Estimating the number,frequency, and dominance of S-alleles in a natural population of Arabidopsis lyrata(Brassicaceae) with sporophytic control of self-incompatibility

In homomorphic plant self-incompatibility (SI) systems, large numbers of alleles may be maintained at a single Mendelian locus. Most estimators of the number of alleles present in natural populations are designed for gametophytic self-incompatibility systems (GSI) in which the recognition phenotype of the pollen is determined by its own haploid genotype. In sporophytic systems (SSI), the recognition phenotype of the pollen is determined by the diploid genotype of its parent, and dominance differs among alleles. We describe research aimed at estimates of S-allele numbers in a natural population of Arabidopsis lyrata (Brassicaceae), whose SSI system has recently been described. Using a combination of pollination studies and PCR-based identification of alleles at a locus equivalent to the Brassica SRK gene, we identified and sequenced 11 putative alleles in a sample of 20 individuals from different maternal seed sets. The pollination results indicate that we have not amplified all alleles that must be present. Extensive partial incompatibility, nonreciprocal compatibility differences, and evidence of weakened expression of SI in some genotypes, prevent us from determining the exact number of missing alleles based only on cross-pollination data. Although we show that none of the theoretical models currently proposed is completely appropriate for estimating the number of alleles in this system, we estimate that there are between 13 and 16 different S-alleles in our sample, probably between 16 and 25 alleles in the population, and discuss the relative frequency of alleles in relation to dominance.     

Population genetic structure of Arabidopsis lyrata in Europe

Population genetic theory predicts that the self-incompatible and perennial herb, Arabidopsis lyrata, will have a genetic structure that differs from the self-fertilizing, annual Arabidopsis thaliana. We quantified the genetic structure for eight populations of A. lyrata ssp. petraea in historically nonglaciated regions of central Europe. Analysis of 20 microsatellite loci for 344 individuals demonstrated that, in accordance with predictions, diploid populations had high genome-wide heterozygosity (H(O) = 0.48; H(E) = 0.52), high within-population diversity (83% of total) compatible with mutation-drift equilibrium, and moderate differentiation among populations (F(ST) = 0.17). Within a single population, the vast majority of genetic variability (92%) was found at the smallest spatial scale (< 3 m). Although there was no evidence of biparental inbreeding or clonal propagation at this scale (F(IS) = 0.003), significant fine-scale spatial autocorrelation indicated localized gene flow presumably due to gravity dispersed seeds (Sp = 0.018). Limited gene flow between isolated population clusters (regions) separated by hundreds of kilometres has given rise to an isolation by distance pattern of diversification, with low, but significant, differentiation among regions (F(ST) = 0.05). The maintenance of geographically widespread polymorphisms and uniformly high diversity throughout central Europe is consistent with periglacial survival of A. lyrata ssp. petraea north of the Alps in steppe-tundra habitats during the last glacial maximum. As expected of northern and previously glaciated localities, A. lyrata in Iceland was genetically less diverse and highly differentiated from central Europe (H(E) = 0.37; F(ST) = 0.27).     

Cross-species microsatellite markers for elucidating population genetic structure in Arabidopsis and Arabis (Brassicaeae)

Species closely related to model organisms present the opportunity to efficiently apply molecular and functional tools developed by a large research community to taxa with different ecological and evolutionary histories. We complied 42 microsatellite loci that amplify under common conditions in four closely related Arabidopsis: A. thaliana; A. halleri; A. lyrata ssp. lyrata; and A. lyrata ssp. petraea, as well as in one more distantly related crucifer; Arabis drummondii. Variation at these loci is amenable to a diversity of applications including population genetics, phylogeographical analyses, mapping of inter and intraspecific crosses, and recombination mapping. Our analysis of microsatellite variation illustrates significant differences in population genetic parameters among three Arabidopsis species. A population of A. thaliana, an inbreeding annual plant associated with disturbed habitats, was highly monomorphic (P = 8% percent polymorphic loci) and only 0.2% heterozygous for 648 locus-by-individual combinations. A population of the self-incompatible perennial herb, A. halleri, was more genetically variable (P = 71%) and had an excess of heterozygosity that may reflect a recent population bottleneck associated with human-mediated founder events. A population of the self-incompatible perennial herb, A. lyrata ssp. petraea, was even more genetically variable (P = 86%) and appeared to be at mutation-drift equilibrium. Population structure estimated from neutrally evolving loci provides an empirical expectation against which hypotheses of adaptive evolution at functional loci can be tested.     

Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata

We study the segregation of variants of a putative self-incompatibility gene in Arabidopsis lyrata. This gene encodes a sequence that is homologous to the protein encoded by the SRK gene involved in self-incompatibility in Brassica species. We show by diallel pollinations of plants in several full-sib families that seven different sequences of the gene in A. lyrata are linked to different S-alleles, and segregation analysis in further sibships shows that four other sequences behave as allelic to these. The family data on incompatibility provide evidence for dominance classes among the S-alleles, as expected for a sporophytic SI system. We observe no division into pollen-dominant and pollen-recessive classes of alleles as has been found in Brassica, but our alleles fall into at least three dominance classes in both pollen and stigma expression. The diversity among sequences of the A. lyrata putative S-alleles is greater than among the published Brassica SRK sequences, and, unlike Brassica, the alleles do not cluster into groups with similar dominance.     

Population structure in Arabidopsis lyrata: evidence for divergent selection on trichome production

Leaf trichomes may serve several biological functions including protection against herbivores, drought, and UV radiation; and their adaptive value can be expected to vary among environments. The perennial, self-incompatible herb Arabidopsis lyrata is polymorphic for trichome production, and occurs in a glabrous and a trichome-producing form. Controlled crosses indicate that the polymorphism is governed by a single gene, with trichome production being dominant. We examined the hypothesis that trichome production is subject to divergent selection (i.e., directional selection favoring different phenotypes in different populations) by comparing patterns of variation at the locus coding for glabrousness and at eight putatively neutral isozyme loci in Swedish populations of A. lyrata. The genetic diversity (He) and allele number at isozyme loci tended to increase with population size and decreased with latitude of origin, whereas genetic diversity at the locus coding for glabrousness did not vary with population size and increased with latitude of origin. The degree of genetic differentiation at the glabrousness locus was much higher than that at isozyme loci. Genetic identity at isozyme loci was negatively related to geographic distance, suggesting isolation by distance. In contrast, there was no significant correlation between genetic identity at the glabrousness locus and at isozyme loci. The results are consistent with the hypothesis that divergent selection contributes to population differentiation in trichome production in A. lyrata.     

The transition to self-compatibility in Arabidopsis thaliana and evolution within S-haplotypes over 10 Myr

A recent investigation found evidence that the transition of Arabidopsis thaliana from ancestral self-incompatibility (SI) to full self-compatibility occurred very recently and suggested that this occurred through a selective fixation of a nonfunctional allele (PsiSCR1) at the SCR gene, which determines pollen specificity in the incompatibility response. The main evidence is the lack of polymorphism at the SCR locus in A. thaliana. However, the nearby SRK gene, which determines stigma specificity in self-incompatible Brassicaceae species, has extremely high sequence diversity, with 3 very divergent SRK haplotypes, 2 of them present in multiple strains. Such high diversity is extremely unusual in this species, and it suggests the possibility that multiple, different SRK haplotypes may have been preserved from A. thaliana's self-incompatible ancestor. To study the evolution of S-haplotypes in the A. thaliana lineage, we searched the 2 most closely related Arabidopsis species Arabidopsis lyrata and Arabidopsis halleri, in which most populations have retained SI, and found SRK sequences corresponding to all 3 A. thaliana haplogroup sequences. Our molecular evolutionary analyses of these 3 S-haplotypes provide an independent estimate of the timing of the breakdown of SI and again exclude an ancient transition to selfing in A. thaliana. Comparing sequences of each of the 3 haplogroups between species, we find that 2 of the 3 SRK sequences (haplogroups A and B) are similar throughout their length, suggesting that little or no recombination with other SRK alleles has occurred since these species diverged. The diversity difference between the SCR and SRK loci in A. thaliana, however, suggests crossing-over, either within SRK or between the SCR and SRK loci. If the loss of SI involved fixation of the PsiSCR1 sequence, the exchange must have occurred during its fixation. Divergence between the species is much lower at the S-locus, compared with reference loci, and we discuss two contributory possibilities. Introgression may have occurred between A. lyrata and A. halleri and between their ancestral lineage and A. thaliana, at least for some period after their split. In addition, the coalescence times of sequences of individual S-haplogroups are expected to be less than those of alleles at non-S-loci.     

