Genetics: Early Online, published on August 7, 2019 as 10.1534/genetics.119.302306 GENETICS | INVESTIGATION

Invasion and extinction dynamics of types under facultative sexual

Peter Czuppon∗,‡ and George W. A. Constable†,1,‡ ∗Center for Interdisciplinary Research in Biology, CNRS, Collège de France, PSL Research University, Paris, France; Institute of Ecology and Environmental Sciences of Paris, Sorbonne Université, UPEC, CNRS, IRD, INRA, Paris, France, †Department of Mathematical Sciences, The University of Bath, ‡Authors contributed equally

ABSTRACT In sexually reproducing isogamous species, syngamy between is generally not indiscriminate, but rather restricted to occurring between complementary self-incompatible mating types. A longstanding question regards the evolutionary pressures that control the number of mating types observed in natural populations, which ranges from two to many thousands. Here, we describe a population genetic null model of this and derive expressions for the stationary probability distribution of the number of mating types, the establishment probability of a newly arising and the mean time to extinction of a resident type. Our results yield that the average rate of in a population correlates positively with the expected number of mating types observed. We further show that the low number of mating types predicted in the rare- regime is primarily driven by low invasion probabilities of new mating type alleles, with established resident alleles being very stable over long evolutionary periods. Moreover, our model naturally exhibits varying selection strength dependent on the number of resident mating types. This results in higher extinction and lower invasion rates for an increasing number of residents.

KEYWORDS Mating types; Isogamy; Facultative sex; Self-incompatibility; Balancing selection; Frequency-dependent selection; Finite populations

n isogamous species, the size differentiation that de- cytoplasmic conflict by promoting uniparental inheritance of I fines the in anisogamous species is absent (Lehtonen organelles (UPI) (Hurst and Hamilton 1992; Hadjivasiliou et al. et al. 2016). Despite their morphological similarity, the gametes 2012). of isogamous species are typically not interchangeable, but Empirical observations of the number of mating types vary rather fall into one of a number of genetically determined self- between species, ranging from 2 to many thousands (Kothe incompatible gamete classes, termed mating types. Syngamy 1996). For example, intermediate numbers of mating types are can only occur between gametes of distinct and complementary reported in of the -species (3 − 9 different mating types. mating types (Doerder et al. 1995; Phadke and Zufall 2009)) and The evolutionary explanation for this self-incompatibility, slime molds (2 − 13 mating types (Bloomfield et al. 2010; Clark which limits the number of potential mates available to an isoga- and Haskins 2010)). Larger numbers can be found in fungal mous organism, has been the subject of debate, with a number populations; in Coprinellus disseminatus the global population of competing hypotheses proposed (Billiard et al. 2011). These is estimated to contain 123 different mating types (James et al. include theories that such self-incompatibility alleles limit in- 2006), while Schizophyllum commune has a staggering 23, 328 breeding depression (Charlesworth and Charlesworth 1979), distinct types (Kothe 1999). This naturally leads to the question increase encounter rates between gamete pairs (Hoekstra 1982; which type of evolutionary pressures govern this diversity in Hadjivasiliou et al. 2015; Hadjivasiliou and Pomiankowski 2019), mating type number? This has been the subject of much debate, allow for ploidy level detection and the instigation of the motivated in part by a discrepancy between these empirical developmental program (Haag 2007; Perrin 2012), or manage observations and simple evolutionary reasoning. From a theoretical standpoint, one might naïvely expect to see doi: 10.1534/genetics.XXX.XXXXXX a very large number of mating types within any given species, Manuscript compiled: Wednesday 7th August, 2019 due to the “rare sex advantage” of novel types (Iwasa and Sasaki 1corresponding author 1987). Since mating types are self-incompatible, rare types have

