details an important development of population genetics theory that can be used to infer past changes in the selfing rate of natural populations. The inference procedure is convincing and represents a substantial improvement upon previous methods. The work will be of broad interest to researchers studying mating system evolution and its consequences and will improve demographic inferences drawn from population genetic approaches.
https://doi.org/10.7554/eLife.82384.sa0Introduction
Hermaphroditism is common in many groups of eukaryotes, especially in plants, and allows uniparental reproduction through selfing. The rate of selffertilization is known to vary widely between species and populations; from exclusive outcrossing, through mixed mating, to predominant selffertilization (Stebbins, 1950; Whitehead et al., 2018). Transition from outcrossing to predominant selffertilization is the most frequent reproductive transition in flowering plants (Barrett, 2010) and is thought to have occurred many hundreds of times. Such a transition has profound ecological and evolutionary consequences affecting the genetic functioning and the demography of a population as well as patterns of dispersal. Taking selfing rate and its possible variations into account is thus key to properly understand the evolution of fate of a species.
In flowering plants, crossfertilization is ensured by diverse molecular and morphological features, some of which being referred to as selfincompatibility (SI) mechanisms (Charlesworth, 2010), which are defined as ‘the inability of a hermaphroditic fertile seed plant to produce zygotes after selfpollination’ (de Nettancourt, 1997). SI mechanisms enable the pistil of a plant to identify and repel selfpollen or pollen of a related genetic type and as a consequence, avoid inbreeding (de Nettancourt, 1997). SI systems are known to be encoded by a small number of genes and can therefore easily be lost. Indeed, genetic disruptions of SI systems through naturally occurring mutations are thought to be a major driver of plant reproductive diversity and have been linked to recent transitions to predominant selffertilization in several species (Shimizu and Tsuchimatsu, 2015; Mattila et al., 2020).
On the short term, whether a new mutation responsible for an SI breakdown will invade the population or be lost depends on the balance between the main advantages of selfing (reproductive assurance and gene transmission advantage) and inbreeding depression, that is the reduced fitness caused by an increased homozygosity under inbreeding (Charlesworth and Charlesworth, 1987; Charlesworth, 2006). On the long term, selfing is predicted to increase extinction rate and to reduce diversification of selfing lineages as it has been observed in a few clades such as Solanaceae (ZenilFerguson et al., 2019), Primulaceae (de Vos et al., 2014); but see Landis et al., 2018. A consequence is the supposed recent origin of selfing species due to an excess of transitions on terminal branches. It thus nicely illustrates how the balance between microevolutionary and macroevolutionary processes can generate the observed distribution of mating systems among species (Igic et al., 2008; Goldberg et al., 2010). This peculiar dynamic is also invoked to explain that the genomic effects of selfing are often detected on withinspecies polymorphism but very rarely on betweenspecies divergence (Glémin et al., 2019). The recent timing of these transitions is thus a central assumption but has not been systematically tested as it remains a challenging task.
Despite the increasing number of available methods to reconstruct the evolutionary history of populations using genomic data (Excoffier et al., 2013; Schiffels and Durbin, 2014; Boitard et al., 2016; Terhorst et al., 2017; Speidel et al., 2019), none of them is currently able to explicitly identify and estimate the age of transitions in reproductive strategies. In flowering plants, previous attempts to estimate the age of transitions to selfing were based on limited genetic variation at a single locus directly controlling the reproductive mode in plants: the Slocus (reviewed in Mattila et al., 2020). For example, naturally occurring lossoffunction alleles at the Slocus were shown to be responsible for the loss of SI in Arabidopsis thaliana (Tsuchimatsu et al., 2010). The steady accumulation of nonsynonymous alleles following loss of constraint at the Slocus was used to estimate that the age of the transition is at most 1.48 million years old, based on current estimates of the mutation rate (Shimizu and Tsuchimatsu, 2015). Note that the original upperbound estimate in Bechsgaard et al., 2006, is 413,000 years owing to the use of a different mutation rate (see Figure 4 in Shimizu and Tsuchimatsu, 2015). This approach is limited by the small number of genetic variants upon which the estimation is conducted and can only be used in species for which, as is the case in A. thaliana, the genetic determinism of the loss of SI is known. However, a shift in reproductive system is expected to strongly impact genomewide polymorphisms patterns, that is not only at the (S)loci controlling it, thereby leaving a potentially characteristic molecular signature. We use this rationale and build upon previous theoretical work to develop two inference tools allowing to use fullgenome polymorphism data of any species in order to (1) reveal the occurrence of past changes in reproduction mode and (2) estimate their age.
A classic way to consider selfing assumes a theoretical population of N diploid individuals which produce offspring through selfing or outcrossing with probability σ and 1 – σ, respectively. Under a model of neutral evolution, the distribution of polymorphic sites in a sample of sequenced individuals, that is the frequency of alleles (single nucleotide polymorphism [SNPs]), is determined by the underlying genealogy of this population. A genealogy has for properties its length measured as the time to the most recent common ancestor (T_{MRCA}) and its topology, that is the order and number of branching processes. Along the genome, genealogies change due to the effect of recombination (the socalled ancestral recombination graph [ARG]; Hudson and Kaplan, 1988; Wiuf and Hein, 1999). Two population parameters determine the distribution and characteristics of genealogies observed in a sample of several genomes: the population mutation rate (θ) and the population recombination rate (ρ). In the presence of predominant selfing, the effective population size (N_{σ}) of a population of N individuals is scaled by the selfing rate (σ) yielding N_{σ}=N/(1+F) (Fu, 1997; Nordborg and Donnelly, 1997) and the recombination rate (r_{σ}) is scaled as r_{σ}=r(1 – F) (Golding and Strobeck, 1980; Nordborg, 2000; Padhukasahasram et al., 2008) at least when r is not too high (Padhukasahasram et al., 2008), where r is the recombination rate (for example per site) in the genome and F is the inbreeding coefficient. When inbreeding is only due to partial selfing, F=σ/(2 – σ) and takes values between 0 and 1 (0 for outcrossing and 1 for fully selfing). As a consequence, the population recombination rate takes now for value in a selfing population:
With μ being the mutation rate in the genome (e.g. per site), the population mutation parameter accounts for the effect of selfing as follows:
We note that the classic ratio of population recombination by population mutation rate ρ/θ=r/μ in outcrossing species becomes now with selfing ρ_{σ}/θ_{σ}=r(1 – F)/μ (Nordborg and Donnelly, 1997; Möhle, 1998; Nordborg, 2000). Taken together, expressions (Equations 1; 2) suggest that selfing does not amount to a simple change in effective population size (from N to N_{σ}), and that the reduction of the effective recombination rate is more severe than the reduction in effective population size.
Following these insights, two key predictions can be derived. First, a characteristic and specific signal of selfing, in contrast to outcrossing, is expected to be present in SNP data due to the joint action of recombination along the genome (rate ρ_{σ}) and of the genealogical (coalescence) process (rate θ_{σ}). The first prediction underlies the previous development of a sequentially Markov coalescent method (eSMC) to estimate a fixed selfing rate using estimations of the ratio ρ_{σ}/θ_{σ} from pairs of genomes (Sellinger et al., 2020). Second, evolutionary changes in reproductive systems (transition from selfing to outcrossing or vice versa) are reflected in variations of the ratio ρ_{σ}/θ_{σ} in time and are identifiable and distinguishable from changes in population sizes alone. The latter suggests that a characteristic signature of the change in selfing rate in time should be observed in polymorphism data along the genome, if genetic variation can be summarized in a way that is informative about the joint effect of genetic drift and recombination. Indeed, it is desirable to (1) estimate changes in population size which occur independently of a transition, for example when species colonize new habitats as facilitated by selfing, and (2) disentangle the possible confounding effect of populationsize changes on the estimation and detection of a transition. To our knowledge no statistical inference method exists that takes advantage of these theoretical predictions to jointly estimate temporal changes in selfing rates and in population sizes.
Two types of modelbased methods are used to draw inference of past demographic events using fullgenome polymorphism data. First, the distributions of the time to the most recent common ancestor (T_{MRCA}) along a pair of chromosomes sampled from a sexually reproducing population can be modeled and approximated assuming the sequentially Markov coalescent (SMC) (McVean and Cardin, 2005). SMCbased methods infer model parameters, such as demographic histories, from fully sequenced genomes and have been implemented in several statistical software used to estimate changes in effective population size through time under the assumption of a WrightFisher (WF) model. Li and Durbin, 2011, developed a pairwise SMC estimating the distribution of T_{MRCA} over two sequences, and Schiffels and Durbin, 2014, extended the framework to consider multiple (more than two) sequences at a time, albeit only estimating the most recent pairwise T_{MRCA}. These SMCbased methods are based on a hidden Markov model (HMM) and the forwardbackward algorithm for estimating the ARG (i.e. the genealogies along the sequence) and a BaumWelch algorithm to estimate the parameters of the model. In our previously developed SMC method, eSMC, we included the effect of constantintime seed banks and selffertilization in order to estimate these parameters (i.e. dormancy and selfing rates) jointly with past population sizes (Sellinger et al., 2020). eSMC uses also pairs of sequences and the estimated transition matrix which summarizes the transition between two consecutive genealogies along the genomes (due to a recombination event), in contrast to the computationally intensive approach based on the actual series of coalescence times (Gattepaille et al., 2016). The SMC methods are characterized by their easy applicability to datasets as these do not require the user to specify a given demographic model. However, the SMC methods rely on an analytical expression for the transition probability between genealogies and do not provide a measure of uncertainty of the parameters or allow for hypothesis testing between different models/scenarios (though in principle the transition matrix can be used to do so, Palacios et al., 2019). Second, approximate Bayesian computation (ABC) is a computational approach to estimate posterior probabilities for models and parameters that is well suited for demographic modeling with many parameters and without any analytically derived likelihood function (Beaumont et al., 2002; Csilléry et al., 2010). Two advantages of the ABC method are that it allows to compare competing demographic hypotheses on the basis of Bayes factors and it does not require bootstrapping the data to generate measures of uncertainty for the inferred parameters. A critical aspect of ABC is that it requires a careful summarization of the genomic data into a set of summary statistics that carry information about the parameters of interest (Beaumont et al., 2002). This step is specially challenging when using genomewide data (Boitard et al., 2016). With the aim to use the information on genetic drift and recombination present in fullgenome polymorphism data, we develop therefore both an SMC and an ABC approach to infer past demographic events and the time of transition to selfing. We first provide analytical and simulation results explicating the consequences of a transition to selfing on genomic variation, thereby confirming our second prediction that temporal changes in selfing rate leave observable specific patterns in polymorphism data across the genome. Then, we analyze the statistical accuracy of the two newly developed methods to identify and estimate the age of transitions to selfing using a small number of sequenced genomes. Third, as a proof of principle, we apply these methods to estimate the age of the transition to selfing in A. thaliana, in which it has been documented and for which fullgenome polymorphism data exist. These new methods are useful toolkits for dating and understanding the evolution of the selfing syndrome and shifts in breeding systems and reproduction modes.
Results
The consequences of a transition to selfing on patterns of genomic variation
We consider a theoretical population of N diploid individuals (equivalent to 2N chromosomes), which produce offspring through selfing or outcrossing with probability σ and 1 – σ, respectively (following the notations in Nordborg, 2000). At some time (t_{σ}) in the past, the previously outcrossing population (with σ=0) undergoes a transition to selfing and remains selfing until present (with a selfing rate σ>0). Independently of the change in selfing rate, the population size can change once from N_{ANC} (ancestral) to N_{PRES} (present) at time t_{N} (measured from the present). We implement the selfing model both in the forward WF framework, in which selfing can be simulated explicitly, and using the coalescentwithselfing, in which selfing is modeled through a rescaling of the effective population size and of the recombination rate at t_{σ} (Nordborg and Donnelly, 1997; Möhle, 1998; Nordborg, 2000) (see Materials and methods). In other words, selfing rate is not constant in time in our model.
