The evolution of selfing from outcrossing ancestors in Brassicaceae: what have we learned from variation at the S-locus?

Evolutionary transitions between mating systems have occurred repetitively and independently in flowering plants. One of the most spectacular advances of the recent empirical literature in the field was the discovery of the underlying genetic machinery, which provides the opportunity to retrospectively document the scenario of the outcrossing to selfing transitions in a phylogenetic perspective. In this review, we explore the literature describing patterns of polymorphism and molecular evolution of the locus controlling self-incompatibility (S-locus) in selfing species of the Brassicaceae family in order to document the transition from outcrossing to selfing, a retrospective approach that we describe as the 'mating system genes approach'. The data point to strikingly contrasted scenarios of transition from outcrossing to selfing. We also perform original analyses of the fully sequenced genomes of four species showing self-compatibility, to compare the orthologous S-locus region with that of functional S-locus haplotypes. Phylogenetic analyses suggest that all species we investigated evolved independently towards loss of self-incompatibility, and in most cases almost intact sequences of either of the two S-locus genes suggest that these transitions occurred relatively recently. The S-locus region in Aethionema arabicum, representing the most basal lineage of Brassicaceae, showed unusual patterns so that our analysis could not determine whether self-incompatibility was lost secondarily, or evolved in the core Brassicaceae after the split with this basal lineage. Although the approach we detail can only be used when mating system genes have been identified in a clade, we suggest that its integration with phylogenetic and population genetic approaches should help determine the main routes of this predominant mating system shift in plants.

Can we continue to neglect genomic variation in introgression rates when inferring the history of speciation? A case study in a Mytilus hybrid zone.

The use of molecular data to reconstruct the history of divergence and gene flow between populations of closely related taxa represents a challenging problem. It has been proposed that the long-standing debate about the geography of speciation can be resolved by comparing the likelihoods of a model of isolation with migration and a model of secondary contact. However, data are commonly only fit to a model of isolation with migration and rarely tested against the secondary contact alternative. Furthermore, most demographic inference methods have neglected variation in introgression rates and assume that the gene flow parameter (Nm) is similar among loci. Here, we show that neglecting this source of variation can give misleading results. We analysed DNA sequences sampled from populations of the marine mussels, Mytilus edulis and M. galloprovincialis, across a well-studied mosaic hybrid zone in Europe and evaluated various scenarios of speciation, with or without variation in introgression rates, using an Approximate Bayesian Computation (ABC) approach. Models with heterogeneous gene flow across loci always outperformed models assuming equal migration rates irrespective of the history of gene flow being considered. By incorporating this heterogeneity, the best-supported scenario was a long period of allopatric isolation during the first three-quarters of the time since divergence followed by secondary contact and introgression during the last quarter. By contrast, constraining migration to be homogeneous failed to discriminate among any of the different models of gene flow tested. Our simulations thus provide statistical support for the secondary contact scenario in the European Mytilus hybrid zone that the standard coalescent approach failed to confirm. Our results demonstrate that genomic variation in introgression rates can have profound impacts on the biological conclusions drawn from inference methods and needs to be incorporated in future studies.

Molecular evolution within and between self-incompatibility specificities.

Genes under multiallelic balancing selection have sharply contrasted evolutionary dynamics across timescales, with much longer coalescence time among functionally distinct allelic lines but much shorter coalescence time among gene copies within allelic lines as compared with the genomic background. In this paper, we combine theoretical and empirical approaches to investigate patterns of molecular evolution within and between self-incompatibility (SI) specificities. We first use numerical simulations to investigate coalescence times within allelic lines in a subdivided population for a sporophytic SI system. We then report on a comprehensive analysis of nucleotide polymorphism among gene copies within five distinct allelic lines in the closely related Arabidopsis halleri and Arabidopsis lyrata. In line with our model predictions, we find that the observed level of polymorphism among gene copies was generally low but differed among allelic lines. The data provide compelling direct evidence for recombination and/or gene conversion not only within the two most recessive allelic lines but also between two closely related but distinct allelic lines, suggesting that recombination at the Arabidopsis SI locus is possible in the absence of large sequence divergence among haplotypes. We observed shared polymorphic sites between the two species in one allelic line and strikingly similar haplotypes in another allelic line. We discuss whether convergent evolution may have led to this pattern and suggest that these observations are consistent with ongoing or very recent introgression, as previously documented.