Genetics 1 Copyright 2019. more opportunities for mating and thus each type experiences recent work has emphasized the relevance of finite population negative-frequency dependent selection. Therefore, each novel size null models (i.e. models in which all mating types are mating type produced by mutation should establish in the pop- phenotypically similar) for addressing the distribution of mating ulation and the number of mating types should consistently type numbers observed in nature (Constable and Kokko 2018; grow. However, this prediction stands in stark contrast to what Czuppon and Rogers 2019). These studies stress that even in is observed in the natural world. Although isogamous species the absence of species-specific biological processes, the number with hundreds, or even thousands, of mating types are possi- of mating types in any real finite population cannot be infinite. ble (Kothe 1996), examples of such species are very rare; the Instead the expected number of types will arise from balance vast majority have very few (typically two) mating types (Hadji- between mutations (which introduce new mating types) and vasiliou 2014). Explaining this discrepancy between theory and extinctions (which decrease the number of types), leading to a empirical observation has been the focus of much work, and number of mating types well below the studied population size. multiple theories have been proposed (Billiard et al. 2011). As an explanation for the low number of mating types often One prominent hypothesis is that UPI drives the evolution of observed in isogamous species, this mutation-extinction balance two mating types (Hadjivasiliou et al. 2012), with larger mating hypothesis may seem at first improbable, particularly in the type numbers becoming less stable with the increased complex- light of a number of classic population genetic studies of self- ity of coordinating an organelle donor-receiver program (Hurst incompatibility (SI) alleles in plants (Wright 1939, 1960, 1964; and Hamilton 1992). While recent modelling work has shown Ewens 1964; Nagylaki 1975; Yokoyama and Nei 1979; Yokoyama that when the frequency-dependent effects of UPI are accounted and Hetherington 1982), see also Clark and Kao (1994) for a for, an invading donor-receiver program does not reduce the review. From a modelling perspective, the dynamics of SI alleles expected number of mating types (Hadjivasiliou et al. 2013), per- in gametophytic species, those where SI is determined at the ceived empirical support for the theory comes from the fact that haploid pollen stage (Bod’ová et al. 2018), closely resemble those many species with more than two mating types have developed of SI mating type alleles in isogamous species. (A comparison mechanisms to ensure homoplasmy without such a program. with the dynamics of SI alleles in sporophytic species, in which For instance, in bursaria, with up to eight mating SI of the haploid pollen is determined by the diploid parent, types (Phadke and Zufall 2009), and S. commune, sexual repro- can be more complicated due to the complex dominance rela- duction is achieved following the exchange of nuclei between tionships that regulate SI in some of these species (Thompson cells without cytoplasmic mixing (Birky 1995). However this in and Taylor 1966; Prigoda et al. 2005; Billiard et al. 2007).) In turn leads to an opposing question; if it really is only a donor- a seminal paper (Wright 1939), Wright predicted that for the receiver program that limits the number of mating types, why model plant Oenothera organensis, a population of around 500 do ciliates and Agaricomycetes, with these alternative methods individuals could sustain approximately 13 SI alleles (see also for ensuring homoplasmy, still feature species with very few Crosby (1966)). As the number of predicted SI alleles would types (as low as two and three respectively, see James (2015))? rise with increasing effective population size (which intuitively A further hypothesis (Hoekstra 1982; Hoekstra et al. 1991), reduces genetic drift and hence extinction rates), many more with renewed attention (Hadjivasiliou and Pomiankowski 2016), SI mating type alleles might naïvely be expected in isogamous suggests that it is cell-cell signalling between gametes that limits species, for which effective population sizes can be of the order 6 the number of mating types. Here co-evolution between two 10 (Baranova et al. 2015). resident types has been shown to potentially limit the evolution- While in the above context the plausibility of the mutation- ary success of a third mutant mating type. If derived from a extinction balance hypothesis might seem doubtful, an impor- resident type, this new mutant must essentially cross a fitness tant biological feature of isogamous species with less promi- valley before it can develop encounter rates with the residents nence in plants is the potential to reproduce asexually. In many comparable to their current pairwise encounter rate, limiting its isogamous species, long periods of asexual reproduction are invasion potential (Hadjivasiliou and Pomiankowski 2016). punctuated by rare bouts of facultative sex. For instance, in In each of these hypotheses, a biologically plausible emergent the single celled green , Chlamydomonas reinhardtii (with mechanism is sought that generates a selective advantage to two mating types (Goodenough et al. 2007)), recent genomic a pair of mating types, thus limiting their number to two. A estimates have placed the rate of sexual reproduction to be once notable exception can be found in Iwasa and Sasaki (1987). Here in every 770 asexual generations (Hasan and Ness 2018). Sex in it was demonstrated that under certain dynamics for the mating yeast, which also typically have two mating types (Butler 2007), type encounter rate, the advantage to rare mating types could appears to be rarer still, with estimates of once in every thou- be suppressed. In the limit of infinitely long-lived gametes (that sand to three thousand asexual generations reported in some can always survive until a suitable partner becomes available) species (Tsai et al. 2008). selection for mating type numbers greater than two could be In Hadjivasiliou et al. (2016), a computational model was eliminated entirely. It was verbally suggested that under such a used to show that long periods of asexual reproduction would scenario, genetic drift would purge new mating types, limiting lead to substantial drift in mating types frequencies. Using a their number. This led to a bimodal prediction for the number population genetic model, Constable and Kokko (2018) demon- of types; populations would either have two (given a particular strated further that as sex becomes increasingly rare, the relative set of encounter rate dynamics and immortal gametes), or have strength of genetic drift over selection for novel mating types infinitely many otherwise (with each at infinitely low frequency). is amplified. This leads to a lower expected number of mating While the conditions required for two mating types in Iwasa types in mutation-extinction balance (a similar observation has and Sasaki (1987) were stringent (and indeed, the possibility been made of Solanaceae gametophytic SI alleles (Vallejo-Marín of intermediate numbers of mating types impossible) it was and Uyenoyama 2008)). This was true even in the absence of notable for suggesting that genetic drift may have a key role any species-specific selective mechanisms. A survey of available to play in determining mating type number. In a similar vein, empirical data further supported the view that the rate of fac-

2 Czuppon and Constable ultative sex is a key predictor of the number of mating types in of the number of mating types, extending the results of Consta- isogamous species. ble and Kokko (2018) (where only bounds of the mode of this Analytic bounds on the number of expected mating types un- distribution were calculated). We then provide an expression for der a mutation-extinction balance were calculated in Constable the establishment probability of a novel mating type in faculta- and Kokko (2018), using the stationary distribution of a sim- tively sexual populations, generalizing the results of Czuppon ple Moran-type model of mating type dynamics. In Czuppon and Rogers (2019). Finally, combining these expressions, we and Rogers (2019) an approximation of the expected number of calculate the mean time to extinction of a resident mating type mating types was calculated under the assumption that sex is allele in a novel way. To be more precise, instead of relying obligate. Muirhead and Wakeley (2009) used a similar model to solely on the one dimensional dynamics of a focal mating type calculate the stationary distribution of the frequency spectrum (in mating type frequency space) we make use of the dynamics of mating type alleles, however estimates on the number of ex- on the number of mating types (in mating type number space). pected mating types were not explicitly calculated. In addition, We conclude by discussing the biological implications of these facultative sex was not modelled (although the mathematical results. analysis employed can account for this factor). While such estimates of the number of mating type alleles Model in the stationary distribution are informative, they obscure the dynamical processes that drive and maintain this equilibrium, We consider a population in which the self-incompatible mating the establishment and extinction of alleles. These quantities are type of an individual is determined by one of an infinite set important for two reasons. of potential alleles at a single locus. Such single locus deter- Firstly, they are both independent of the arrival rate of new mination systems are for example found in the social amoebae SI alleles. Null models of mating type dynamics often equate Dictyostelium discoideum (with three mating types (Bloomfield the arrival rate of new SI alleles with an effective mutation rate, et al. 2010)) and Didymium squamulosum (with up to 12 mating and in doing so take a course grained approach to the genetic, types (Clark and Haskins 2010)). The evolutionary dynamics are molecular and physiological mechanisms underlying mating determined by a Moran process; generations are overlapping type determination. However this effective mutation rate is a and the population is assumed to be at an ecological equilibrium confounding parameter, as it is difficult to estimate empirically. at which the number of individuals, N, is constant over time. (A similar problem was encountered by Wright, who conceded We denote the number of individuals carrying mating type i by ∞ = that there could be argument about what a “reasonable” rate ni, such that ∑i=1 ni N. should be (Wright 1939)). In contrast, the effective mutation rate Asexual reproduction happens with probability c ∈ [0, 1]. In affects neither the establishment probability of a mutant mating this case a randomly chosen individual produces a clone which type nor the expected extinction time of a resident type. These replaces one of the individuals in the population, also chosen quantities thus provide mutation rate independent measures of uniformly at random. the evolutionary dynamics of mating types, that can be used to During sexual reproduction, which occurs with probability test the feasibility of the mutation-extinction hypothesis. 1 − c, two randomly chosen individuals mate if they express Secondly, whereas the stationary distribution of mating type different mating types. In the case of a successful mating, an number can only be observed over evolutionary time periods, offspring individual is produced that replaces one of the other the establishment probability of new mating types and the mean individuals in the population. The mating type of the offspring extinction time of mating types can be observed over signifi- is chosen at random from among the two parental alleles. cantly shorter time periods. These quantities are thus important Additionally, we consider the emergence of novel mating for providing empirically testable insight into the evolution of types through mutations which occur at rate m. We imple- mating type number. ment mutations according to the infinitely many alleles model (Kimura and Crow 1964), where the mutated individual ex- Extinction rates have previously been studied in related sys- presses a completely new mating type not previously present tems featuring negative frequency dependent selection, which in the population. For simplicity we consider the case in which results in high levels of polymorphism. In Takahata (1990) a mutation events are decoupled from reproduction. Mutants one-dimensional stochastic diffusion was used to identify the possess the same characteristics as any other mating type in the dynamics of the polymorphic major histocompatibility complex population; that is they are self-incompatible and mate with (MHC) system with a time re-scaled neutral coalescent. This al- non-self types at the same per-capita rate as the resident types. lowed for estimating the extinction rates of MHC alleles. These results were verified numerically in Takahata and Nei (1990) Mathematically implementing the model described above, and later in Slatkin and Muirhead (1999). The same theoretical the probability per unit time for a type i to increase by one and a approach was adapted to gametophytic SI in plants obtaining re- type j to decrease by one through a birth-death event is given by sults showing that the time to the most recent common ancestor     ni (1 − c) ni ∑k6=i nk nj of a sex allele might even exceed the speciation time (Vekemans Tij = c + , for ni > 0. (1) and Slatkin 1994). In the specific context of mating type alleles, N 2 N N N | {z } | {z } such results have thus far been absent. However recently in reproduction death Czuppon and Rogers (2019) the establishment probability of newly arising mating type was calculated for populations in The reproduction term is split into an asexual component (the which sex is obligate. We will rely on this quantity to obtain our first term) and a sexual component (the second term). We note, estimate on the mean extinction time of resident mating types. that the sum in the sexual reproduction term goes over all non-i In this paper we complement this literature by exploring sce- mating types present in the population, generating a reproduc- narios of self-incompatibility under facultative sex. We begin by tive advantage to rare mating types. The probability per unit calculating an analytic expression for the stationary distribution time that a novel mating type i is generated from an ancestral