In the spirit of the SMC approaches, we obtain for our model a first analytical result extending the previous theoretical work to consider the transition between two consecutive genealogies along the genome due to a recombination event (here reduced to the coalescence time of a sample of size two). In contrast to the classic assumption of SMC methods (Li and Durbin, 2011; Schiffels and Durbin, 2014; Palacios et al., 2019) the ages of recombination events do not follow here a uniform distribution along the genealogy, but rather are functions of the selfing and recombination rates at each time point. As recombination and selfing rates are assumed nonconstant through time, the probability for a recombination event to occur follows an inhomogeneous Poisson process along the coalescent tree. We thus compute the probability for an effective recombination event to occur in a sample of size two (p(recs)), conditioned to the current coalescence time s, and with the selfing and recombination rates at time k being, respectively, σ_{k} and r_{k}. We find (using Equation 1 for recombination above):
Note that this value depends in an inhomogeneous way on the current coalescent time, which requires to integrate along the branch length of the coalescent tree. In other words, the variation of population size through time does not affect the probability of a recombination to occur conditioned on the coalescence time, that is the variation of population size affects the coalescence rate only. In Equation 3 it is visible that the probability for a recombination event to occur and to modify the genealogy is affected by the selfing rate (σ_{k}). Thus, this result opens the possibility to jointly infer piecewise functions of selfing or recombination rates and population sizes through time simultaneously. In practice, a change in selfing rate can be detected when the span of the genealogy along the genome does not decrease homogeneously with increasing coalescence time as demonstrated in Figure 1—figure supplement 1 (based on Equation 3). A further formal description of the coalescence process with variable recombination and/or selfing rates through time in the context of the SMC is provided in Appendix 1, especially providing the required expressions for the transition and emission probabilities needed to compute the transition matrix.
Following the spirit of SMC methods, we now turn to simulations to explicit the importance of Equation 3 for inference. We thereafter verify the second prediction  temporal changes in selfing rate leave observable specific patterns in polymorphism data across the genome  by quantifying the expected distributions of T_{MRCA}segments (hereafter TLdistribution) under our model. These segments are defined as successive and adjacent sets of nucleotidic positions sharing the same time to the most recent common ancestor (T_{MRCA}) and are separated by the breakpoints of ancestral recombination events (McVean and Cardin, 2005). Segments are summarized by their lengths and T_{MRCA} (Figure 1A and B). When the selfing rate is constant the TLdistribution has a negative constant covariance, because, on average, older segments are exposed, and shortened by a larger number of recombination events compared to younger segments (Figure 1A). In the case of a transition from outcrossing to predominant selfing, the rate at which the segments shorten with age is dramatically increased in the outcrossing phase compared to the selfing phase (Figure 1B), leading to a characteristic change in the covariance of the joint distribution at t_{σ}. This behavior of the model is invariably observed, using our coalescent model rescaling ρ and θ, or by explicitly using forward WF simulations (Figure 1—figure supplement 2). Importantly, this specific genomic signature of a transition to selfing is not observed when the selfing rate is constant and only the populationsize changes from N_{σ} to N, where N_{σ} would be the effective population size of a population with selfing rate σ (Figure 1A). The simulations confirm thus the results from Equation 3 and Figure 1—figure supplement 1.
Although the same increase in ρ could in principle be accounted for by a very large ancestral population size (i.e. N_{ANC} = N_{PRES}/(1 – F)), such a model would also have a much larger ancestral θ compared to the selfing model, and thus cannot produce the same TLdistribution as a transition to selfing (and thus is not a confounding scenario for a transition to predominant selffertilization). The TLdistribution is determined by the probability of recombination events, which, as explicated in Equation 3, is affected by the change in selfing (σ) when conditioning on a given θ. Simulated TLdistributions under a range of values for t_{σ} illustrate the dependency of the change in the covariance between the age and length of T_{MRCA}segments on the age of the transition (Figure 1—figure supplement 3). Thus, this suggests that genomewide polymorphism data contains information about shifts to selfing when the age of the transition falls well within the distribution of T_{MRCA}.
We also made the important observation that all the segments that coalesce in the outcrossing phase (T_{MRCA} older than t_{σ}) are spatially clustered along simulated chromosomes (Figure 1C). This effect can also be captured by inspecting the occurrences of transitions between different T_{MRCA} for a large number of successive and adjacent fragments (Figure 1D and E). In the case of a transition to selfing, the T_{MRCA} transition matrix distinctly shows that segments with T_{MRCA} older than t_{σ} are more likely to be followed by segments that are also older than this time. Although a similar dependency between successive T_{MRCA} also exists when selfing is constant, the magnitude of the effect is more pronounced in the case of a shift to selfing. The specific genomic signatures of a transition to selfing and its equivalence to two simultaneous temporal changes in effective recombination rate and population size motivated us to design two new statistical methods to estimate the age of transitions to selfing.
Statistical methods to estimate the age of a transition to selfing: teSMC
Based on Equation 3, we extend eSMC into teSMC, allowing the estimation of varying selfing or recombination rates through time, jointly with varying population size (see Materials and methods and Appendix 1). In order to account for prior knowledge, two modes are implemented for parameter inference: (1) the free mode, in which each hidden state has its own independent selfing/recombination rate, and (2) the singletransition mode in which teSMC estimates only three parameters (the current and ancestral rates, and the transition time between both rates), a constraint greatly reducing the number of inferred parameters and well suited for the analysis of recent and sudden shifts from outcrossing to predominant selffertilization.
First, to demonstrate the theoretical accuracy of our model and inference method, we analyze its performance when sequences of T_{MRCA} are given as input (the age of coalescent trees for two samples). This is termed the bestcase convergence of teSMC (Sellinger et al., 2021). We simulate data from a population undergoing a strong bottleneck and simultaneously a transition to selfing or a change in recombination rate. In both cases the population size and the past selfing or recombination values are recovered with high accuracy (Figure 2—figure supplement 1). Second, to understand the convergence properties of teSMC, we analyze its performance under a simple scenario assuming a constant population size and a constant selfing value of 0.9 given different amount of data. We compare the eSMC method, which estimate a constant rate of selfing in time with teSMC, which estimates varying selfing through time (Figure 2—figure supplement 2). When selfing is known to be constant (eSMC), the value of this parameter is recovered with high accuracy and low variance even with the lowest amount of given data (Figure 2—figure supplement 2A). When each hidden state can have its own selfing value (teSMC), constant selfing rate is indeed correctly recovered but a greater amount of data is required to reduce the variance in the estimation (Figure 2—figure supplement 2B).
We now evaluate the statistical accuracy of teSMC on polymorphism data from genome pieces of 5 Mb, simulated under a model with constant population size (N=40,000) with mutation (μ) and recombination (r) rates of 1×10^{–8}, and with an instantaneous change from outcrossing (σ_{ANC} = 0.1) to predominant selfing (σ_{PRES} = 0.99) at time t_{σ} (see Materials and methods). Both the singletransition mode and the free mode estimation procedures perform well over the complete range of t_{σ} values (Figure 2A).
Population sizes estimated under the assumption of a constant selfing rate are consistently larger than the true value in the outcrossing phase and display large fluctuations in the selfing phase, which could be mistaken for past populationsize bottlenecks (Figure 2—figure supplement 3). However, when teSMC is allowed to account for the change in selfing rate, populationsize estimates (N) remain close to the true values. We note that the increased variance in N in the recent selfing phase are likely caused by a smaller number of available T_{MRCA}segments.
Finally, we evaluate the ability of teSMC to jointly estimate the age of a transition to predominant selfing and the time of a stepwise change in population size. To do this we use simulated data produced as above, except with the addition of a single stepwise populationsize reduction (Figure 2B and C) or expansion (Figure 2D and E). In both cases our results indicate that teSMC, especially the free mode inference method, is able to precisely estimate the age of the shift to selfing, regardless of the relative timing of the populationsize change and the transition to selfing. Also, in most cases, the population sizes inferred by teSMC are close to the true simulated values (Figure 2—figure supplement 4). However, when the transition is recent and the present population size is low, the precision of the populationsize estimates decreases (Figure 2—figure supplement 4O). We note that teSMC fails, in the latter case, to recover the population size, suggesting a lack of data (coalescent events) (Sellinger et al., 2021). These results demonstrate that transitions to predominant selffertilization and more generally large changes in recombination rate through time can be captured by teSMC and the estimations can be disentangled from changes in population sizes.
Statistical methods to estimate the age of a transition to selfing: tsABC
In the case of a transition to selfing, we require that the summary statistics used in the ABC are informative about coalescence and recombination rates, in order to make changes in selfing rates and population size distinguishable by the ABC model choice (Figure 3). Note that while the lengths of T_{MRCA}segments are straightforward to calculate on simulated genealogies (Figure 1A and B), a main task is to estimate the length and age of these segments based on genomic diversity data. Following the results from teSMC using the highly informative transition matrix which summarizes the distribution of recombination events and of T_{MRCA} for two successive coalescent trees along the genome (Figure 2—figure supplement 1), we derive new summary statistics for our ABC. Namely, we sum the number of nucleotide differences (i.e. SNPs) between pairs of sampled chromosomes using nonoverlapping genomic windows of size ω (set to 10 kb throughout the whole study) and construct a transition matrix for pairwise diversity (displayed in Figure 1D and E). This means that we evaluate the proportion of genomic windows with a given diversity value (π) transitioning to the adjacent window with another (or similar) value of diversity. In effect, this ABC transition matrix should tend to the input of teSMC when ω tends to 1. We refer to this summarization as TM_{win} (Figure 3—figure supplement 1, see Materials and methods). The rationale behind this summarization is that, if the window size is smaller than some of the clusters of T_{MRCA}segments in the outcrossing phase, then, TM_{win} will capture both the expected excess of diversity of those segments and their clustering (Figure 1C). For comparison we also considered classic summarization based on the site frequency spectrum (SFS) and a discretized distribution of the decay in linkage disequilibrium (LDdecay), as these carry information about temporal changes in population size and selfing rates (Tang et al., 2007; Boitard et al., 2016) based on the theory outlined in the introduction. We therefore evaluated the efficiency of three sets of summary statistics: SFS/LD, TM_{win}, and SFS/LD/TM_{win} (see Materials and methods).
To test whether our approach can distinguish a transition to selfing from a reduction in population size, we conduct an ABC model choice analysis using two competing models (Figure 3A and B): model 1 where the selfing rate changes from σ_{ANC} to σ_{PRES} at t_{σ} and with N constant, and model 2 with population size changes from N_{ANC} to N_{PRES} and σ constant. We simulate datasets under model 1 and evaluated the ability of tsABC to identify the correct model for transitions of varying ages, using different sets of summary statistics to summarize the genetic data (see Appendix 2). Our results show that our method recovers the correct model for transitions as old as 2.5N_{σ} generations (Figure 3C–E), with N_{σ}=N/(1+F) being the effective population size for a selfing population. Interestingly, summarizing the genetic data using the SFS and LDdecay yields better performance for recent shifts to selfing (Figure 3C and D), while using TM_{win} performs better for shifts occurring between (0.5N_{σ} and 3N_{σ}); such that combining both set of summary statistics yields the best performance. This analysis confirms that shifts to selfing as old as approximatively 2.5N_{σ} can be detected and can be disentangled from changes in population size using the ABC model choice procedure if appropriate summary statistics are used.
Next, we evaluate the accuracy of our method for estimating the age of a transition to selfing (t_{σ}). We simulate 100 datasets under model 1 (Figure 3A) with values of t_{σ} ranging from 1000 to 200,000 generations, and used tsABC to reestimate posterior distributions for t_{σ} and the other parameters of the model (Figure 3F–H, Figure 3—figure supplement 2). Estimations are obtained using the same three summarization strategies used for the model choice (SFS/LD, TM_{win}, SFS/LD+TM_{win}). The age of a shift to selfing could be well estimated using the TM_{win} approach, while the SFS+LD approach overestimated t_{σ} almost over the complete range of values (Figure 3F and G). Combining SFS, LD, and TM_{win} does not further improve the accuracy of the estimations (Figure 3H). We note that the parameters N and σ_{PRES} (i.e. the population size and the current selfing rate) are both better estimated with TM_{win} than with SFS/LD, except for transitions younger than 10^{4} generations ago where σ_{PRES} is slightly better estimated with SFS/LD (Figure 3—figure supplement 2D). However, no set of summary statistics could estimate the ancestral selfing rate (Figure 3—figure supplement 2G–I).