Effect of balancing selection on spatial genetic structure within populations: theoretical investigations on the self-incompatibility locus and empirical studies in Arabidopsis halleri.

The effect of selection on patterns of genetic structure within and between populations may be studied by contrasting observed patterns at the genes targeted by selection with those of unlinked neutral marker loci. Local directional selection on target genes will produce stronger population genetic structure than at neutral loci, whereas the reverse is expected for balancing selection. However, theoretical predictions on the intensity of this signal under precise models of balancing selection are still lacking. Using negative frequency-dependent selection acting on self-incompatibility systems in plants as a model of balancing selection, we investigated the effect of such selection on patterns of spatial genetic structure within a continuous population. Using numerical simulations, we tested the effect of the type of self-incompatibility system, the number of alleles at the self-incompatibility locus and the dominance interactions among them, the extent of gene dispersal, and the immigration rate on spatial genetic structure at the selected locus and at unlinked neutral loci. We confirm that frequency-dependent selection is expected to reduce the extent of spatial genetic structure as compared to neutral loci, particularly in situations with low number of alleles at the self-incompatibility locus, high frequency of codominant interactions among alleles, restricted gene dispersal and restricted immigration from outside populations. Hence the signature of selection on spatial genetic structure is expected to vary across species and populations, and we show that empirical data from the literature as well as data reported here on three natural populations of the herb Arabidopsis halleri confirm these theoretical results.

High paternal diversity in the self-incompatible herb Arabidopsis halleri despite clonal reproduction and spatially restricted pollen dispersal.

The number of sires fertilizing a given dam is a key parameter of the mating system in species with spatially restricted offspring dispersal, since genetic relatedness among maternal sibs determines the intensity of sib competition. In flowering plants, the extent of multiple paternity is determined by factors such as floral biology, properties of the pollen vector, selfing rate, spatial organization of the population, and genetic compatibility between neighbours. To assess the extent of multiple paternity and identify ecological factors involved, we performed a detailed study of mating patterns in a small population of a self-incompatible clonal herb, Arabidopsis halleri. We mapped and genotyped 364 individuals and 256 of their offspring at 12 microsatellite loci and jointly analysed the level of multiple paternity, pollen and seed dispersal, and spatial genetic structure. We found very low levels of correlated paternity among sibs (P(full-sib) = 3.8%) indicating high multiple paternity. Our estimate of the outcrossing rate was 98.7%, suggesting functional self-incompatibility. The pollen dispersal distribution was significantly restricted (mean effective pollen dispersal distance: 4.42 m) but long-distance successful pollination occurred and immigrating pollen was at most 10% of all pollination events. Patterns of genetic structure indicated little extent of clonal reproduction, and a low but significant spatial genetic structure typical for a self-incompatible species. Overall, in spite of restricted pollen dispersal, the multiple paternity in this self-incompatible species was very high, a result that we interpret as a consequence of high plant density and high pollinator service in this population.

Unequal allelic frequencies at the self-incompatibility locus within local populations of Prunus avium L.: an effect of population structure?

In this paper, we investigated the genetic structure and distribution of allelic frequencies at the gametophytic self-incompatibility locus in three populations of Prunus avium L. In line with theoretical predictions under balancing selection, genetic structure at the self-incompatibility locus was almost three times lower than at seven unlinked microsatellites. Furthermore, we found that S-allele frequencies in wild cherry populations departed significantly from the expected isoplethic distribution towards which balancing selection is expected to drive allelic frequencies (i.e. identical frequency equal to the inverse of the number of alleles in the population). To assess whether this departure could be caused either by drift alone or by population structure, we used numerical simulations to compare our observations with allelic frequency distributions expected : (1) within a single deme from a subdivided population with various levels of differentiation; and (2) within a finite panmictic population with identical allelic diversity. We also investigated the effects of sample size and degree of population structure on tests of departure from isoplethic equilibrium. Overall, our results showed that the observed allele frequency distributions were consistent with a model of subdivided population with demes linked by moderate migration rate.