Evolutionary dynamics of mating types 3 mating type j is given by where n (1 − c) n N − n nj ( ) = i + i i ≥ T ∝ m , for n = 0, (2) b ni c , if ni 1 , ij N i N 2 N N b(ni) ∝ m , if ni = 0 , (5) which can only occur when type i is not already present in nj d(n ) = , for all n . the population. As described in Eq. (S6) in the Supplementary j N j Information, the full expression for the probability transition rate Under this decomposition, the stationary distribution of mating given in Eq. (2) features an additional normalization constant, type alleles takes the exact form included to ensure that the mutation rate is independent of the ∞ | = ↓ population composition (i.e. that ∑i=1 Tij ni =0 m). n −1 1 M−1 i b(k)d (Φ − k) The deterministic limit (N → ∞) of this model is described by Pst = i (6) n N ∏ ∏ ( − ( + )) ( + ) a system of ordinary differential equations where the dynamics i=1 k=0 b Φi k 1 d k 1 (neglecting mutations) of a the ith mating type, with frequency ni ↓ xi = limN→∞ , is given by where n is the vector n reordered with its largest entries first, N st N is a normalization constant that enforces ∑n Pn = 1 and Φi is dxi (1 − c) defined as = xi xj(xj − xi) . (3) dt 2 ∑ i−1 j6=i ↓ Φi = N − ∑ nj . (7) j=1 This system has been studied in Iwasa and Sasaki (1987) (see their Mating kinetics I). The dynamical system possesses an The full derivation of Eq. (6) is given in Constable and Kokko internal stable fixed point where all mating types are present at (2018). In terms of birth-death processes, it is interesting to note equal frequencies. Analogous finite population size models lead that Eq. (6) is the product of standard stationary distributions to stochastic differential equations and have been analyzed in obtained for a two-allele (single variable) population (Karlin Czuppon and Rogers (2019). Note that in that paper mutation and Taylor 1975), moderated by a series of effective population was implemented in a distinct manner, with the assumption sizes. A similar observation has been made in a related model that mutation occurs with reproductive events. While that of multi-allelic selection (Muirhead and Wakeley 2009). implementation is biologically more reasonable, the choice leads to only minor quantitative differences in the dynamics. The stationary distribution of the number of mating type alle- les Data availability: The code and simulation outputs can be The stationary distribution of populations with M mating types found on github: st is defined as PM. This gives the probability of finding a popula- https://github.com/gwaconstable/InvExtDynMatTypes. tion with a given set of parameters (N, c and m) in a state with st M mating types at long times. PM is related to the distribution st Results Pn through the following summation; st st In this section we mathematically analyze the null model just PM = ∑ Pn , (8) presented. We begin by characterizing the long-time equilibrium n∈S(M) behavior of the model, which is described by its stationary distri- where S(M) is the set of all vectors n that represent a population bution. This allows us to answer the key question of how many with M present mating types (that is, all vectors n with M non- mating types, M, are predicted by the null model as a function of zero elements). the population size N, mutation rate m and, importantly, relative While Eq. (8) can be expressed neatly, evaluating this quantity rate of asexual reproduction, c. However, while this results in an is problematic as it involves summations of Eq. (6) over sets of important benchmark for the expected number of mating types the infinite vector n. To make analytic progress we note that in real populations, it provides little insight into the dynamics when the population size N is large and the per-generation of mating type number. To this end we further calculate the mutation rate m = mN is small, at intermediate times the probability that a novel mutant mating type establishes in the g population resides in a quasi-stationary distribution in the region population and the expected extinction time of a resident mating of a fixed point of the dynamics in the deterministic limit. More type allele. These quantities provide a deeper insight into the precisely, when the population is comprised of M mating types, ongoing dynamics of mating type number in real populations. this fixed point is given by ni ≈ N/M for each of the i present mating types, and ni = 0 otherwise. Following an extinction or The stationary distribution of individuals carrying each mating mutation event (which changes the number of mating types in type allele the population) the population quickly relaxes to a new quasi- st We denote by Pn the stationary distribution of the number of stationary distribution in the region of an alternate fixed point individuals of each mating type. In Constable and Kokko (2018) with M ± 1 mating types, i.e. ni ≈ N/(M ± 1). Within the limit st st it was shown that an analytic solution for Pn is accessible as the of large N and small mg then, Pn can be approximated by a probability transition rates Tij in Eqs. (1-2) can be decomposed superposition of these quasi-stationary distributions. into the product of a birth function b(ni) and death function Briefly, the quasi-stationary distribution of the population in d(nj) that each depend only on the number of each mating type the region of a fixed point can be calculated by conducting a reproducing and dying respectively; diffusion approximation on the underlying Moran model and linearizing the resultant advection-diffusion equation about a Tij = b(ni)d(nj) , (4) deterministic fixed point (the van Kampen approximation). The