Robustness of inference to model violations
We here provide two analyses to demonstrate the robustness of our inference method to two violations of the model assumptions: first recombination and mutation rates may vary along the genome, and second, background selection (BGS) in combination with selfing may affect the inference of transition to selfing. First, to assess the potential limits of our approach, we analyze the performance of teSMC when mutation and recombination rates are potentially nonconstant along the genome (Figure 2—figure supplements 5–6). When mutation and recombination rates are constant along the genome, teSMC recovers a constant population size and accurate selfing rates (Figure 2—figure supplements 5A and 6A). When recombination rate varies by a twofold factor along the genome, the estimation of population size is accurate (Figure 2—figure supplement 5B) but the variance of selfing rates inference increases (Figure 2—figure supplement 6B). Variation of the mutation rate by a twofold factor along the genome biases inference through time, leading to erroneously high inferred selfing rates and population size (Figure 2—figure supplement 2C, D). Yet, these results are reassuring as they demonstrate that no spurious transition from outcrossing to selfing in the past is inferred under variation of the recombination rate or mutation rate along the genome.
BGS refers to the effect of deleterious alleles on linked neutral diversity (Charlesworth et al., 1993; Irwin et al., 2016). Recently, several studies highlighted that neglecting the effect of BGS in demographic analyses can lead to statistical biases and potential missidentification of populationsize changes (Ewing and Jensen, 2016; Johri et al., 2021). Because transitions to selfing result in strong reduction of the effective population size and recombination rate (Equations 1; 2), a corresponding increase of linkage between deleterious and neutral alleles occurs (Charlesworth et al., 1993). Selfing indeed drastically magnifies the effect of BGS (Roze, 2016), so we test whether selfing and BGS could affect the accuracy of our inference methods. Because both the teSMC and the tsABC methods ignore the effect of selection, we evaluate their performance when applied to data simulated under a model with both a transition to selfing and BGS. We use slim3 (Haller and Messer, 2019) to simulate genomic data with the same distribution of exonic sequences as in five predefined regions of 1 Mb from the genome of A. thaliana (exact coordinates in methods) and model negative selection on exonic sequences according to the distribution of fitness effects (DFEs) published by Hämälä and Tiffin, 2020. We found that when exonic sequences are not excluded from the analysis (unmasked), teSMC slightly underestimate the time of transition to selfing. However, when coding regions simulated with selection are masked the date of transition is accurately recovered (Figure 4A) and the performance is similar to the case without BGS (Figure 2A). We note that the tsABC estimations remain accurate even without masking exonic regions (Figure 4B, Figure 4—figure supplement 1) and the performance similar to the case without BGS (Figure 3H). These results suggest that our approach is generally robust to the effect of negative selection on linked neutral sites, even in compact genomes such as the one of A. thaliana (Figure 4).
Application to A. thaliana
Deactivation of the SI mechanism through mutations knocking out the genes SCR and SRK is known to be responsible for the transition to predominant selffertilization in the model species A. thaliana (Tsuchimatsu et al., 2010). This shift to selfing is the focus of several studies and estimates of its age have been obtained using different types of data and statistical approaches. Rescaling the original estimate with the more recent mutation rate of Ossowski et al., 2010, Bechsgaard et al., 2006, estimation of the oldest possible age of the transition is 1.48 million years (Shimizu and Tsuchimatsu, 2015); and was obtained using phylogenetic analyses of the Slocus in A. thaliana and Arabidopsis lyrata. Tang et al., 2007, analyzed the genomewide decay in LD but could not detect the expected signature of a transition to selfing, and therefore concluded that the shift must have been older than the oldest coalescent events in their sample (older than 1 million years approximately). Here, estimations obtained with teSMC and tsABC range from 592,321 to 756,976 depending on which method and population samples are used (Figure 5A, Table 1, Supplementary file 1, Table S6). Our estimates are younger than the one proposed by Bechsgaard et al., 2006, and Tang et al., 2007. The probability that Bechsgaard’s maximum age of the transition is sampled from the posterior distributions for t_{σ} obtained with tsABC is equal to zero, as it is much older than the upper boundary of the corresponding credibility intervals (Table 1). We find the estimates of transition to selfing to be robust to the geographical origin of the population samples (Iberian nonrelicts, Iberian relicts, or central European, Figure 5, Supplementary file 1, Tables S5 and S6). Note that we are also able to jointly estimate the demography of each A. thaliana population and the transition to predominant selffertilization (Figure 5B). Genetic variants used for these estimations were taken from genomic intervals outside of pericentromeric regions, which are associated with aboveaverage levels of nucleotide diversity in A. thaliana without elucidated reason yet (Schmid et al., 2005; Clark et al., 2007). Replicating this inference with a different set of loci resulted in very similar results (Figure 5—figure supplement 2). For application of tsABC and teSMC to other species, we therefore recommend to first investigate whether a similar association between pericentromeric regions and levels of diversity is observed and more generally whether some genomic regions exhibit peculiar patterns of diversity.
Compared to previous results ignoring the transition to selfing (Sellinger et al., 2020), we find a populationsize decline starting at the time of transition and not continuously declining since the far past, as in previous studies (Sellinger et al., 2020). Our results suggest that populationsize decline could be linked to the transition to selfing and that our demographic inference using teSMC should be more reliable than those from eSMC (and other SMC approach ignoring the selfing transition). A way forward would be to improve our inference framework using the new theoretical results by Upadhya and Steinrücken, 2022, which focuses on modeling the time to the most recent common ancestor of a sample size bigger than two. This study demonstrates that the inference in the far past can be improved, at the cost of losing accuracy in recent times. The estimated effective population sizes inferred by teSMC are comparable to the ones obtained by Durvasula et al., 2017 and Fulgione and Hancock, 2018 between present and 400,000 generations in the past; pending that their msmc2 results are rescaled by 1/(1+F), which is approximately two in this case. For times older than 400,000, around the age of the transition, Durvasula et al., 2017 reports smaller effective sizes than teSMC.
Discussion
While the biological importance of shifts in mating systems or reproductive modes is long recognized as a key evolutionary and ecological process, no method based on genomewide polymorphism data was available to estimate the age of a change in reproductive mode, while accounting for demographic history. In this study we show that by accounting for both the frequency distribution of SNPs and the distribution of historical recombination events, it is possible to recover shifts in reproductive modes based on a small number of fully sequenced genomes. The key idea underlying our approach is to leverage the properties of genomic segments delimited by recombination events (T_{MRCA}segments). Our simulations show that the molecular signature of a transition to selfing is twofold. The first effect is a change in the relation between the age and the length of genomic segments that occurs at the time of the transition (Figure 1, Figure 1—figure supplements 1–3), and the second is a characteristic spatial distribution of the segments along the chromosome, where segments older than the transition tend to occur as clusters (Figure 1C–E). Several SMCbased methods already take advantage of both the distribution of age and lengths of T_{MRCA}segments to estimate past demographic history (Li and Durbin, 2011; V Barroso et al., 2019; Steinrücken et al., 2019), spatial structure (Wang et al., 2020), heterogeneous recombination rates along the genome (V Barroso et al., 2019), or lifehistory traits such as dormancy (Sellinger et al., 2020). SMCbased as well as other inference methods (Kerdoncuff et al., 2020) rely on estimating changes in the ratio of population recombination rate by population mutation rate along the genome (the ratio ρ/θ). Yet, none of them attempted to leverage the information on the distribution of T_{MRCA}segments to also estimate temporal changes of the ratio ρ/θ, which in our case amounts to estimate variation of the selfing rate. Interestingly, as most inference methods are developed with a main application to human data in mind, the estimation of a changing ρ/θ ratio has not yet been a relevant and pressing question. Deng et al., 2021, described how temporal recombination rates could be estimated from reconstructed ARGs, but only evaluated the performance of this approach on simulations with a constant population size and rate.
In this study we develop two approaches to estimate change in selfing in time: one building upon the Markovian assumption of coalescence events along the genome (teSMC) and another one by considering the dependencies between the genetic diversity observed in successive genomic windows of fixed size (tsABC). The advantage of the teSMC approach is that it is most effective in identifying the boundaries and the ages of T_{MRCA}segments and therefore allows capturing the characteristic effect of a shift to selfing (as shown in Figure 1A and B). teSMC is also efficient in the way it optimizes the likelihood such that the method can be executed on a single desktop computer when applied to empirical data. The drawback of the teSMC method is that extending it to more complicated demographic scenario is mathematically and computationally difficult (e.g. to selection or admixture). Conversely, the tsABC approach can easily be extended to more complicated demographic and nonneutral scenarios but suffers from a lower statistical performance caused by the approximation made in the summarization of the data; and by the requirement to conduct many simulations that can only be realistically obtained on a highperformance computing cluster. Our results suggest our approaches to be robust to the presence of BGS, and to a lesser extent to variation of recombination and mutation rate along the genome. We nevertheless show that a spurious change in selfing rate is not generated by variation in recombination rate along the genome (Figure 2—figure supplement 6). We finally highlight the novelty in the design of the tsABC approach, which introduces a new summary statistic, the transition matrix for heterozygosity levels, which contains similar information as in the transition matrix computed by the teSMC method and which could be incorporated as a useful summary statistics in other ABC methods. Both methods belong thus to the same conceptual framework and are complementary.
Our results indicate that genomic distributions of T_{MRCA} estimates are more informative about the age of transitions to selffertilization than approaches relying on the rate of pseudogenization of SI loci (Bechsgaard et al., 2006). Furthermore, Slocusbased inferences of shifts to selfing are limited to species for which such transitions have been caused by a lossoffunction mutation in the Slocus and for which the Slocus has been identified and properly assembled. This information is only partially available for other plant species, and the genetic determinism of selfing also varies between genera (FranklinTong, 2008). Thus, our inference methods offer the opportunity to test for the existence and the timing of changes in mode of reproduction in potentially any species providing fullgenome polymorphism data are available. They also pave the way for addressing longstanding questions on the evolution of reproductive systems which cannot be directly tested from field experimental or even phylogenetic approaches. How frequent and how recent are transitions from outcrossing to selfing is the main question, and the one that motivated our work. Phylogenetic methods can at best infer that a transition has occurred at some time on a given branch of a phylogeny. For example, considering the case of A. thaliana, knowing that the two sister species, A. lyrata and Arabidopsis halleri, are selfincompatible indicates that the shift to selfing occurred after the divergence between the two lineages, that is between present time and 13 millions years (Beilstein et al., 2010), which is poorly informative. In addition, from phylogenetic character mapping, when two or more sister species share the same character state, most of the time the shift to this state is inferred before the divergence of the species. However, it may not be the case for very labile traits with high extinction rate as selffertilization. In the fungus genus Neurospora, although a clade of species shares a homothallic mating system (equivalent to selfing), a detailed molecular analysis of the mat locus that controls mating system revealed that the breakdown of this locus occurred several times independently (Gioti et al., 2012). Reversion from selfing to outcrossing is supposed to be very rare but this question is still debated (Barrett, 2013). Our methods provide the adequate tool to tackle this question and their systematic application to various species may help discovering such possible reversion or even more complex scenarios. As an example, we apply teSMC to estimate scenarios of transition from outcrossing to selfing followed by reversion, or the reverse, as well as more gradual (stepwise) transitions (Figure 2—figure supplements 7–8). The method performed well and is thus promising for detecting more complex histories.