Haplotype structure of the stigmatic self-incompatibility gene in natural populations of Arabidopsis lyrata

We describe analyses of almost full-length sequences (including both the kinase domain and the S-domain) of the putative SRK incompatibility gene of the self-incompatible plant Arabidopsis lyrata. In A. lyrata, the SRK S-domain controls the pistil recognition specificity, as in self-incompatible Brassica species. In alleles from plants derived from natural A. lyrata populations, nonsynonymous and synonymous site diversity values are very high in both domains; even in exons 3 to 7 of the kinase domain, which probably have no recognition functions, 39% of the amino acids are polymorphic. Within populations, diversity between alleles is high, as expected for an incompatibility locus, which should be under frequency-dependent selection within populations, whereas within the different putative allelic classes polymorphism is very low, as predicted from theoretical models when recombination is rare. Nonsynonymous site variability declines in the kinase domain with increasing distance from the S-domain border, although synonymous diversity remains high, and the introns are unalignable. A decline in nonsynonymous diversity is expected due to selective constraints in the kinase domain, in combination with recombination (allowing diversity to decrease at sites distant from those under balancing selection). However, it is unclear whether recombination occurs in the SRK locus, and interpretation of the observed diversity pattern is complicated by apparent gene conversion with a paralogous gene (or genes). Patterns of linkage disequilibrium in our SRK sequences do not support the conclusion that recombination occurs, which was suggested from previous analyses based on Brassica SLG sequences.     

Comparing the linkage maps of the close relatives Arabidopsis lyrata and A. thaliana

We have constructed a genetic map of Arabidopsis lyrata, a self-incompatible relative of the plant model species A. thaliana. A. lyrata is a diploid (n = 8) species that diverged from A. thaliana (n = 5) approximately 5 MYA. Mapping was conducted in a full-sib progeny of two unrelated F(1) hybrids between two European populations of A. lyrata ssp. petraea. We used the least-squares method of the Joinmap program for map construction. The gross chromosomal differences between the two species were most parsimoniously explained with three fusions, two reciprocal translocations, and one inversion. The total map length was 515 cM, and the distances were 12% larger than those between corresponding markers in the linkage map of A. thaliana. The 72 markers, consisting of microsatellites and gene-based markers, were spaced on average every 8 cM. Transmission ratio distortion was extensive, and most distortions were specific to each reciprocal cross, suggesting cytoplasmic interactions. We estimate locations and most probable genotype frequencies of transmission ratio distorting loci (TRDL) with a Bayesian method and discuss the possible reasons for the observed distortions.     

Breakdown of self-incompatibility in the perennial Arabidopsis lyrata (Brassicaceae) and its genetic consequences