4 Czuppon and Constable full calculation is conducted in the Supplementary Information, where we show that these quasi-stationary distributions are 1.0 Gaussian (see Eqns. (S18)-(S22)). 0.8 Each of the quasi-stationary distributions can now be renor- malized, using Eq. (6) to ‘pin’ the height of each quasi-stationary 0.6 distribution to the height of Eq. (6) in the region of the determin- istic fixed point (see Hufton et al. (2016) and Vasconcelos et al. 0.4 (2017) for similar approaches). The full calculation is detailed in Section S3 in the Supplementary Information. Substituting the 0.2 st resulting approximation for Pn into Eq. (8) and taking the limit 0.0 of large N we find 5 10 15

1/2 M −1  M−1 M− 1 " M−1 # (2π) 2 2m M 2 θ Figure 1 Stationary distribution of mating types Pst Pst ≈ × , M. For M M 1 + c M! θ − M−1 any given rate of asexual reproduction, c, larger populations (larger N) contain more mating types on average. Meanwhile "  Mθ#N   θ increasing the rate of asexual to sexual reproduction (increas- N(M−1)/2 θ − M−1 , (9) θ − M−1 ing c) decreases the expected number of mating types. An- alytic results (solid lines) are obtained by evaluating Eq. (9). ∞ st where M is a normalization constant such that ∑M=1 PM = 1 Simulation results (corresponding shaded regions) are ob- and tained using the Gillespie algorithm (Gillespie 1976), averaged 1 + c over 5 × 106 generations, sampled every 102 generations and θ = . (10) 3 1 − c following a relaxation period of 10 generations from initial conditions with one more mating type than the mode number Note that when sexual reproduction is obligate (c = 0), θ = 1, predicted analytically by the model (see Eq. (9)). The mutation while when sex is facultative and rare θ becomes large. rate is m = 10−8 in all plots. The analytic description can be Comparing our approximate expression for the distribution seen to capture the simulated behavior of the model with very of the number of mating types, Eq. (9), against the results of high accuracy. stochastic simulation of the population, we find excellent agree- ment (see Figure 1). We note that in a facultatively sexual popu- lation, where the role of genetic drift is amplified by high clonal reproduction rates, c, extinctions can even lower the number of mating types to one (see Supplementary Information, Section S8). In this scenario no further sexual reproduction events can take place, and the population reproduces purely asexually until a new mating type is generated by mutation. 600 st The mode number of mating types Since the distribution PM is unimodal, obtaining an estimate for its mode is straightforward 100 numerically. We define by rM the ratio of probabilities of having M and M − 1 mating types; Pst = M 10 rM st . (11) PM−1 For any given set of parameters, this ratio is independent of the M normalization constant . Substituting Eq. (9) into Eq. (11), 1 we obtain a simplified analytic expression for rM (see Supple- st mentary Information, Eq. (S42)). Since the distribution PM is unimodal, finding its mode is equivalent to finding the value of st M for which PM starts to decrease. The approximate mode of st PM, which we denote Mo, can then be obtained as the solution to the equation Figure 2 Analytic results on the mode number of mating r = 1 . (12) Mo types, Mo, as a function of the population size, N and the per- generation mutation rate, mg = Nm, under differing rates of The function rM − 1 has a single root, and thus solving rM = 1 o asexual reproduction, c. As the rate of sexual to asexual repro- for Mo is numerically straightforward. In Figure 2 we plot the mode number of mating types as a duction is decreased [panels (a-d)] so too does the expected number of mating types. When sex is obligate [panel (a)], of function of the per-generation mutation rate, mg and the popu- lation size, N, for four different rates of asexual to sexual repro- the order of hundreds of mating types are expected. When duction. Here we see that facultative sex has a strong influence sex is facultative and very rare, occurring approximately once over the number of mating types, with even a ratio of 9 : 1 to every thousand asexual reproduction events [panel (d)] asexual to sexual divisions (panel (b)) reducing the expected far fewer mating types are observed. Results are obtained by number of mating types by an order of magnitude compared to numerically solving Eq. (12). the obligately sexual scenario (panel (a)).

Evolutionary dynamics of mating types 5 The mean time until the extinction of a mating type allele 1 Our final goal is to calculate the mean extinction time of a mating 0.100 type allele. We assume that initially the population is close to 0.010 its equilibrium (i.e. all mating types are approximately at equal frequencies). We then model the arrival of mating types (through 0.001 mutation events) and their extinction (due to genetic drift) as a - 4 10 birth-death process on the number of mating types. Let βM be

10- 5 the probability per unit time that a population with M resident mating types gains a new mating type, and δM be the probability 2 4 6 8 10 per unit time that a mating type goes extinct. Although we are interested in calculating the time between Figure 3 Establishment probability of a novel mating type as extinction events, we first turn our attention to the stationary a function of M for varying rates of asexual reproduction, c. probability distribution of this effective ‘birth-death’ process Analytic results are obtained by evaluating Eq. (13). Simula- st in the number of mating types; PM. Solving the equations for tion results are obtained from Gillespie simulations; in each the stationary distribution of the effective birth-death process, 5 3 plot N = 10 and results are averaged over 10 runs (c = 0), we obtain the well-known result (see for instance (Allen 2011, 4 5 10 runs (c = 0.9 and c = 0.99), and 10 runs (c = 0.999). Chapter 6)) Error bars are ±2 times the standard-deviation of the sample binomial distribution. st βM−1 st PM = PM−1 . (14) δM