Gene and allelic genealogies at a gametophytic self-incompatibility locus.

The properties of gene and allelic genealogies at a gametophytic self-incompatibility locus in plants have been investigated analytically and checked against extensive numerical simulations. It is found that, as with overdominant loci, there are two genealogical processes with markedly different time scales. First, functionally distinct allelic lines diverge on an extremely long time scale which is inversely related to the mutation rate to new alleles. These alleles show a genealogical structure which is similar, after an appropriate rescaling of time, to that described by the coalescent process for genes at a neutral locus. Second, gene copies sampled within the same functional allelic line show genealogical relationships similar to neutral gene genealogies but on a much shorter time scale, which is on the same order of magnitude as the harmonic mean of the number of gene copies within an allelic line. These results are discussed in relation to data showing trans-specific polymorphisms for alleles at the gametophytic self-incompatibility locus in the Solanaceae. It is shown that population sizes on the order of 4 x 10(5) and a mutation rate per locus per generation as high as 10(-6) could account for estimated allelic divergence times in this family.

Evolutionary dynamics of sporophytic self-incompatibility alleles in plants.

The stationary frequency distribution and allelic dynamics in finite populations are analyzed through stochastic simulations in three models of single-locus, multi-allelic sporophytic self-incompatibility. The models differ in the dominance relationships among alleles. In one model, alleles act codominantly in both pollen and style (SSIcod), in the second, alleles form a dominance hierarchy in pollen and style (SSIdom). In the third model, alleles interact codominantly in the style and form a dominance hierarchy in the pollen (SSIdomcod). The SSIcod model behaves similarly to the model of gametophytic self-incompatibility, but the selection intensity is stronger. With dominance, dominant alleles invade the population more easily than recessive alleles and have a lower frequency at equilibrium. In the SSIdom model, recessive alleles have both a higher allele frequency and higher expected life span. In the SSIdomcod model, however, loss due to drift occurs more easily for pollen-recessive than for pollen-dominant alleles, and therefore, dominant alleles have a higher expected life span than the more recessive alleles. The process of allelic turnover in the SSIdomcod and SSIdom models is closely approximated by a random walk on a dominance ladder. Implications of the results for experimental studies of sporophytic self-incompatibility in natural populations are discussed.

Allelic genealogies in sporophytic self-incompatibility systems in plants.

Expectations for the time scale and structure of allelic genealogies in finite populations are formed under three models of sporophytic self-incompatibility. The models differ in the dominance interactions among the alleles that determine the self-incompatibility phenotype: In the SSIcod model, alleles act codominantly in both pollen and style, in the SSIdom model, alleles form a dominance hierarchy, and in SSIdomcod, alleles are codominant in the style and show a dominance hierarchy in the pollen. Coalescence times of alleles rarely differ more than threefold from those under gametophytic self-incompatibility, and transspecific polymorphism is therefore expected to be equally common. The previously reported directional turnover process of alleles in the SSIdomcod model results in coalescence times lower and substitution rates higher than those in the other models. The SSIdom model assumes strong asymmetries in allelic action, and the most recessive extant allele is likely to be the most recent common ancestor. Despite these asymmetries, the expected shape of the allele genealogies does not deviate markedly from the shape of a neutral gene genealogy. The application of the results to sequence surveys of alleles, including interspecific comparisons, is discussed.

Patterns of allozyme variation in diploid and tetraploid Centaurea jacea at different spatial scales.