Another useful application of the methods is for demographic inferences when shifts in mating systems are suspected. As they alter the distribution of age and lengths of T_{MRCA}segments, they can lead to spurious shifts in population size if not taken into account (Figure 2—figure supplement 3). For example, using an SMC approach, ancestral drops in population sizes have been inferred in the selfers Capsella orientalis and C. bursapastoris but not in the outcrosser Coreopsis grandiflora (Kryvokhyzha et al., 2019). This could correspond to real changes in population size but also to transition toward selfing. This issue is especially worth being considered in cultivated species. The demographic history associated with domestication is a central question for the study of crop species and demographic scenarios have been inferred in many species. However, shifts, or at least variations, in mating system occurred quite frequently during plant domestication (e.g. African rice, tomato, grapevine, melon) (Glémin and Bataillon, 2009; Meyer et al., 2012) and taking such variations into account would help refining demographic scenarios.
The methods we developed focus on outcrossing/selfing transitions. However, they could be extended to other reproductive modes such as sex/asex transitions by adapting the relevant population parameters using existing works on coalescence with facultative sexual reproduction (Hartfield et al., 2018). Overall, our methods may open new ways to answer the old riddle of why so many species do reproduce sexually (Barton and Charlesworth, 1998).
Materials and methods
Modeling of a transition to selfing using forward and coalescent simulations
Request a detailed protocolTo model a transition from outcrossing to predominant selfing, we considered a single population composed of N diploid individuals. At each generation, each offspring is generated by selffertilization of a single individual or by outcrossing with probabilities σ and 1–σ, respectively, where σ is the selfing rate. Unless stated otherwise, transitions to predominant selfing were modeled by allowing the selfing rate to change instantaneously from σ_{ANC} to σ_{PRES} at time t_{σ}. The mutation and recombination were set to 1×10^{–8} events per generation per nucleotide. When needed the population size was allowed to change instantaneously from N_{ANC} to N_{PRES} at t_{N}. This model was implemented using the WF simulation mode in slim3 (Haller and Messer, 2019); scripts to simulate genetic data using this model are available on our git repository (https://github.com/laurentlabmpipz/struett_and_sellinger_et_al.git). We used this model to generate genetic variation for a sample of n=20 haploid genomes, sampled from 20 different individuals, and composed of five DNA sequences of 1 Mb each.
The same model was implemented in a coalescent framework using msprime (Kelleher et al., 2016). Following Nelson et al., 2020, who showed that continuoustime coalescent simulations of large sequences cause biases in patterns of identitybydescent and LD, we implemented a hybrid model in which the first 1000 generations were simulated using a discretetime coalescent process and the following generations were modeled using the SMC′ algorithm (Marjoram and Wall, 2006). The coalescent implementation, which runs significantly faster than the forward WF implementation, was used for the ABC and performance analyses (see below), while the WF forward implementation was used to assess the quality of the coalescentwithselfing approximation proposed by Nordborg and Donnelly, 1997; Nordborg, 2000; Figure 1—figure supplement 1, and was extended to study the consequences of negative selection on inference with the tsABC and teSMC methods (see below).
Analysis of T_{MRCA}segments in simulated data
Request a detailed protocolFor the forward and coalescent simulations, we wrote functions to obtain the lengths and the time to the most recent ancestor (T_{MRCA}) of T_{MRCA}segments. T_{MRCA}segments are sets of contiguous nucleotides in a sample of size two that share the same MRCA. The joint distribution of T_{MRCA} and lengths of those segments (TLdistributions) was used to describe the consequences of a transition to selfing at the genomic level and how it differs from a change in population size (Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 3). T_{MRCA}segments were analyzed by identifying consequential recombination events in the history of the sample (i.e. events that lead to the inclusion of a new MRCA) and the corresponding breakpoints represented the boundaries of the successive segments. Then, the T_{MRCA} of each segment was obtained by identifying the MRCA of each segment.
We also calculated the transition matrix of T_{MRCA} of successive T_{MRCA}segments along the genome (TM_{true}, Figure 1D and E). For this, we discretized T_{MRCA} values and counted the frequencies of segment transitions along simulated sequences for each combination of discrete T_{MRCA} values. T_{MRCA} were discretized using a similar approach as in MSMC (Schiffels and Durbin, 2014) with the lower boundary of bin i given by, –8N × log(1–i/m), where N is the population size and m the total number of bins. The relevant code can be found at https://github.com/laurentlabmpipz/struett_and_sellinger_et_al.git, (Sellinger, 2023; Strütt, 2023).
Calculation of summary statistics of polymorphism data
Request a detailed protocolWhile TLdistributions carry the characteristic signature of shifts to selfing, they are also challenging to infer from empirical genetic data. Therefore, we used three summarization approaches to capture this signal using polymorphism data: (1) The unfolded SFS, which is the distribution of absolute derived allele frequencies in the sample and is known to carry information about past populationsize changes. (2) A discretized distribution of LD decay inspired from the approach taken by Boitard et al., 2016, who used it jointly with the SFS to estimate past changes in population sizes. Unlike the SFS, which only carries information about N but not the recombination rate (r), LDdecay depends on the product of N and r. Combining both distributions therefore allows to capture the signature of changes in N and r. LD was calculated as ${r}^{2}$ from a subset of 10,000 randomly chosen SNPs and discretized into discrete physical distances with following breakpoints: 6105; 11,379; 21,209; 39,531; 73,680; 137,328; 255,958; 477,066; 889,175 bp. (3) Windowbased transition matrix (TM_{win}): While TM_{true} carries a characteristic signal to estimate shifts to selfing, it is not straightforward to calculate it using polymorphism data. This is because the boundaries of T_{MRCA}segments are not directly observable and need to be inferred themselves. TM_{win} captures some of the information in TM_{true} by computing the pairwise diversity in nonoverlapping successive windows of 10 kb for a sample of size two.
Simulations with BGS
Request a detailed protocolSimulations with BGS were conducted with slim3. We used the DFE estimated by DFEalpha for A. thaliana published by Hämälä and Tiffin, 2020. The DFE was used to assign negative selection coefficients to simulated coding nonsynonymous genetic variants only (i.e. we did not simulate negative selection on functional noncoding regions). We took care of simulating realistic proportions and spatial distributions of coding sequences by using the positional information of CDS from the annotation of the reference genome of A. thaliana (Arabidopsis Genome Initiative, 2000). Except for the DFEs and genetic structure, all other parameters and dataset dimensions were identical to the simulations without negative selection.
Application to A. thaliana
Request a detailed protocolTo calculate summary statistics on the A. thaliana dataset, we used the imputed genotype matrix provided on the 1001 genomes website (https://1001genomes.org/data/GMIMPI/releases/v3.1/SNP_matrix_imputed_hdf5/). We used samples from three separate genetic clusters: central European (CEU), Iberian nonrelicts (IBnr), and Relicts. Only accessions with cluster membership >95% were included; because our model does not account for gene flow between ancestry groups (Supplementary file 1, Table S4). The final sample sizes are provided in Table 1. We conducted a genomewide slidingwindow analysis of nucleotide diversity with vcftools (Danecek et al., 2011) and identified large genomic regions with elevated nucleotide diversity, centered around pericentromeric regions (Underwood et al., 2018). Estimations were conducted using five 1Mblong genomic regions (Supplementary file 1, Table S5) that did not overlap with these elevated diversity regions (Figure 5—figure supplement 1). We resampled 12 haplotypes multiple times and calculated the combined summary statistics, SFS, LD, and TM_{win}. We centralized and normalized the statistics and calculated the first 20PLS (see Appendix 2). tsABC was conducted on the A. thaliana data, by using 12 samples for 5 independent loci of 1 Mb length. The mutation and recombination rates were set to 6.95e9 (Ossowski et al., 2010) and 3.6e9. The recombination rate is the genomewide average provided by Salomé et al., 2012. We simulated a total set of 130,000 vectors of summary statistics. Parameter estimates were conducted as described for the ABC performance analysis. We provided the mode of the average posterior distributions as a final result. In addition, we used a subsample of 20 A. thaliana sequences per genetic cluster to estimate demography and the transition to selfing using teSMC in the onetransition mode. Data and scripts can be found at https://github.com/laurentlabmpipz/struett_and_sellinger_et_al.git and https://github.com/TPPSellinger/eSMC2.
Data availability
Request a detailed protocolA complete detailed description of the teSMC and tsABC methods is in the SI text Appendices 1 and 2. The tsABC method can be found on GitHub (https://github.com/sstruett/tsABC). Scripts for all figures, simulations, and the specific tsABC workflow used in this study can be found on a separate repository (https://github.com/laurentlabmpipz/struett_and_sellinger_et_al.git). The teSMC method and a tutorial to simulate and analyze data are available on GitHub (https://github.com/TPPSellinger/eSMC2).
Appendix 1
Description of teSMC
teSMC
Note that teSMC also includes the effect of dormancy (or seed banks) although not discussed in the manuscript. To define our HMM we need to define:
The hidden states,
The signal (observed data) to be used as input,
A transition matrix (probability of switching from one state to another),
An emission matrix (probability of observing the data given the current hidden state),
An initial probability (probability of hidden states at the first position of the sequence).
Notations and assumptions
We here define the different notations used and their meaning:
${\sigma}_{t}$: self fertilization rate (between 0 and 1) at time t,
${\beta}_{t}$: dormancy rate (between 0 and 1) at time t,
r_{t}: recombination rate per nucleotide (at time t) per units of coalescence time,
μ: mutation rate per nucleotide per units of coalescence time,
${\mu}_{b}$: ratio of mutation rate during the dormant stage over the mutation rate during the active stage,
u: time at which the recombination occurs (follows a piecewise uniform distribution),
L: sequence length in bp,
${N}_{t}$: population size at time t,
${\chi}_{t}$: scaling factor for the population size at time t (${N}_{t}={\chi}_{t}{N}_{e}$).
The assumptions of the model are:
Piecewise constant population size,
Piecewise constant selfing, dormancy, and recombination rate in time,
Constant mutation rate in time,
Constant mutation and recombination rate along the sequence.
Observations
Our observations, also called signal, is a sequence of 1 and 0. This sequence is build from phasing the DNA sequences of two individuals. When going along the sequence, at a given position, if both nucleotides are similar then the signal is 0 (no mutation occurred). If both nucleotides are different, then a mutation occurred, and the signal is 1.
Hidden states
We define our hidden states at one position on the genome as the coalescent time between the two individuals. This is denoted as the coalescent time t (t>0). More precisely, because the state space must be finite, we discretize time in several (n) intervals. At one interval, the hidden state takes a value $\alpha $ if $t\in [{T}_{\alpha},{T}_{\alpha +1}]$, where $\alpha \in [0,(n1)]$. We define ${T}_{\alpha}$ as:
In addition, a transition from a coalescent time $s$ to time $t$ ($t\ne s$) at the next sequence position can only occur if a recombination has happened inbetween the two positions. We describe this event below.
Initial probability
We use the equilibrium probability as initial probability. The equilibrium probability is defined as the probability that the first coalescent happens at each time interval and is thus given by:
Transition matrix
A transition to state $t$ from state $s$ (t ≠ s) only occurs if there is a recombination event. Because recombination, dormancy, and selfing are assumed nonconstant through time, recombination events along the coalescence tree follow an inhomogeneous Poisson process. Hence, the probability for a recombination to occur conditioned to the current coalescence time $s$ is:
We assume that a recombination event occurred at time $u$ ($u<s$), where $u$ follows a piecewise uniform distribution between 0 and $s$, that is uniform in each hidden state but the density between hidden state can change. Then, we can define three possible scenarios, namely the new coalescent time of the coalescent tree can be smaller ($t<s$), bigger ($t>s$), or unchanged ($t=s$) compared to the initial tree. These computations are also found in previous PSMC approaches (Li and Durbin, 2011) and in Sellinger et al., 2020.
t<s
The resulting floating branch of the recombination event coalesces at time $t<s$. This means that the coalescence did not occur before time $t$ (including itself). In addition we have $u<t$. The transition probability is therefore obtained as:
t=s
The resulting floating branch of the recombination event (self) coalesces before time $t$. We therefore obtain the transition probability:
t>s
The resulting floating branch of the recombination event does not coalesce before time $s$, and no coalescent event happens before time $t$. We therefore obtain the transition probability:
Transition probability in continuous time
The transition probability conditioned to the occurrence of a recombination event occurring at time can be summarized as:
If all ${\sigma}_{k}=0,{\beta}_{k}=1$, we fall back on the probability from PSMC′. To find $p(ts)$ we use the total probability formula which is given as:
As explained above, time is discretized in $n$ small intervals. The hidden state is $\alpha $ if $t\in [{T}_{\alpha},{T}_{\alpha +1}]$, where $\alpha \in [0,(n1)]$ with ${T}_{\alpha}$ defined as:
The transition matrix summarizes the probability of transition from one hidden state to another. Therefore, we need to compute the probability of the coalescent time at the previous sequence position (which is here $s$) to belong to the state $\gamma $. To do this we replace $s$ by the expected coalescent time knowing the coalescence events to be occurring in the hidden state $\gamma $. We write this expected value as ${t}_{\gamma}$ which is defined as:
With the following definitions:
From the previous equation, we can define our transition probability:
Calculation of $p(\alpha \gamma )$
To compute $p(\alpha \gamma )$, we first need $p(t{t}_{\gamma})$, which is obtained under three possible cases.
Case 1: $\alpha <\gamma$
where we define:
We can now calculate $p(\alpha \gamma )$.
Case 2: $\alpha >\gamma$
We first need $p(t{t}_{\gamma})$ when $\alpha >\gamma$, which is obtained as:
We can now calculate $p(\alpha \gamma )$.
Case 3: $\alpha =\gamma $
Because the sum of probabilities for the three cases sum up to one, we obtain the following formula:
Emission matrix
Note as highlighted in Sellinger et al., 2020, that seed banking can significantly lengthen the coalescent tree. In this case the infinite site model hypothesis might be violated, and therefore we adapt the formula for the emission matrix:
where μ is the mutation rate per nucleotide per N generations, ${\mu}_{b}$ ratio of mutation rate during the dormant stage over the one in the active stage, ${\beta}_{\gamma}$ the dormancy rate in state $\gamma $, and $t\gamma $ the average coalescent time in state $\gamma $.
Appendix 2
ABC method
Approximate Bayesian computation
ABC offers a statistical framework for modeling, widely used for population genetics (Beaumont et al., 2002). ABC addresses the two crucial modeling questions (1) which is the most supported model given the observed data, and (2) which are the most supported parameter values given the observed data from natural populations. Likelihood functions or total probabilities may be analytically complicated or computationally intractable, whereas, in the ABC framework, a posterior probability distribution is approximated using simulations. In addition, since ABC is Bayesian, it allows the implementation of prior knowledge. For this purpose, instead of using the full complexity of genetic diversity as in fulllikelihood methods, the observation must be summarized into a scalar vector of summarizing statistics. The same summarizing statistics can be calculated from both simulated and natural populations. The Euclidean distance between the two simulated and observed statistics vectors determines the similarity between the simulated and true models. By defining a similarity threshold ε, we identify a set of simulations to explain the observed results. The distribution of parameters of those simulations approximates the posterior probability distribution. For this purpose, the ABC method could be optimized in a plethora of ways. The optimal performance of the ABC depends on the Bayesian sufficiency criterion of the summarizing statistic and the extensive exploration of the likelihood surface. Thus, we use PLS not only to reduce the dimensionality of the summary statistics vectors but also to reduce the influence of noise in the data. Moreover, we also apply multinomial regression (Wegmann et al., 2010) for the model choice and local linear regression for estimating the parameters. Both allow an increment of ε, causing an increase in the marginal posterior densities. This increase in the marginal density causes reduced suffering of the curse of dimensionality to improve approximation to the likelihood function.
Models
We propose a transitiontoselfing model (Figure 3A) and a confounding populationsize model (Figure 3B) to distinguish these two effects. The transitiontoselfing model represents a diploid, random mating population of constant size, which undergoes a transition to selfing at a given time. However, the populationsize model assumes a constant selfing rate.
In contrast to A, the populationsize model B undergoes one single change in population size, that is we propose two possible demographic models explaining a reduced genetic diversity after a single event in the past. In both models, A and B, we define equal prior probabilities of current population sizes; however, the effective population size in the ancestral state of the scenario of A could increase to the twofold, only, but in B, we define the identical prior probabilities of population sizes compared to the current population size. Thus, we could identify whether a transition to selfing is inferred specifically or confounded with a simple reduction in population size.
Simulation of genetic data
If not indicated otherwise, from 20 individuals, we sampled 5 independent haplotypes of 1 Mb length mimicking five chromosomes. For the calculation of the SFS and LD we use the 20 haplotypes. The other summary statistics are based on pairwise comparison of sequences (TL_{true}, TM_{true}, TM_{win}). If not indicated differently we compare all possible 20 choose 2 pairs. Thus, the total pairwise length results in 950 Mb. We used mutation and recombination rates of μ=r = 10^{–8}. For the ABC we created 100,000 datasets for each model. We used corresponding prior parameters for both models (Supplementary file 1, Table S1). We tested the performance of the ABC under the assumption of neutrality. The corresponding PODs were simulated for different transition times under the shifttoselfing model (Supplementary file 1, Table S1). Thus, we obtain a time series for the performance analysis. We tested the performance for following transitioning times in generations: 1000; 2000; 3000; 4000; 5000; 6000; 7000; 8000; 9000; 10,000; 12,000; 16,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; 200,000. That translates to coalescent time units ranging from 0.05 to 10. For each condition, we created 100 independent PODs. We tested whether the assumptions of our ABC are robust if the PODs were simulated under BGS. We used the DFEs estimated by Hämälä and Tiffin, 2020, for A. thaliana. We simulated sets of five pseudogenomes of 1 Mb and used the annotation file of TAIR10 to determine the spatial distribution of exons. We simulated the BGSPODs forwardintime under WF assumptions. We created 100 independent burnins of 10N generations. Starting from those, we simulated a time series of transitions to explicit selfing under the same parameters as used for the neutral PODs. Each set of five simulations was aggregated and summarized into a single POD. The same set of PODS were used to test the statistical performance of teSMC.
Implementation and software
Except for BGS, we simulated the genetic data using the coalescent implemented in msprime version 0.7.4 (Kelleher et al., 2016). We simulated the most recent 1000 generations under the discretetime WF to avoid biases in IBD and the following times under the SMCprime. We used the rescaled coalescentwithselfing (Nordborg and Donnelly, 1997) to simulate transitions to selfing for a recent selfing past. Backwardintime, our simulations underwent a transition to outcrossing.
For the BGS simulations, we wrote a forwardintime simulator using slim version 3.6 (Haller and Messer, 2019). We forbid accidental selfing. We used the treesequencerecording option. A few lineages were not coalesced after the simulation. Thus, we recapitated the obtained treesequences from the pyslimpackage version 0.6, which in turn utilizes msprime.
We implemented the whole pipeline into Snakemake 5.13. We have run all simulations on the highperformance cluster of the MPIPZ.
Summarization of genetic data
SFS/LD
For each sampled set of 20 haplotypes, calculated the folded SFS and the LD. The LD was calculated as r² from a subset of 10,000 randomly chosen SNPs and discretized into discrete physical distances with following breakpoints: 6105; 11,379; 21,209; 39,531; 73,680; 137,328; 255,958; 477,066; 889,175 bp. Because recombination rates are not constant through time, we cannot relate physical distance to coalescent times. Thus, we set the discretization boundaries to fixed values.
TM_{win}
For each pairwise comparison, we counted the number of SNPs for a sliding nonoverlapping window of 10,000 bp. The ABC depends on vectors of scalar summary statistics of finite size. Thus, we discretized the diversity into m segments, each reflecting the expected diversity under a given coalescent time. The discretization was derived from the quantiles of the exponential distribution in order to obtain an equal information distribution per discretized diversity window via ${f*log}_{e}\frac{1i}{m}$ . If $f=1$, the diversity scales in coalescent times with mutation rate 1. To scale the discretization breakpoints we used $f=8\cdot {L}_{\mathrm{W}\mathrm{I}\mathrm{N}}\cdot N\cdot \mu$ with μ, $N,{L}_{WIN}$ being the per generation per bp mutation rate, the population size, and the window size, respectively. We usually choose $f$ to provide Bayesian sufficiency within the time frame of expected estimates of times of transition to selfing. For the performance analysis of the ABC, we chose $N=\mathrm{40,000}$. Further, i and m were defined as $i\u03f5\left\{1\dots m\right\}$ and $m=20$ bins. We only kept discretization breakpoints with at least a single integer value inbetween neighboring breakpoints. Further, we obtained the transition proportion of the $i$th to the $\left(i+1\right)$ th window for each of the m discrete diversity bins. Thus, we obtained a m² transition matrix. We flattened the matrix into a onedimensional vector of maximal length m².
Dimensionality reduction
The dimensionality of our summary statistics TLdistribution, TM_{true}, and TM_{win} is up to 400 for each. To overcome the curse of dimensionality in our ABC, first, we centralized, normalized, and Box Cox transformed each calculated statistic to obtain the orthogonal independent variation. Then, we applied the PLS analysis to our data as suggested from Wegmann et al., 2010. According to the chosen combination of summary statistics, we chose an appropriate number of model selection and parameter estimation components. SFS and LD are summarizations of lower dimension. Thus, we did not necessarily apply a dimensionality reduction on them.
Model choice
The Bayesian framework naturally allows obtaining a posterior density for each proposed model. The Bayes factor is simply defined as the ratio of the marginal densities of two models. In ABC, we approximate those marginal densities by the posterior density. Thus, the Bayes factor $B}_{AB}=\frac{den{s}_{A}\left(stat{s}_{obs}\right)}{den{s}_{B}\left(stat{s}_{obs}\right)$ provides an approximation to the model support given the observed data (compare Wegmann et al., 2010). We categorize the Bayes factors into negative, barley worth mentioning, substantial, strong, very strong, and decisive (Jeffreys, 1998).
We calculated the Bayes factor for different sets of summarizing statistics, performed a model choice using a multinomial regression analysis between proposed models A and B. The calculations were done using the R package abc (Csilléry et al., 2012). We accepted 1% of the total number of simulations for the model choice.
Parameter inference
We tested the performance of parameter inference under model 1 (Figure 3) described in the model selection section (Statistical methods to estimate the age of a transition to selfing: tsABC) to estimate the date of a transition to selfing. We conducted the estimation using the R package abc (Csilléry et al., 2010). We accepted the closest 1% of the simulations of the transitioning model. For each corresponding set of summarizing statistics, we estimated the average posterior distribution for the 100 PODs. We show the average quantiles for the following credible intervals: 99%, 95%, 90%, 80%, 50%, 25%, 10% and the median for the whole time series. We show the performance for each parameter of the model using following sets of summary statistics: SFS/LD, TM_{win}, and both combined. For each we use an individual set of PLS components: no dimensionality reduction for SFS/LD, 20PLS for TM_{win}, and the combined set.
Performance/accuracy analysis
Transitions to selfing result in a reduction of diversity. To test whether our ABC approach identifies transitions to selfing against a model of population census reduction, we conduct a model choice experiment. We approximate and compare the posterior densities of the transition to selfing model with constant population size (Figure 3C–E) to a model which undergoes a change in population size but is constant in selfing (Figure 3). We calculated the Bayes factors using multinomial logistic regression. The results (Figure 3) depict the proportion of the correct model estimations. Depending on our summarizing statistics, our results indicate that we can precisely detect transitions to selfing for times up to 2.5N_{e} generations in the past if r/μ=1.
Interestingly, when using SFS/LD, we precisely detect the correct model for very recent times and outperform any other combination of parameters. However, TM_{win} enables us to detect also older transitions to selfing. With the combination of both summarizations, we maintain a consistently high performance to model selection. The power and specificity of our model selection are more uncertain if the ratio r/μ increases.
After determining the correct model, we estimated the actual parameter of that model: When did a population undergo a transition to selfing? To test the performance of estimating the parameters, we reused the previously simulated PODs and calculated several credibility intervals for each different time point.
Data availability
A complete detailed description of the teSMC and tsABC methods is in the SI text Appendix 1 and 2. The tsABC method can be found on GitHub (https://github.com/sstruett/tsABC, copy archived at swh:1:rev:370866ae9f0de08582c86f34edbdf3193ca7bb2c). Scripts for all figures, simulations, and the specific tsABC workflow used in this study can be found on a separate repository (https://github.com/laurentlabmpipz/struett_and_sellinger_et_al.git). copy archived at The teSMC method and a tutorial to simulate and analyze data are available on GitHub (https://github.com/TPPSellinger/eSMC2, copy archived at swh:1:rev:3cb705d7fc9f2e0918bb488eec1da30d4e08a2ec).