The establishment probability of a new mating type allele Rearranging for δM, we find

Having approximated the mode of the number of mating types st P β − we now proceed to study the evolutionary dynamics of the δ = β M−1 = M 1 , (15) M M−1 Pst r number of mating types. We begin by computing the probability M M of a successful establishment, QEst, of a newly arising mutant M where we have used the definition of r given in Eq. (11). The Est M in a population of M resident mating types. We define QM as mean time between extinction events is the inverse of this effec- the probability that this novel mating type allele (initially at a tive death rate, i.e. frequency 1/N) reaches the new stationary frequency of mating types (1/(M + 1)) before any of the resident mating types goes Ext 1 1 rM TM = = , (16) extinct. N δM NβM−1 For c < 1 the deterministic equilibrium is an internal fixed point. This is crucial for our approximation of the establishment where the factor N accounts for the fact that we are measuring probability since we will identify it by the survival probability the time until an extinction event in time units of generations. Ext Pst of a corresponding branching process (see Section S5 in the Further, we note that since TM is determined using M (which Supplementary Information). Furthermore, we assume that itself accounts for covariances in allelic frequencies in the sta- Ext N  M, i.e. the population size is sufficiently large that the tionary state), our expression for TM captures the effect of allele resident mating types are not quickly lost through genetic drift. frequency fluctuations in all M types. Under these assumptions we can extend the computation from As we have already analytically calculated rM (see Eq. (11)), Czuppon and Rogers (2019) for obligate sexual reproduction all we now need to evaluate the mean time until the extinction and find of a mating type allele is an expression for βM (see Eq. (16)). We assume that the effective ‘probability birth rate’, βM (the  1 − c  1 probability per unit time that the number of mating types in QEst = . (13) M 1 + c M the system increases) is given by the product of the mutation rate and the establishment probability of a mutant allele (see Eq. (13)): Est In the case of obligate sex (c = 0), QM reduces to 1/M, re- covering the result found in Czuppon and Rogers (2019). For  1 − c  1 β ≈ mQEst = m . (17) facultative sex (0 < c < 1) we see that the establishment proba- M M 1 + c M bility is reduced. Since asexual reproduction increases the time to reach the stationary state in the deterministic system (i.e. the This approximation relies on the frequencies of the resident selection strength for even mating type ratios is reduced, see types being close to their deterministic equilibrium (an assump- Est also Eq. (3)), newly arising mutants spend more time at low fre- tion made in the derivation of QM ). In general this will be true quencies where they are susceptible to purging by genetic drift. if the state containing M types is sufficiently stable and if muta- Est This is reflected by QM decreasing with c (see Figure 3). Our tions are occurring sufficiently infrequently that the population definition of the establishment probability includes that none has time to relax to this quasi-stationary state. If these conditions of the resident mating types become extinct during the inva- are not met, it is possible that a new mutant mating type may sion process. This impedes a straightforward comparison with arise in a population with a highly uneven mating type distribu- Est the case of obligate asexual reproduction, c = 1 (i.e. a neutral tion, where QM will no longer provide an accurate estimate of Moran model). The corresponding establishment probability the establishment probability. We discuss these points further might naïvely be assumed to be 1/(M + 1); however this value below. ignores the survival of all resident mating types and therefore Inserting this effective birth rate into Eq. (16) and substitut- overestimates the actual establishment probability. ing rM from Eq. (11) (see Eq. (S42)), we can express the mean

6 Czuppon and Constable breaks down. The true distribution becomes increasingly flat, 107 while the Gaussian approximation does not respect the bound- ary conditions requiring non-negative number of mating types 105 st ni. Since this results in erroneous approximations of PM, (the crucial ingredient calculating Eq. (14)) when the number of mat- 1000 ing types is highly unstable (i.e. when M  Mo), we do not expect our analytically derived mean extinction time to provide 10 reasonable results in this regime. This reasoning is assessed in 0 50 100 150 more detail and validated in the Supplementary Information (see Section S6). 107 We now seek a more quantitative measure of when we ex- Ext pect Eq. (18) to remain valid. We first note that naturally TM 105 should increase monotonically with the population size, and decrease monotonically with the rate of asexual reproduction 1000 and the number of resident mating types. However, on evalu- ating Eq. (18), we find that these expectations are violated as 10 the relative strength of genetic drift increases (i.e. as the initial 0 500 1000 1500 2000 number of mating types, N/M, becomes small) or, conversely, as the strength of selection for equal numbers of mating types decreases (i.e. c becomes large). In these regimes, the dynamics Figure 4 Mean extinction time of the first mating type allele, of the mating type alleles approach neutrality, and the extinction TM, as a function of N for populations with initially M = 2, time is better approximated by standard results on the neutral M = 3 and M = 4 mating types. Analytic results (see Eq. (20)) multi-allelic Moran model Baxter et al. (2007) (see also Eq. (S51) are plotted as solid lines. Simulation results are averaged over in the Supplementary Information); 103 Gillespie simulations, with error bars indicating the stan- dard deviation of results. Vertical dashed lines indicate the M−1 M s  s  TNeutral = −N (−1)s−1 log . (19) transition between the approximation of Eq. (18) (right of M ∑ s=1 s M M dashed line) and the neutral limit of Eq. (19) (left of dashed Ext Neutral line), defined by Eq. (20). As the rate of asexual reproduction Combining TM and TM , the mean time to extinction of a is increased (from c = 0.1 in panel (a) to c = 0.9 in panel (b)), mating type allele can then be approximated by the mean extinction time drops rapidly (note differing scales ( ∂TExt ∂TExt ∂TExt TExt while M > 0, M < 0, M < 0, on x-axis). T = M ∂N ∂c ∂M M Neutral TM otherwise . (20) extinction time as In Figure 4, we can see that this captures the results of simula- 1 3 !− 2 tions very well. √  M− 2 1 Ext 2(M − 1) 3 M θ − M T = 2π θ 2 In Figure 5 we plot predicted extinction times for regions M 1 + c M − 1 1 θ − M−1 of parameter space that are prohibitively time consuming to  N investigate numerically. The gray regions which dominate the !1−Mθ − 1 θ − 1  1  θ parameter space for low asexual reproduction rates depict extinc- × θθ M θ −  . 3 1 M − 1 tion rates larger than 1011 generations. To set this into context, N 2 θ − M−1 we compare this to the evolutionary history of fungi. Fungi (18) first evolved around 1.5 billion years ago (Wang et al. 1999). As- We first note that as we would expect, Eq. (18) is independent suming approximately 100 generations a year, a conservative of the mutation rate of novel mating type alleles (the mean time estimate considering the doubling time of yeast which is around until the extinction of a mating type allele should not depend 90 minutes, this leads to an order of magnitude guess of 1011 on the time until a new mating type allele arrives). Further, generations for the entire evolutionary history of fungi. Hence, we can see that when the population size, N, is large the mean in the context of obligate sex (see Figure 5 top-left panel), mating time to extinction is dominated by the term raised to the power types alleles are expected to remain fixed in a population while of N in Eq. (18) (explaining the linear growth of log(TExt) for M their number remains less than 6. This condition is severely re- large N in Figure 4). Although Eq. (18) is lengthy, it is useful laxed in populations in which sex is facultative (see for example as it allows us to quickly evaluate the mean extinction time Figure 5 bottom-right panel). Even though extinction times re- for arbitrary parameter values. We can then rapidly explore main high for realistic effective population sizes, some turnover parameter regimes that would be prohibitively time consuming of mating types might be expected over long time periods, e.g. to simulate (see Figure 5). the evolutionary history of fungi. Moreover, this observation The expression derived in Eq. (18) becomes increasingly ac- indicates that mating types loss is most likely be driven by ex- curate with increasing population size, N, decreasing rates of treme population bottlenecks decreasing the effective population asexual reproduction, c, and smaller numbers of initial mating size N. types, M. The approximation can break down however when either N is small, or c or M are large. In this latter range, as the Discussion population dynamics become increasingly dominated by genetic drift, various assumptions involved in the derivation of Eq. (18) The evolutionary mechanisms that drive the number of mating can become invalid. Most importantly the Gaussian approxima- type alleles observed across species has been the topic of numer- tion for the quasi-stationary distribution of a focal mating type ous theoretical studies. Our analysis in the context of haploid