Mating systems in plants are known to be highly labile traits, with frequent transitions from outcrossing to selfing. The genetic basis for breakdown in self-incompatibility (SI) systems has been studied, but data on variation in selfing rates in species for which the molecular basis of SI is known are rare. This study surveyed such variation in Arabidopsis lyrata (Brassicaceae), which is often considered an obligately outcrossing species, to examine the causes and genetic consequences of changes in its breeding system. Based on controlled self-pollinations in the greenhouse, three populations from the Great Lakes region of North America included a minority of self-compatible (SC) individuals, while two showed larger proportions of SC individuals and all populations contained some individuals capable of setting selfed seeds. Loss of SI was not associated with particular haplotypes at the S-locus (as estimated by alleles amplified at the SRK locus, the gene controlling female specificity) and all populations contained similar numbers of SRK alleles, suggesting that some other genetic factor is responsible for modifying the SI reaction. The loss of SI has resulted in an effective shift in the mating system, as the two populations with a high frequency of SC individuals showed significantly lower microsatellite-based multilocus outcrossing rates and higher inbreeding coefficients than the other populations. Based on microsatellites, observed heterozygosities and genetic diversity were also significantly depressed in these populations. These findings provide the unique opportunity to examine in detail the consequences of mating system changes within a species with a well-characterized SI system.     

Selection at work in self-incompatible Arabidopsis lyrata: mating patterns in a natural population

Identification and characterization of the self-incompatibility genes in Brassicaceae species now allow typing of self-incompatibility haplotypes in natural populations. In this study we sampled and mapped all 88 individuals in a small population of Arabidopsis lyrata from Iceland. The self-incompatibility haplotypes at the SRK gene were typed for all the plants and some of their progeny and used to investigate the realized mating patterns in the population. The observed frequencies of haplotypes were found to change considerably from the parent generation to the offspring generation around their deterministic equilibria as determined from the known dominance relations among haplotypes. We provide direct evidence that the incompatibility system discriminates against matings among adjacent individuals. Multiple paternity is very common, causing mate availability among progeny of a single mother to be much larger than expected for single paternity.     

Population size, self-incompatibility and genetic rescue in diploid and tetraploid races of Rutidosis leptorrhynchoides (Asteraceae)

Self-incompatibility systems function to prevent inbreeding, and work effectively in large, genetically diverse populations. However, a decrease in population size can reduce genetic diversity at the self-incompatibility locus, which leads to a reduction in mate availability and has important demographic implications for small populations. Currently, little is known about the response of self-incompatible polyploid species to a reduction in population size. In Rutidosis leptorrhynchoides there was a significant decrease in the within-population probability of fertilization with a decline in population size for diploid populations and a marginally significant relationship for tetraploid populations, suggesting that in small populations of both chromosome races fertilization success is reduced due to a decrease in self-incompatibility allele (S-allele) diversity. There was no significant difference between the slopes of the fertility-population size relationship for diploid and tetraploid populations which indicates a similar rate of decline in fertilization success with population size for both chromosome races. Fertilization success increased when crosses were undertaken between populations and this was significantly related to population size for diploid and tetraploid populations, indicating that small populations gain the greatest benefit to fertilization success from crossing between populations. For tetraploid populations the benefits of crossing between populations tended to decline more rapidly with increasing population size. These results suggest that for small populations that have reduced fertilization success, genetic rescue by introducing new genetic material from other populations is an important means of ameliorating mate limitation issues associated with reduced S-allele diversity in both diploid and tetraploid races.     

Selection at work in self-incompatible Arabidopsis lyrata. II. Spatial distribution of S haplotypes in Iceland

We survey the distribution of haplotypes at the self-incompatibility (SI) locus of Arabidopsis lyrata (Brassicaceae) at 12 locations spread over the species' natural distribution in Iceland. Previous investigations of the system have identified 34 functionally different S haplotypes maintained by frequency-dependent selection and arranged them into four classes of dominance in their phenotypic expression. On the basis of this model of dominance and the island model of population subdivision, we compare the distribution of S haplotypes with that expected from population genetic theory. We observe 18 different S haplotypes, recessive haplotypes being more common than dominant ones, and dominant ones being shared by fewer populations. As expected, differentiation, although significant, is very low at the S locus even over distances of up to 300 km. The frequency of the most recessive haplotype is slightly larger than expected for a panmictic population, but consistent with a subdivided population with the observed differentiation. Frequencies in nature reflect effects of segregation distortion previously observed in controlled crosses. The dynamics of the S-locus variation are, however, well represented by a 12-island model and our simplified model of dominance interactions.     