The extent and spatial patterns of genetic variation at allozyme markers were investigated within and between diploid and autotetraploid knapweeds (Centaurea jacea L. sensu lato, Asteraceae) at contrasted geographic scales: (1) among populations sampled from a diploid-tetraploid contact zone in the northeastern part of the Belgian Ardennes, and (2) within mixed populations from that zone where diploids and tetraploids coexist. Our data were also compared with a published dataset by Sommer (1990) describing allozyme variation in separate diploid and tetraploid knapweeds populations collected throughout Europe. Genetic diversity was higher in tetraploids. In the Belgian Ardennes and within the mixed populations, both cytotypes had similar levels of spatial genetic structure, they were genetically differentiated, and their distributions of allele frequencies were not spatially correlated. In contrast, at the European scale, diploids and tetraploids did not show differentiated gene pools and presented a strong correlation between their patterns of spatial genetic variation. Numerical simulations showed that the striking difference in patterns observed at small and large geographic scales could be accounted for by a combination of (1) isolation by distance within cytotypes; and (2) partial reproductive barriers between cytotypes and/or recurrent formation of tetraploids. We suggest that this may explain the difficulty of the taxonomic treatment of knapweeds and of polyploid complexes in general.

Allozyme diversity and genetic structure in South-Western populations of heather, Calluna vulgaris.

Genetic diversity, population genetic structure and gene flow in Calluna vulgaris (L.) Hull were assessed by means of seven allozyme loci scored in 18 populations from the South-Western area of the species' range. Genetic diversity was lower (HT = 0.20) than reported for long-lived widespread species but was characterized by a high number of alleles per locus (5.60 at the species level) of which more than 70% were rare. More than 95% of genetic variation was found at the intrapopulation level (GST = 0.047). High levels of past gene flow were inferred, based on the allozyme data (Nm = 5.2 from GST . Nm = 10.2 from the 'private allele' method). Calluna vulgaris exhibited several geographic patterns of genetic variation. Both cluster analysis, constructed with various genetic distances and principal components analysis showed that Spanish and Pyrenean populations were clearly different from those collected in the Massif Central and Belgium. Also, a trend for decreasing genetic diversity towards Northern populations was detected. These patterns might be related to the post-glacial history of Calluna. In addition, it is shown that isolation by distance has played a role in the geographic shaping of genetic variation in this species.

Genetic structure and mating systems of metallicolous and nonmetallicolous populations of Thlaspi caerulescens.

• Genetic variation structure and breeding system were investigated in metallicolous (MET) and nonmetallicolous (NONMET) populations of the heavy-metal hyperaccumulator Thlaspi caerulescens from Belgium, Luxembourg and the French Mediterranean region. • Allozyme variation showed a clear differentiation between the two ecotypes in Belgium and Luxembourg but not in southern France, in line with the lower degree of geographical isolation between the two ecotypes in this region. • In both regions inbreeding coefficient (FIS ) of NONMET populations was significantly higher compared to MET populations. Pollen/Ovule ratios were significantly higher in MET compared with NONMET populations. • These results suggest that NONMET populations of T. caerulescens are more self-fertile than their MET counterparts. This contrasts with earlier studies on other metal-tolerant species in which selfing rates were higher in MET populations. This pattern may be explained by the fact that both ecotypes are not in sympatry in the populations studied, and therefore reproductive isolation has not been selected to maintain the adaptations to metal-contaminated soils. In addition, higher selfing rates in NONMET populations may have evolved as a mechanism of reproductive assurance, because these populations generally are at low densities.

Patterns of allozymic variation within calluna vulgaris populations at seed bank and adult stages

We investigated the spatial genetic structure within and between two plots of Calluna vulgaris and the extent to which the soil seed bank differed genetically from adults at seven allozyme loci. Averaged over the two plots, the seed bank and adult populations contained very similar levels of genetic diversity. Moreover, seeds contained in a single soil core (100 cm3) exhibited similar mean allozyme diversity to the surrounding adult population, indicating that the seed bank preserves genetic diversity at a very local scale. Few differences in allelic frequencies were found between the seed bank and its surrounding adult population in each plot. Mean GST indicated a lack of differentiation between the two plots at adult (GST = 0.008) and seed bank (GST = 0.002) stages. Low interplot differentiation is consistent with the outcrossing mating system of the population (tm = 0.91 in one plot) and its history of human disturbance. In contrast, spatial autocorrelation analysis of adults indicated a genetic structure at a very local scale, with positive autocorrelation for all alleles below 2 m in one plot and with a pattern of positive autocorrelation below 8 m in the two plots. Current limitation to seed dispersal rather than spatial extension of clones is thought to be responsible for local genetic structure.