Evolutionary dynamics of mating types 7 esting open problem is to explore other null models that are capable of explaining these discrepancies. Essentially this task translates to identifying additional mechanisms that decrease selection for more mating types (where their number is overesti-

1011 mated) or increase this selection strength (where their number 109 is underestimated). There exist a number of biologically reason-

107 able potential candidates. In Eq. (1) we have assumed mass action encounter rate dy- 105 namics between gametes, leading to a linear relationship be- tween a mating type’s frequency and its probability of finding a 103 sexual partner (i.e (1 − xi)). This implementation neglects active mate search. In reality, in species such as C. reinhardtii or the 2 10 Ditylum brightwellii, that have developed active methods to increase the encounter rate between complementary mating types (Snell and Goodenough 2009; Waite and Harrison 1992), this term might be more accurately described by a decreasing concave up function (see for example Ashby and Gupta (2014)). This would qualitatively recapitulate the results of Iwasa and Sasaki (1987) (Mating Kinetics 3 and 4) and lead to a decrease in the predicted number of mating types. Figure 5 Analytic predictions for the mean extinction time Further, our model assumes that a mating type is determined of a resident mating type allele as a function of N and M by a single locus. Thus, our model is restricted to bipolar mating (see Eq. (20)). The blue dashed line indicates the parameter type systems, as opposed to tetrapolar systems where the mat- regime in which extinction rates become approximately neu- ing type is determined by alleles at two loci (Nieuwenhuis et al. tral (Eq. (19)). Extinction times in the gray shaded region ex- 2013). We expect that additional loci may lead to larger numbers 11 ceed 10 generations. As these time are approximately longer of mating types maintained in the population (as mating type than the evolutionary history of fungi, they have been omitted allele combinations can be regenerated through recombination, for clarity. the extinction rate of mating types will be reduced). Indeed, this would be consistent with the larger number of mating types empirically observed in the tetrapolar S. commune, as well as the self-incompatibility adds new results and perspectives on the general observation that tetrapolarity is associated with an in- evolutionary dynamics of this type of balancing selection. crease in mating type diversity (Nieuwenhuis et al. 2013). While A specificity of mating type SI, as studied here, when com- it is interesting to note that the number of alleles predicted by pared to gametophytic SI, as prevalent in plants, is the possibility our model capture the number of A and B self-incompatibility to reproduce asexually. Our findings support and extend the complexes in S. commune separately (see Table 1), theoretically previous result that switching between an asexual and a sexual addressing the mutation-extinction hypothesis for tetrapolar sys- life cycle significantly reduces the number of mating types in a tems in a systematic way, along with the effects of encounter population (Constable and Kokko 2018). Empirically, available rate dynamics, will be interesting areas for future investigations. data appears to support the view that more frequent sex is corre- Thus far we have focused on modelling considerations that lated with more mating types (see Table 1 and again Constable may well strengthen the mutation-extinction balance hypothesis. and Kokko (2018)). However we are hampered from making Of course, non-neutral differences between mating types must any strong empirical claims in this area as a result of a paucity be considered as well, and a pluralistic view that incorporates of empirical estimates of rates of sex in natural populations. the remaining hypotheses for observed mating type numbers In most species such estimates are absent, however with new may be necessary. For instance, we have not accounted for the methodologies and understanding for estimating the rate of sex fact that newly arising mutants are unlikely to be fully compat- arising increasingly frequently (Nieuwenhuis et al. 2018; Ennos ible with (or equally as fit as) resident mating types, a biologi- and Hu 2018; Hartfield et al. 2018), it is our hope that this gap in cally important consideration (Power 1976; Hadjivasiliou and the literature will soon be filled. Pomiankowski 2016; Krumbeck et al. 2019) that would lower the In Table 1 we test our model quantitatively against four number of types relative to our idealized estimate. In addition, species where estimates for the rate of sex are available. We we have not considered how forms of homothalism (i.e. the evo- compare the number of mating types observed empirically with lution of self-compatibility) may affect our results. For instance the mode number predicted theoretically. Since estimates for the most yeasts have evolved the ability to switch mating types mutation rate of new mating type alleles and the effective pop- between sexual generations (Nieuwenhuis and Immler 2016; ulation size parameters are difficult to obtain, we considered a Nieuwenhuis et al. 2018), while among ciliates some species ex- range of values. We find that while facultative sex explains much hibit probabilistic mating type expression (Paixao et al. 2011). of the variation in mating type number, there are quantitative Indeed, as demonstrated in Hadjivasiliou et al. (2016), the rapid disagreements. In particular, for a range of parameters (particu- loss of mating type diversity when sex is rare and local popula- larly large effective population size) the number of mating types tion sizes are low (see Figure 5) can drive selection for mating is overestimated in Saccharomyces cerevisiae, C. reinhardtii and type switching in order to maintain the capacity for sexual re- Tetrahymena, while it is underestimated in S. commune. production. Such factors make a comparison of our model with Given the simplicity of the model that we have proposed, these species problematic. Further mechanisms that may limit these quantitative disagreements are not unexpected. An inter- the number of mating types that are not included in our model,