1001genomes1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana.https://doi.org/10.1016/j.cell.2016.05.063
References

Understanding plant reproductive diversityPhilosophical Transactions of the Royal Society B 365:99–109.https://doi.org/10.1098/rstb.2009.0199

The evolution of plant reproductive systems: how often are transitions irreversible?Proceedings of the Royal Society B 280:20130913.https://doi.org/10.1098/rspb.2013.0913

The transition to selfcompatibility in Arabidopsis thaliana and evolution within SHaplotypes over 10 MYRMolecular Biology and Evolution 23:1741–1750.https://doi.org/10.1093/molbev/msl042

Inbreeding depression and its evolutionary consequencesAnnual Review of Ecology and Systematics 18:237–268.https://doi.org/10.1146/annurev.es.18.110187.001321

Evolution of plant breeding systemsCurrent Biology 16:R726–R735.https://doi.org/10.1016/j.cub.2006.07.068

Approximate Bayesian computation (ABC) in practiceTrends in Ecology & Evolution 25:410–418.https://doi.org/10.1016/j.tree.2010.04.001

Abc: an R package for approximate Bayesian computation (ABC)Methods in Ecology and Evolution 3:475–479.https://doi.org/10.1111/j.2041210X.2011.00179.x

The variant call format and VcftoolsBioinformatics 27:2156–2158.https://doi.org/10.1093/bioinformatics/btr330

Incompatibility in AngiospermsSexual Plant Reproduction 10:185–199.https://doi.org/10.1007/s004970050087

The distribution of waiting distances in ancestral Recombination graphsTheoretical Population Biology 141:34–43.https://doi.org/10.1016/j.tpb.2021.06.003

Heterostyly accelerates diversification via reduced extinction in primrosesProceedings of the Royal Society B 281:20140075.https://doi.org/10.1098/rspb.2014.0075

BookSelfIncompatibility in Flowering PlantsBerlin, Heidelberg: Springer.https://doi.org/10.1007/9783540684862

Archaic lineages broaden our view on the history of Arabidopsis ThalianaThe New Phytologist 219:1194–1198.https://doi.org/10.1111/nph.15244

Unidirectional evolutionary transitions in fungal mating systems and the role of Transposable elementsMolecular Biology and Evolution 29:3215–3226.https://doi.org/10.1093/molbev/mss132

A comparative view of the evolution of grasses under DomesticationThe New Phytologist 183:273–290.https://doi.org/10.1111/j.14698137.2009.02884.x

Genome evolution in Outcrossing vs. Selfing vs. Asexual speciesMethods in Molecular Biology 1910:331–369.https://doi.org/10.1007/9781493990740_11

Slim 3: forward genetic simulations beyond the wright–fisher modelMolecular Biology and Evolution 36:632–637.https://doi.org/10.1093/molbev/msy228

Loss of selfincompatibility and its evolutionary consequencesInternational Journal of Plant Sciences 169:93–104.https://doi.org/10.1086/523362

The impact of purifying and background selection on the inference of population history: Problems and prospectsMolecular Biology and Evolution 38:2986–3003.https://doi.org/10.1093/molbev/msab050

Efficient Coalescent simulation and Genealogical analysis for large sample sizesPLOS Computational Biology 12:e1004842.https://doi.org/10.1371/journal.pcbi.1004842

Testing for population decline using maximal linkage disequilibrium blocksTheoretical Population Biology 134:171–181.https://doi.org/10.1016/j.tpb.2020.03.004

Evolution of floral traits and impact of reproductive mode on diversification in the Phlox family (Polemoniaceae)Molecular Phylogenetics and Evolution 127:878–890.https://doi.org/10.1016/j.ympev.2018.06.035

Population Genomics of transitions to Selfing in Brassicaceae model systemsMethods in Molecular Biology 2090:269–287.https://doi.org/10.1007/9781071601990_11

Approximating the coalescent with recombinationPhilosophical Transactions of the Royal Society B 360:1387–1393.https://doi.org/10.1098/rstb.2005.1673

A convergence theorem for Markov chains arising in population Genetics and the Coalescent with SelfingAdvances in Applied Probability 30:493–512.https://doi.org/10.1239/aap/1035228080

The coalescent process with selfingGenetics 146:1185–1195.https://doi.org/10.1093/genetics/146.3.1185

Limits and convergence properties of the Sequentially Markovian CoalescentMolecular Ecology Resources 21:2231–2248.https://doi.org/10.1111/17550998.13416

Evolution of Selfing: Recurrent patterns in molecular adaptationAnnual Review of Ecology, Evolution, and Systematics 46:593–622.https://doi.org/10.1146/annurevecolsys112414054249

A method for genomewide Genealogy estimation for thousands of samplesNature Genetics 51:1321–1329.https://doi.org/10.1038/s415880190484x

BookVariation and Evolution in PlantsNew York, United states: Columbia University Press.https://doi.org/10.7312/steb94536

Robust inference of population size histories from Genomic sequencing dataPLOS Computational Biology 18:e1010419.https://doi.org/10.1371/journal.pcbi.1010419

Plant mating systems often vary widely among populationsFrontiers in Ecology and Evolution 6:38.https://doi.org/10.3389/fevo.2018.00038

Interaction among Ploidy, breeding system and lineage diversificationThe New Phytologist 224:1252–1265.https://doi.org/10.1111/nph.16184
Decision letter