New insights from fine-scale spatial genetic structure analyses in plant populations.

Many empirical studies have assessed fine-scale spatial genetic structure (SGS), i.e. the nonrandom spatial distribution of genotypes, within plant populations using genetic markers and spatial autocorrelation techniques. These studies mostly provided qualitative descriptions of SGS, rendering quantitative comparisons among studies difficult. The theory of isolation by distance can predict the pattern of SGS under limited gene dispersal, suggesting new approaches, based on the relationship between pairwise relatedness coefficients and the spatial distance between individuals, to quantify SGS and infer gene dispersal parameters. Here we review the theory underlying such methods and discuss issues about their application to plant populations, such as the choice of the relatedness statistics, the sampling scheme to adopt, the procedure to test SGS, and the interpretation of spatial autocorrelograms. We propose to quantify SGS by an 'Sp' statistic primarily dependent upon the rate of decrease of pairwise kinship coefficients between individuals with the logarithm of the distance in two dimensions. Under certain conditions, this statistic estimates the reciprocal of the neighbourhood size. Reanalysing data from, mostly, published studies, the Sp statistic was assessed for 47 plant species. It was found to be significantly related to the mating system (higher in selfing species) and to the life form (higher in herbs than trees), as well as to the population density (higher under low density). We discuss the necessity for comparing SGS with direct estimates of gene dispersal distances, and show how the approach presented can be extended to assess (i) the level of biparental inbreeding, and (ii) the kurtosis of the gene dispersal distribution.

Isolation by distance in a continuous population: reconciliation between spatial autocorrelation analysis and population genetics models.

Analysis of the spatial genetic structure within continuous populations in their natural habitat can reveal acting evolutionary processes. Spatial autocorrelation statistics are often used for this purpose, but their relationships with population genetics models have not been thoroughly established. Moreover, it has been argued that the dependency of these statistics on variation in mutation rates among loci strongly limits their interest for inferential purposes. In the context of an isolation by distance process, we describe relationships between a descriptor of the spatial genetic structure used in empirical studies, Moran's I statistic and population genetics parameters. In particular, we point out that, when Moran's I statistic is used to describe correlation in allele frequencies at the individual level, it provides an estimator of Wright's coefficient of relationship. We also show that the latter parameter, as a descriptor of genetic structure, is not influenced by selfing rate or ploidy level. Under specific finite population models, numerical simulations show that values of Moran's I statistic can be predicted from analytical theory. These simulations are also used to estimate the time taken to approach a structure at equilibrium. Finally, we discuss the conditions under which spatial autocorrelation statistics are little influenced by variation in mutation rates, so that they could be used to estimate gene dispersal parameters.

Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size.

We investigate the distribution of sizes of fragments obtained from the amplified fragment length polymorphism (AFLP) marker technique. We find that empirical distributions obtained in two plant species, Phaseolus lunatus and Lolium perenne, are consistent with the expected distributions obtained from analytical theory and from numerical simulations. Our results indicate that the size distribution is strongly asymmetrical, with a much higher proportion of small than large fragments, that it is not influenced by the number of selective nucleotides nor by genome size but that it may vary with genome-wide GC-content, with a higher proportion of small fragments in cases of lower GC-content when considering the standard AFLP protocol with the enzyme MseI. Results from population samples of the two plant species show that there is a negative relationship between AFLP fragment size and fragment population frequency. Monte Carlo simulations reveal that size homoplasy, arising from pulling together nonhomologous fragments of the same size, generates patterns similar to those observed in P. lunatus and L. perenne because of the asymmetry of the size distribution. We discuss the implications of these results in the context of estimating genetic diversity with AFLP markers.