8 Czuppon and Constable Est Species Estimated c Empirical M Null model Mo QM TM (Generations) ∗ Est −4 274 6 S. cerevisiae 1 − 1/2000 [1] 2 [5] 9 ≥ Mo ≥ 1 Q2 = 1.25 × 10 10 ≥ T2 ≥ 10 Est −4 707 9 C. reinhardtii 1 − 1/770 [2] 2 [6] 14 ≥ Mo ≥ 1 Q2 = 3.25 × 10 10 ≥ T2 ≥ 10 Est −3 1822 20 Tetrahymena † 1 − 1/100 [3] 3 − 9 [7] 38 ≥ Mo ≥ 4 Q3 = 1.68 × 10 10 ≥ T3 ≥ 10 Est −4 153 4 Q9 = 5.58 × 10 10 ≥ T9 ≥ 10 Est −5 S. commune 0 [4] 23, 328 ‡ [8] 510 ≥ Mo ≥ 48 Q23,328 = 4.29 × 10 41 ? ≥ T23,328 ≥ 0.5 ? Est −3 26 Q288 = 3.47 × 10 10 ≥ T288 ≥ 58 ? Est −2 337 4 Q81 = 1.23 × 10 10 ≥ T81 ≥ 10 ∗ Considering only strains of S. cerevisiae that do not feature mating type switching, for which our model is appropriate. † Considering only Tetrahymena species with synclonal inheritance, for which our model is appropriate. ‡ Tetrapolar with mating type compatibility determined by genetic complexes A (with 288 variants) and B (with 81 variants). ? Lower-bound extinction time is effectively neutral (see Eq. (20)). Table 1 A comparison of empirical values for the number of mating types in various isogamous species with the predictions of the 7 5 Est null model. We assume an effective population size of between 10 ≥ N ≥ 10 in our calculations of M and TM (note that QM is −6 −8 independent of N). In our calculation for M, we further assume a per-generation mutation rate, mg = mN, of 10 ≥ mg ≥ 10 . For references to empirical data, see the list at the end of the manuscript. For a visualization of the full theoretical distributions of st M, PM, for each parameter set, see Section S8 in the Supplementary Information. including UPI and costly mate search are reviewed in Billiard selective strength enters in both the establishment probability et al. (2011). and the stationary distribution, our computed extinction time Our model for haploid SI is conceptually similar to exten- accounts for the fluctuating selective pressure. sively studied gametophytic SI systems observed in plants. Yet These explicit estimates on establishment probabilities and we take a novel mathematical approach in estimating the num- typical extinction times yield new insight into the dynamics that ber of mating types supported by a finite population. Instead drive the low number of mating types predicted by our model of using the extinction boundaries of the one-dimensional diffu- in the rare sex regime. One might initially suspect that these sion approximation of a focal mating type as in most previous numbers are the result of a high turnover of mating type alleles, studies (Wright 1939, 1960, 1964; Ewens 1964; Yokoyama and i.e. frequent extinction and invasion events. However we find Nei 1979; Yokoyama and Hetherington 1982; Vallejo-Marín and that in fact they are a result of very low invasion probabilities Uyenoyama 2008), we study a birth-death process on the total for novel mating types, combined with rapidly decreasing ex- number of mating types. Utilizing just the local description of tinction times as a function of resident mating type diversity. It the stationary distribution around an interior stable fixed point, is worth mentioning that although our approximations for the we circumvent the problem of the diffusion approximation for extinction time break down when the resident state becomes systems with a stable deterministic behavior being inaccurate at highly unstable (i.e. when M  Mo), these represent states that the boundaries (Assaf and Meerson 2017). This leads to robust would be very short lived in nature, and thus are not relevant st predictions for the stationary probability distribution PM (Fig- from a biological perspective. ure 1), and, importantly, the mean extinction time of a mating Finally, in Table 1 we link our theoretical observation of small type allele, TM (Figure4). numbers of mating type alleles yielding very large extinction In calculating this mean extinction time, we deviate from the times (see Figure 4) to some species examples. Indeed, the large approaches taken before which rely on the one-dimensional extinction times found by the quantitative study are in line with dynamics of a focal mating type (Takahata 1990; Vekemans previous empirical observations of long terminal branches in al- and Slatkin 1994). This previous method compares the one- lelic genealogies under negative-frequency dependent selection, dimensional diffusion to a time-rescaled neutral coalescent, thus e.g. in fungi (May et al. 1999) and Solanaceae (Uyenoyama 1997). obtaining estimates for the diversification rate (i.e. the establish- Similar to previous simulation studies (Slatkin and Muirhead ment rate of a novel mating type). This comparison is conducted 1999; Gervais et al. 2011) we find that the number of resident by assuming constant selective strength. In our model, this mating types strongly influences the diversification rate; the larger the resident number, the lower the diversification rate. would be equivalent to assuming that the term ∑j xixj is con- stant. However, with a varying number of present mating types This slowdown is ubiquitously observed in natural systems (and indeed, also with fluctuations in the mating type frequen- under balancing selection, such as self-incompatibility in cies themselves) this value is not constant in time. Hence, a fungi (May et al. 1999) or Solanaceae (Uyenoyama 1997) and comparison with a time-rescaled neutral coalescent does not MHC-systems (Solberg et al. 2008). We find that long terminal seem appropriate since the selection strength would need to be branches in self-incompatibility systems, corresponding to re-assessed after each coalescence event which is associated with old mating type allele ages (with some being older than the a loss of a mating type. This variation in selection strength has corresponding species age (Richman 2000)) emerge naturally been empirically observed by analyzing the genome of various under balancing selection for two reasons: (i) the stability of the Coprinus cinereus (mushroom fungus) populations worldwide internal equilibrium can lead to extremely large extinction times that show that selective forces in the mating type dynamics are of alleles thus enabling such long branches and (ii) the lower dependent on the number of distinct lineages (May et al. 1999); establishment rate due to a large number of resident SI alleles see also Richman (2000) for other examples in the context of slows down the diversification rate and hence decreases the balancing selection. Our technique, explicitly computing the number of newly established mating types. mean extinction time by the effective birth-death process on the number of mating types, avoids this problem. Since the varying In conclusion, we analyzed the evolutionary dynamics of self-