Vincent CastricReviewing Editor; Université de Lille, France

Molly PrzeworskiSenior Editor; Columbia University, United States

Takashi TsuchimatsuReviewer; Chiba University, Japan
Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.
Decision letter after peer review:
Thank you for submitting your article "Inference of evolutionary transitions to selffertilization using wholegenome sequences" for consideration by eLife. We apologize for the uncharacteristic delay in the handling of your manuscript, which was due to the absence of an editor. Your article has now been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Molly Przeworski as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Takashi Tsuchimatsu (Reviewer #1).
The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.
Essential revisions:
Both reviewers appreciated an important contribution to the study of mating systems transitions, and the welcome addition of promising new methods to detect ancestral changes in the rate of selfing. In spite of these promises, however, they also judged that the current manuscript did not reach its full potential and needed major revisions on two main aspects:
1. The comparison with the estimate of the time since A. thaliana has been a selfer by Bechsgaard et al. (2006) needs to be more careful and comprehensive. This includes in particular the lack of a proper test beyond the comparison of point estimates, and the use of different mutation rates. The importance of this latter point was already noted in a previous review (Shimizu and Tsuchimatsu 2015; Ann Rev Eco Evo Syst), which is missing from the current version. They also called for an evaluation of the consequences of the (arbitrary) choice of a limited number of genomic regions, and a more formal comparison of the ancestral population size estimated by their method with those available from the literature for that species.
2. Reviewer 3 noted a number of missing references that are required to better anchor the present developments in the previous theoretical literature, and made a series of precise suggestions with that regard. Along this line, the manuscript should better highlight that the « core » process upon which the estimation procedure is based (the effect of selfing on the association between segment length and TMRCA) is currently evaluated mostly through computational approximations. The theoretical treatment of this relationship is currently relegated to an appendix, the presentation of which could be improved greatly.
Reviewer #1 (Recommendations for the authors):
1) L6571: I would suggest citing Stebbins' work because he is the pioneer who studied the frequent transition to selfing and the evolutionary deadend.
2) Always italicize S of "Slocus".
3) L93: This would be 413,000 years instead of 430,000 years.
4) It is an important finding that population size is more accurately inferred when the outcrossingtoselfing transition is considered. As discussed, this would be potentially important for other organisms including crops, as the domestication process often involves a shift in mating systems. In that sense, authors could discuss how the estimated population size change of Arabidopsis thaliana (Figure 5B) is (or is not) consistent with previous estimates of population size.
Reviewer #2 (Recommendations for the authors):
As the authors did not even provide page numbers, I use line numbers to indicate the start of sections.
With regard to Theme (1), equations [1] and [2] are attributed to Nordborg, but Fu (1997 Genetics) and especially Golding and Strobeck (1980 Genetics) should also be cited.
The authors use [1] and [2] to develop a sequential Markov coalescent algorithm for estimating ancestral population size and the time since a change in the level of selfing.
Once again, the writing suggests Theme (1). In the Results section (L 202), a rain of references to sequential Markov methods appears, with little discussion of their relationship to the authors' work. Such scholarly discussion might be expected to appear in the Introduction or Discussion sections, but do not. For example, Palacios et al. (2015 Genetics) are relevant to the establishment of a solid inferential framework for approaches of this kind.
The authors address teSMC in a manner that appears to relegate it to mainly theoretical interest because the age of the MRCA of a chromosomal segment (TMRCA) is not in general known. Even so, the authors detect a possibly significant trend in the course of forward simulations of the process: a change in selfing rate induces a change in the magnitude of the negative association between segment length and TMRCA. They seek to use this phenomenon as a signature of a shift in the mating system apart from a shift in population size.
With regard to Theme (2), the paper would have been improved if they had undertaken to explore the basis of this relationship theoretically. The paper only provides an opaque description:
"the probability of a recombination event is not increasing linearly with time" (L 178).
The rate of recombination (r) is in fact not changing at all, and "time" here may (or may not) mean TMRCA. The ambiguity of this sentence leaves considerable room for guessing, and the reader should not have to guess at all. It is ρ_\σ (which is proportional to the probability that the next evolutionary event back in time is a recombination event rather than a coalescence event) that is affected by selfing. Both ρ_\σ and TMRCA decline as selfing increases, but the change in selfing rate has less effect on TMRCA. Is this what L 178 means? In any case, it should surely be stated more clearly.
A few lines later, we have
"We also made the important observation that all the segments that coalesce in the outcrossing phase, trace back their ancestry to a subset of segments that do not coalesce more recently
than t_\σ…." (L 188)
Are the authors saying that segments that coalesce before t_\σ do not coalesce after t_\σ? These are just a few examples (on the same page) of what are NOT minor grammatical lapses but major barriers to communication. They give the impression of sloppiness or indifference to the reader.
The authors note (L 275)
"Unfortunately, while the lengths of TMRCAsegments are straightforward to calculate on simulated genealogies (Figures 1A, B), it is more difficult to estimate them based on genomic diversity data alone."
Unfortunate or not, observing genetic diversity and not TMRCAs is of course the relevant case. While statistical uncertainty does not appear to be addressed for teSMC, it is addressed in the authors' ABC implementation of their approach (tsABC). As this section does deal with basing inferences on observations of genetic diversity (rather than TMRCA), it has greater relevance to the analysis of real data.
Even so, the description (L 288) in the main text suggests only that results were good, but not why or even exactly what the results were. In Figure 3, the reader must glean from the caption exactly what was done and what is being shown. Exactly what is on the Xaxis is unclear. The t_\σ at the far right might suggest that the Xaxis represents the true time since the switch to selfing, but it isn't clear whether the units are in generations or years or some multiple of those units. Figure 3 C/D/E appears to depict support for the true t_\σ, but not support for incorrect values. Figure 3 F/G/H does speak to whether t_\σ can be inferred, but the posterior ranges shown seem to be quite wide. After all this eyeballing of the Figure, a reader might be less entirely convinced that the results are as supportive of tsABC as the text suggests.
The next section (L 316) goes off on a tangent regarding background selection. This aspect, while important, might be reserved for a separate study: one in which a rigorous exploration might be conducted. As presented, it is not entirely clear what was done. It appears that genomic data were simulated under a background selection model. What the authors refer to as "robustness" appears to correspond to obtaining similar results from teSMC or tsABC using masked or unmasked data. The term "masking" seems to suggest that only sites NOT under selection were given to teSMC or tsABC, even though those sites were subject to background selection. If this is correct, then the finding that masking versus unmasking gives about the same results does not address the question of model misspecification: both masked and unmasked data sets could give equally bad inferences.
It seems that a more appropriate test would involve simulating data with and without BGS. The question is then whether the masked BGS data give similar results to the full nonBGS data. It is difficult to ascertain whether or not this is what the authors did.
The description of the analysis of real data is confined to a single paragraph (L 349). That the method suggested that selfcompatibility arose in the Arabidopsis thaliana lineage sometime between previous estimates (413 KYA and 1000 KYA) seems a rather low bar. The authors claim "remarkable" (L 413) agreement between their results and the 413 KYA figure, which was obtained from a modelbased analysis of variation at the Slocus. However, the authors do not even give credible intervals for their estimates. Giving the authors the benefit of the doubt, one might guess that the Xaxis in Figure 5 is in units of 10^4 years and that the 95% credible interval is perhaps (55 KYA, 60 KYA).
Does this mean that the Bechsgaard estimate of 41 KYA lies outside the authors' credible range? Note that the Bechsgaard figure assumes the rough estimate of 5 MY for the divergence of A. thaliana from its SI relatives, so that a slight revision of this figure could bring their estimate closer to (or farther from) those of the authors. That no comment or discussion about the results of real data appears bolsters the impression of Themes (1) and (3).
The Discussion begins around L 367. Once again, I am supportive of this submission as primarily theoretical, with the very short treatment of the Arabidopsis data intended only as a worked example. However, the authors seem to have (much) greater ambitions. If the authors actually wish to characterize their work as a breakthrough that opens up longstanding questions to rigorous analysis, then they need to address in detail inferences made on the basis of real data.
For the Arabidopsis analysis (the only real data application), do the authors regard their estimate as superior to the modelbased estimate of Bechsgaard? How seriously should the estimates of the changes in population size (Figure 5B) be taken? If the authors wish to argue that the changes in population size are real, they might at least provide credible intervals for the estimates and explore what is known about the ecological history of Arabidopsis.
Authors concede (L 312) that their ABC implementation could not infer the ancestral selfing rate. In the Arabidopsis case, this rate is assumed to be zero, as appropriate for a functional selfincompatibility (SI) system. Does this mean, then, that the method is useful mainly for cases in which the ancestral selfing rate is somehow known? Is their method actually only applicable to Arabidopsis?
Rather than addressing such points, the Discussion appears to cast their method permitting access to a very wide range of questions, including the evolution of sex (Barton and Charlesworth 1998). Exactly what the basis for this claim is unclear, especially since the breeding system shift modeled is wholly sexual (meiotic).
Perhaps the most germane suggestion is for the authors to determine whether this submission is primarily whatever the Discussion is maintaining or a theoretical exploration with a bit of illustration using real data.
If the latter (my view), then the theory sections might be improved. They presently appear in the Appendices as dumps of notes, with little effort invested in concise exposition. With regards to Theme (2), when presented with an opportunity for theoretical exploration, the authors appear to choose to rely on computation: the lack of theoretical exploration of the trends noted in Figure 1 and resorting to ABC over a modelbased approach come to mind. I suggest that taking a different tack would facilitate deeper insight.
https://doi.org/10.7554/eLife.82384.sa1Author response
Essential revisions:
Both reviewers appreciated an important contribution to the study of mating systems transitions, and the welcome addition of promising new methods to detect ancestral changes in the rate of selfing. In spite of these promises, however, they also judged that the current manuscript did not reach its full potential and needed major revisions on two main aspects:
1. The comparison with the estimate of the time since A. thaliana has been a selfer by Bechsgaard et al. (2006) needs to be more careful and comprehensive. This includes in particular the lack of a proper test beyond the comparison of point estimates, and the use of different mutation rates. The importance of this latter point was already noted in a previous review (Shimizu and Tsuchimatsu 2015; Ann Rev Eco Evo Syst), which is missing from the current version. They also called for an evaluation of the consequences of the (arbitrary) choice of a limited number of genomic regions, and a more formal comparison of the ancestral population size estimated by their method with those available from the literature for that species.
We thank the editor and reviewers to acknowledge the relevance of the manuscript topic as well as for their constructive criticism of our work. We have a addressed this issues in the revised manuscript (see our answers below)
2. Reviewer 3 noted a number of missing references that are required to better anchor the present developments in the previous theoretical literature, and made a series of precise suggestions with that regard. Along this line, the manuscript should better highlight that the « core » process upon which the estimation procedure is based (the effect of selfing on the association between segment length and TMRCA) is currently evaluated mostly through computational approximations. The theoretical treatment of this relationship is currently relegated to an appendix, the presentation of which could be improved greatly.
We thank reviewer 2 for the sharp comments, which improved the focus of the manuscript. We now increased the first result section by describing the theoretical results and their implication in more depth (was before in the Appendix) on page 7. We also improve the literature part by citing ad hoc references. We also moved (as suggested by reviewer 2) some literature part on SMC and ABC to the introduction (before in the result section) to streamline our result part.
Reviewer #1 (Recommendations for the authors):
1) L6571: I would suggest citing Stebbins' work because he is the pioneer who studied the frequent transition to selfing and the evolutionary deadend.
Done on p. 3 l.6,
2) Always italicize S of "Slocus".
Corrected.
3) L93: This would be 413,000 years instead of 430,000 years.
Corrected at p.4 l.9.
4) It is an important finding that population size is more accurately inferred when the outcrossingtoselfing transition is considered. As discussed, this would be potentially important for other organisms including crops, as the domestication process often involves a shift in mating systems. In that sense, authors could discuss how the estimated population size change of Arabidopsis thaliana (Figure 5B) is (or is not) consistent with previous estimates of population size.
We now compare our estimation of effective population sizes for A. thaliana with previously published estimates. P. 13 l. 1631
Reviewer #2 (Recommendations for the authors):
As the authors did not even provide page numbers, I use line numbers to indicate the start of sections.
We now include page and line numbers.
With regard to Theme (1), equations [1] and [2] are attributed to Nordborg, but Fu (1997 Genetics) and especially Golding and Strobeck (1980 Genetics) should also be cited.
Citations were added at p. 4 l. 31 – p. 5 l. 4
The authors use [1] and [2] to develop a sequential Markov coalescent algorithm for estimating ancestral population size and the time since a change in the level of selfing.
Once again, the writing suggests Theme (1). In the Results section (L 202), a rain of references to sequential Markov methods appears, with little discussion of their relationship to the authors' work. Such scholarly discussion might be expected to appear in the Introduction or Discussion sections, but do not. For example, Palacios et al. (2015 Genetics) are relevant to the establishment of a solid inferential framework for approaches of this kind.
Reply: We cite now the key references by Palacios et al. (2015) and Gattepaille et al. (2016)(p. 6 l. 10, p. 6 l. 5), which provide indeed a solid foundation for the SMC and interpretation of TMRCA. We have improved the introduction to explicit the difference between our method and those from the literature. (p. 5 l. 34 – p. 6 l. 10). We had discussed the link and convergence of results between our approach and the work by Gattepaille et al. in a previous article (Sellinger et al. Mol Ecol Res 2020).
The authors address teSMC in a manner that appears to relegate it to mainly theoretical interest because the age of the MRCA of a chromosomal segment (TMRCA) is not in general known. Even so, the authors detect a possibly significant trend in the course of forward simulations of the process: a change in selfing rate induces a change in the magnitude of the negative association between segment length and TMRCA. They seek to use this phenomenon as a signature of a shift in the mating system apart from a shift in population size.
We thank the reviewer for this summary of our work and the suggestion to explore in more depth the theoretical basis for our inference. We now enhance the theoretical content of the first part of the results. We start now from the theory and then provide the results from simulations, see lines p. 7 l. 6 p.8 l. 33
With regard to Theme (2), the paper would have been improved if they had undertaken to explore the basis of this relationship theoretically. The paper only provides an opaque description:
"the probability of a recombination event is not increasing linearly with time" (L 178).
We thank the reviewer for this suggestion. We now rephrased the section to clearly state our new theoretical results (p. 7 l. 6 p.8 l. 33) and updated Figure 1—figure supplement 1 for visual support. The transitions in Figure 1—figure supplement 1 are now computed from the equation 3 in the main text.
The rate of recombination (r) is in fact not changing at all, and "time" here may (or may not) mean TMRCA. The ambiguity of this sentence leaves considerable room for guessing, and the reader should not have to guess at all. It is ρ_\σ (which is proportional to the probability that the next evolutionary event back in time is a recombination event rather than a coalescence event) that is affected by selfing. Both ρ_\σ and TMRCA decline as selfing increases, but the change in selfing rate has less effect on TMRCA. Is this what L 178 means? In any case, it should surely be stated more clearly.
We apologize for the confusion and changed vocabulary to be more rigorous. We admit that we did not find previously the right balance between in depth precise mathematical explanations and an enhanced readability of our paper for a biological audience (as required for a journal as eLife). What we meant was the rate at which a break in the genealogy occurs. This section and others have been rewritten to be precise in our notations (p. 7 l. 6 p.8 l. 33). The first part of the results is now dedicated to this aspect with the introduction of equation 3.
A few lines later, we have
"We also made the important observation that all the segments that coalesce in the outcrossing phase, trace back their ancestry to a subset of segments that do not coalesce more recently
than t_\σ…." (L 188)
Are the authors saying that segments that coalesce before t_\σ do not coalesce after t_\σ? These are just a few examples (on the same page) of what are NOT minor grammatical lapses but major barriers to communication. They give the impression of sloppiness or indifference to the reader.
We have rewritten few sentences to add precision. P. 8 l. 114
The authors note (L 275)
"Unfortunately, while the lengths of TMRCAsegments are straightforward to calculate on simulated genealogies (Figures 1A, B), it is more difficult to estimate them based on genomic diversity data alone."
Unfortunate or not, observing genetic diversity and not TMRCAs is of course the relevant case. While statistical uncertainty does not appear to be addressed for teSMC, it is addressed in the authors' ABC implementation of their approach (tsABC). As this section does deal with basing inferences on observations of genetic diversity (rather than TMRCA), it has greater relevance to the analysis of real data.
It is true that the uncertainty of TMRCA inference is not seen in SMC methods, as they rely on multiple observations to estimate the time of the hidden states (and thus provide by likelihood estimation one value and not a confidence interval). The uncertainty is addressed in our case by running several analyses on different datasets with either the same simulation parameters or on several regions of the A. thaliana genome as statistical replicates. The reviewer is correct that this is a key point of our study and why we provide both complementary approaches here.
Even so, the description (L 288) in the main text suggests only that results were good, but not why or even exactly what the results were. In Figure 3, the reader must glean from the caption exactly what was done and what is being shown. Exactly what is on the Xaxis is unclear. The t_\σ at the far right might suggest that the Xaxis represents the true time since the switch to selfing, but it isn't clear whether the units are in generations or years or some multiple of those units. Figure 3 C/D/E appears to depict support for the true t_\σ, but not support for incorrect values. Figure 3 F/G/H does speak to whether t_\σ can be inferred, but the posterior ranges shown seem to be quite wide. After all this eyeballing of the Figure, a reader might be less entirely convinced that the results are as supportive of tsABC as the text suggests.
We have updated Figure 3 and its legend to clarify the meaning of the axes and the units. Figure 3 C/D/E show the statistical support for the right model (i.e. transition to selfing) when compared to a stepwise change in N in an ABC model choice procedure. Figure 3 F/G/H shows average quantiles for 100 posteriors on t_\σ and panel H indeed indicates the precision that can be expected for t_σ. We note that the average mode of the 100 posteriors tracks the true value (solid black line in panel H) over the whole range of t_\σ values, that a large proportion of the probability density is centered around the true value (i.e. the posterios are peaky), and that the precision is improved when using TM_win verus a combination of SFS and LD.
The next section (L 316) goes off on a tangent regarding background selection. This aspect, while important, might be reserved for a separate study: one in which a rigorous exploration might be conducted. As presented, it is not entirely clear what was done. It appears that genomic data were simulated under a background selection model. What the authors refer to as "robustness" appears to correspond to obtaining similar results from teSMC or tsABC using masked or unmasked data. The term "masking" seems to suggest that only sites NOT under selection were given to teSMC or tsABC, even though those sites were subject to background selection. If this is correct, then the finding that masking versus unmasking gives about the same results does not address the question of model misspecification: both masked and unmasked data sets could give equally bad inferences.
It seems that a more appropriate test would involve simulating data with and without BGS. The question is then whether the masked BGS data give similar results to the full nonBGS data. It is difficult to ascertain whether or not this is what the authors did.
The simulations used in Figure 4 have indeed been done with BGS. Details about this analysis can be found in the legend of Figure 4, in the main text (methods p. 18 l. 917), and in Appendix 2 (section: simulation of genetic data). What we refer to as robustness does not relate to the comparison between the masked and unmasked data, but rather, as you suggest, to the comparison of the performance of both methods when applied to data simulated with and without BGS. The case without BGS can be seen in Figure 2A for teSMC and figure 3H for tsABC. We have updated the main text to make this clearer (p. 12 l. 324).
The description of the analysis of real data is confined to a single paragraph (L 349).
That the method suggested that selfcompatibility arose in the Arabidopsis thaliana lineage sometime between previous estimates (413 KYA and 1000 KYA) seems a rather low bar. The authors claim "remarkable" (L 413) agreement between their results and the 413 KYA figure, which was obtained from a modelbased analysis of variation at the Slocus. However, the authors do not even give credible intervals for their estimates. Giving the authors the benefit of the doubt, one might guess that the Xaxis in Figure 5 is in units of 10^4 years and that the 95% credible interval is perhaps (55 KYA, 60 KYA).
This part has been revised following criticism from reviewer 1 (see answers to Reviewer 1, and the section “Application to A. thaliana” (p. 12 l. 26)). Credibility intervals for t_\σ are given in table 1. Labelling of figure 5 has been updated.
Does this mean that the Bechsgaard estimate of 41 KYA lies outside the authors' credible range? Note that the Bechsgaard figure assumes the rough estimate of 5 MY for the divergence of A. thaliana from its SI relatives, so that a slight revision of this figure could bring their estimate closer to (or farther from) those of the authors. That no comment or discussion about the results of real data appears bolsters the impression of Themes (1) and (3).
The Discussion begins around L 367. Once again, I am supportive of this submission as primarily theoretical, with the very short treatment of the Arabidopsis data intended only as a worked example. However, the authors seem to have (much) greater ambitions. If the authors actually wish to characterize their work as a breakthrough that opens up longstanding questions to rigorous analysis, then they need to address in detail inferences made on the basis of real data.
Bechsgaard indeed used a different mutation rate than the one assumed in our study. This issue was raised by reviewer 1 as well and we have addressed this in the revised version. (p. 12 l. 26)
For the Arabidopsis analysis (the only real data application), do the authors regard their estimate as superior to the modelbased estimate of Bechsgaard? How seriously should the estimates of the changes in population size (Figure 5B) be taken? If the authors wish to argue that the changes in population size are real, they might at least provide credible intervals for the estimates and explore what is known about the ecological history of Arabidopsis.
This issue was raised by reviewer 1 as well and was considered in the revised version (see answer to reviewer 1 and p. 13 l. 1631 of the main text).
Authors concede (L 312) that their ABC implementation could not infer the ancestral selfing rate. In the Arabidopsis case, this rate is assumed to be zero, as appropriate for a functional selfincompatibility (SI) system. Does this mean, then, that the method is useful mainly for cases in which the ancestral selfing rate is somehow known? Is their method actually only applicable to Arabidopsis?
The method is wellsuited for cases in which the transition occurs owing to a breakdown of a selfincompatibility mechanism. In such cases the selfing rate before the breakdown should be close to zero because of the selfincompatibility mechanism. Breakdown of SI is very common beyond Brassicaceae, as well documented for example in Solanaceae (Igic and Kohn 2006, Goldberg et al. 2010) or Asteraceae (Ferrer and GoodAvila 2006) So, the method is a priori suitable for many species.
Rather than addressing such points, the Discussion appears to cast their method permitting access to a very wide range of questions, including the evolution of sex (Barton and Charlesworth 1998). Exactly what the basis for this claim is unclear, especially since the breeding system shift modeled is wholly sexual (meiotic).
Perhaps the most germane suggestion is for the authors to determine whether this submission is primarily whatever the Discussion is maintaining or a theoretical exploration with a bit of illustration using real data.
If the latter (my view), then the theory sections might be improved. They presently appear in the Appendices as dumps of notes, with little effort invested in concise exposition. With regards to Theme (2), when presented with an opportunity for theoretical exploration, the authors appear to choose to rely on computation: the lack of theoretical exploration of the trends noted in Figure 1 and resorting to ABC over a modelbased approach come to mind. I suggest that taking a different tack would facilitate deeper insight.
Thank you for the suggestion. We hope our changes and addition of a result section with theoretical results has improved our message. We also worked on the Appendix to provide better explanation for the formulae.
https://doi.org/10.7554/eLife.82384.sa2Article and author information
Author details
Funding
Max Planck Institute for Plant Breeding Research (open access funding)
 Stefan Strütt
 Stefan Laurent
Open access funding provided by Max Planck Society.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project 462181533 to SL and projects 317616126, 254587930, and 447121532 to AT.
Senior Editor
 Molly Przeworski, Columbia University, United States
Reviewing Editor
 Vincent Castric, Université de Lille, France
Reviewer
 Takashi Tsuchimatsu, Chiba University, Japan
Version history
 Preprint posted: August 1, 2022 (view preprint)
 Received: August 2, 2022
 Accepted: May 8, 2023
 Accepted Manuscript published: May 11, 2023 (version 1)
 Version of Record published: June 28, 2023 (version 2)
Copyright
© 2023, Strütt, Sellinger et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics

 659
 Page views

 103
 Downloads

 1
 Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading

 Ecology
 Evolutionary Biology
Groups of animals inhabit vastly different sensory worlds, or umwelten, which shape fundamental aspects of their behaviour. Yet the sensory ecology of species is rarely incorporated into the emerging field of collective behaviour, which studies the movements, populationlevel behaviours, and emergent properties of animal groups. Here, we review the contributions of sensory ecology and collective behaviour to understanding how animals move and interact within the context of their social and physical environments. Our goal is to advance and bridge these two areas of inquiry and highlight the potential for their creative integration. To achieve this goal, we organise our review around the following themes: (1) identifying the promise of integrating collective behaviour and sensory ecology; (2) defining and exploring the concept of a ‘sensory collective’; (3) considering the potential for sensory collectives to shape the evolution of sensory systems; (4) exploring examples from diverse taxa to illustrate neural circuits involved in sensing and collective behaviour; and (5) suggesting the need for creative conceptual and methodological advances to quantify ‘sensescapes’. In the final section, (6) applications to biological conservation, we argue that these topics are timely, given the ongoing anthropogenic changes to sensory stimuli (e.g. via light, sound, and chemical pollution) which are anticipated to impact animal collectives and grouplevel behaviour and, in turn, ecosystem composition and function. Our synthesis seeks to provide a forwardlooking perspective on how sensory ecologists and collective behaviourists can both learn from and inspire one another to advance our understanding of animal behaviour, ecology, adaptation, and evolution.

 Evolutionary Biology
Changes in an organism’s environment, genome, or gene expression patterns can lead to changes in its metabolism. The metabolic phenotype can be under selection and contributes to adaptation. However, the networked and convoluted nature of an organism’s metabolism makes relating mutations, metabolic changes, and effects on fitness challenging. To overcome this challenge, we use the longterm evolution experiment (LTEE) with E. coli as a model to understand how mutations can eventually affect metabolism and perhaps fitness. We used mass spectrometry to broadly survey the metabolomes of the ancestral strains and all 12 evolved lines. We combined this metabolic data with mutation and expression data to suggest how mutations that alter specific reaction pathways, such as the biosynthesis of nicotinamide adenine dinucleotide, might increase fitness in the system. Our work provides a better understanding of how mutations might affect fitness through the metabolic changes in the LTEE and thus provides a major step in developing a complete genotype–phenotype map for this experimental system.