The signature of balancing selection: fungal mating compatibility gene evolution.

A key problem in evolutionary biology has been distinguishing the contributions of current and historical processes to the maintenance of genetic variation. Because alleles at self-recognition genes are under balancing selection, they exhibit extended residence times in populations and thus may provide unique insight into population demographic history. However, evidence for balancing selection and extended residence times has almost exclusively depended on identification of transspecific polymorphisms; polymorphisms retained in populations through speciation events. We present a broadly applicable approach for detecting balancing selection and apply it to the b1 mating type gene in the mushroom fungus Coprinus cinereus. The comparison of neutral molecular variation within and between allelic classes was used to directly estimate the strength of balancing selection. Different allelic classes are defined as encoding different mating compatibility types and are thus potentially subject to balancing selection. Variation within an allelic class, where all alleles have the same mating compatibility type, provided an internal standard of neutral evolution. Mating compatibility in this organism is determined by the complex A mating type locus, and b1 is one of several redundantly functioning genes. Consequently, we conducted numerical simulations of a model with two subloci and varying levels of recombination to show that balancing selection should operate at each sublocus. Empirical data show that strong balancing selection has indeed occurred at the b1 locus. The widespread geographic distribution of identical b1 alleles suggests that their association with differing A mating types is the result of recent recombination events.

The effect of subdivision on variation at multi-allelic loci under balancing selection.

Simulations are used to investigate the expected pattern of variation at loci under different forms of multi-allelic balancing selection in a finite island model of a subdivided population. The objective is to evaluate the effect of restricted migration among demes on the distribution of polymorphism at the selected loci at equilibrium, and to compare the results with those expected for a neutral locus. The results show that the expected number of alleles maintained, and numbers of nucleotide differences between alleles, are relatively insensitive to the migration rate, and differentiation remains low even under very restricted migration. However, nucleotide divergence between copies of functionally identical alleles increases sharply when migration decreases. These results are discussed in relation to published surveys of allelic diversity in MHC and plant self-incompatibility systems, and to the possibility of inferring ancient population genetic events and processes. In addition, it is shown that, for sporophytic self-incompatibility systems, it is not necessarily true in a subdivided population that recessive alleles are more frequent than dominant ones.

The effect of hitch-hiking on genes linked to a balanced polymorphism in a subdivided population.

The effect of multi-allelic balancing selection on nucleotide diversity at linked neutral sites was investigated by simulations of subdivided populations. The motivation is to understand the behaviour of self-recognition systems such as the MHC and plant self-incompatibility. For neutral sites, two types of subdivision are present: (1) into demes (connected by migration), and (2) into classes defined by different functional alleles at the selected locus (connected by recombination). Previous theoretical studies of each type of subdivision separately have shown that each increases diversity, and decreases the relative frequencies of low-frequency variants, at neutral sites or loci. We show here that the two types of subdivision act non-additively when sampling is at the whole population level, and that subdivision produces some non-intuitive results. For instance, in highly subdivided populations, genetic diversity at neutral sites may decrease with tighter linkage to a selected locus or site. Another conclusion is that, if there is population subdivision, balancing selection leads to decreased expected FST values for neutral sites linked to the selected locus. Finally, we show that the ability to detect balancing selection by its effects on linked variation, using tests such as Tajima's D, is reduced when genes in a subdivided population are sampled from the total population, rather than within demes.

Higher impact of female than male migration on population structure in large mammals.

We simulated large mammal populations using an individual-based stochastic model under various sex-specific migration schemes and life history parameters from the blue whale and the Asian elephant. Our model predicts that genetic structure at nuclear loci is significantly more influenced by female than by male migration. We identified requisite comigration of mother and offspring during gravidity and lactation as the primary cause of this phenomenon. In addition, our model predicts that the common assumption that geographical patterns of mitochondrial DNA (mtDNA) could be translated into female migration rates (Nmf) will cause biased estimates of maternal gene flow when extensive male migration occurs and male mtDNA haplotypes are included in the analysis.