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Evolutionary dynamics of mating types 11 Tsai, I. J., D. Bensasson, A. Burt, and V. Koufopanou, 2008 Pop- sexual (Nieuwenhuis and Aanen 2012). We therefore assume ulation genomics of the wild yeast Saccharomyces paradoxus: a rate of asexual reproduction of c = 0 for S. commune. We Quantifying the life cycle. Proc. Natl. Acad. Sci. 105: 4957– note however that the life-cycle of S. commune features more 4962. complicated dynamics than accounted for by our model; it is Uyenoyama, M. K., 1997 Genealogical structure among alleles multicellular and can exhibit vegetative growth as a haploid regulating self-incompatibility in natural populations of flow- mycelium. ering plants. Genetics 147: 1389–1400. [5] The mating type of Saccharomyces species is determined by Vallejo-Marín, M. and M. K. Uyenoyama, 2008 pp. 53–71 in On one of two alleles at the MAT locus, termed α and a (Butler 2007). the Evolutionary Modification of Self-Incompatibility: Implications We note that often these variants are referred to as idiomorphs of Partial Clonality for Allelic Diversity and Genealogical Structure, (rather than alleles) because of large differences in their size and Springer Berlin Heidelberg. genetic composition (Butler et al. 2004). Vasconcelos, V. V., F. P. Santos, F. C. Santos, and J. M. Pacheco, [6] C. reinhardtii has two mating types, denoted mt+ and mt−, 2017 Stochastic dynamics through hierarchically embedded each determined by one of two alleles at the MT locus. Similar markov chains. Phys. Rev, Lett. 118: 058301. mating type determination is found among other isogamous Vekemans, X. and M. Slatkin, 1994 Gene and allelic genealogies algae (Hamaji et al. 2013; Sekimoto 2017). at a gametophytic self-incompatibility locus. Genetics 137: [7] Tetrahymena species T. americanis, T. hegewischi, T. hyperan- 1157–1165. gularis and T. pigmentosa have mating type numbers nine, eight, Waite, A. and P. J. Harrison, 1992 Role of sinking and ascent four and three respectively (Phadke and Zufall 2009). We take during sexual reproduction in the marine diatom Ditylum care to focus only on these synclonal species, defined as those brightwellii. Mar. Ecol. Prog. Ser. 87: 113–122. in which mating type is inherited deterministically based on Wang, D. Y.-C., S. Kumar, and S. B. Hedges, 1999 Divergence parental genotype. In caryonidal species, such as T. thermophila, time estimates for the early history of animal phyla and the the mating type of progeny is determined stochastically (Phadke origin of plants, animals and fungi. Proceedings of the Royal and Zufall 2009), and thus a comparison of our model with these Society of London. Series B: Biological Sciences 266: 163–171. species is not appropriate. Wright, S., 1939 The distribution of self-sterility alleles in popu- [8] The mating type of haploid S. commune is determined by lations. Genetics 24: 538–552. two complementary pathways, controlled by unlinked genetic Wright, S., 1960 On the number of self-incompatibility alleles complexes A and B. Each of these regions consists of two weakly maintained in equilibrium by a given mutation rate in a popu- recombining loci, leading to a total of four mating type loci. lation of a given size: a re-examination. Biometrics 16: 61–85. These loci, denoted Aα, Aβ, Bα, and Bβ, have respectively 9, 32, Wright, S., 1964 The distribution of self-incompatibility alleles 9 and 9 alleles (Stankis et al. 1992). Full compatibility between in populations. Evolution 18: 609–619. two mating types is achieved when both the A and B complexes Yokoyama, S. and L. E. Hetherington, 1982 The expected num- of the mates are of different specificities; semi-compatibility ber of self-incompatibility alleles in finite plant populations. occurs when the only one of these complexes is of different Heredity 48: 299–303. specificity (Raudaskoski and Kothe 2019). Yokoyama, S. and M. Nei, 1979 Population dynamics of sex- determining alleles in honey bees and self-incompatibility alleles in plants. Genetics 91: 609–626.

Citations for Table 1. [1] As addressed within the discussion, our model is only strictly appropriate for populations in which the mating type does not switch between generations. Mating type switching is prevalent among many Saccharomyces, however most populations feature non-switching strains (Nieuwenhuis et al. 2018). We assume S. cerevisiae has a similar ecology to S. paradoxus, where sex has been estimated to occur between once every thousand and three thousand generations (Tsai et al. 2008) (indeed, estimates for the rates of outcrossing in both species are comparable). [2] The rate of sex in C. reinhardtii has been estimated to once in every 770 generations (Hasan and Ness 2018). [3] We assume that synclonal Tetrahymena species (such as T. americanis, see [7]) feature similar rates of sex to T. thermophila. Our estimate on the rate of sex is based on a minimal sexually immature period of 60 − 100 fissions in T. thermophila (Doerder et al. 1995). We note however that the frequency of cellular conjugation events observed in other ciliates indicates that lower rates of sex may be appropriate (Lucchesi and Santangelo 2004). [4] Molecular analysis suggests that S. commune features some of the highest rates of sex within the fungal kingdom (Nieuwenhuis and James 2016). In addition, most of the Agaricomycotina (the class to which S. commune belongs) are known to be obligately

12 Czuppon and Constable