Sporophytic self-incompatibility in Senecio squalidus (Asteraceae): S allele dominance interactions and modifiers of cross-compatibility and selfing rates

Understanding genetic mechanisms of self-incompatibility (SI) and how they evolve is central to understanding the mating behaviour of most outbreeding angiosperms. Sporophytic SI (SSI) is controlled by a single multi-allelic locus, S, which is expressed in the diploid (sporophyte) plant to determine the SI phenotype of its haploid (gametophyte) pollen. This allows complex patterns of independent S allele dominance interactions in male (pollen) and female (pistil) reproductive tissues. Senecio squalidus is a useful model for studying the genetic regulation and evolution of SSI because of its population history as an alien invasive species in the UK. S. squalidus maintains a small number of S alleles (7–11) with a high frequency of dominance interactions. Some S. squalidus individuals also show partial selfing and/or greater levels of cross-compatibility than expected under SSI. We previously speculated that these might be adaptations to invasiveness. Here we describe a detailed characterization of the regulation of SSI in S. squalidus. Controlled crosses were used to determine the S allele dominance hierarchy of six S alleles and effects of modifiers on cross-compatibility and partial selfing. Complex dominance interactions among S alleles were found with at least three levels of dominance and tissue-specific codominance. Evidence for S gene modifiers that increase selfing and/or cross-compatibility was also found. These empirical findings are discussed in the context of theoretical predictions for maintenance of S allele dominance interactions, and the role of modifier loci in the evolution of SI.     

The population genetics of sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae): the number, frequency, and dominance interactions of S alleles across its British range

Sporophytic self-incompatibility (SSI) was studied in 11 British Senecio squalidus populations to quantify mating system variation and determine how its recent colonization of the United Kingdom has influenced its mating behavior. S allele number, frequency, and dominance interactions in populations were assessed using full diallels of controlled pollinations. A mean of 5.1 S alleles per population was observed, and no population contained more than six S alleles. Numbers of S alleles within populations of S. squalidus declined with increasing distance from the center of its introduction (Oxford). Cross-classification of S alleles allowed an estimate of approximately seven and no more than 11 S alleles for the entire British S. squalidus population. The low number of S alleles observed in British S. squalidus compared to other SI species is consistent with the population bottleneck associated with S. squalidus' introduction to the Oxford Botanic Garden and subsequent colonization of Britain. Extensive S allele dominance interactions were observed to be a feature of the S. squalidus SSI system and may represent an adaptive response to improve limited mate availability imposed by the presence of so few S alleles. Multilocus allozyme genotypes were also identified for individuals in all populations and geographic patterns of S locus and allozyme loci variation investigated. Less interpopulation structure was observed for the S locus than for allozyme diversity--a finding indicative of the effects of negative frequency-dependent selection at the S locus maintaining equal S phenotypes within populations and enhancing effective migration between populations.     

Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana

As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana approximately 5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis--Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.     

Isolation and characterization of 13 microsatellite loci for Neochamaelea pulverulenta (Cneoraceae)

We report 13 polymorphic nuclear microsatellite loci for Neochamaelea pulverulenta (Cneoraceae) using an enriched-library approach. Although this plant species is tetraploid, expected patterns for tetrasomic segregation were completely absent, and all loci analysed showed a diploid pattern of inheritance. We detected a total of 102 alleles in 57 individuals genotyped (mean number of alleles per locus was 7.85). The values of observed and expected heterozygosities ranged from 0.193 to 0.737 and 0.425 to 0.812 respectively. Disomic segregation, levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for parentage assignment of dispersed seeds, and for analyses of spatial genetic structure and population connectivity.     

Patterns of genetic diversity in outcrossing and selfing populations of Arabidopsis lyrata

Arabidopsis lyrata is normally considered an obligately outcrossing species with a strong self-incompatibility system, but a shift in mating system towards inbreeding has been found in some North American populations (subspecies A. lyrata ssp. lyrata). This study provides a survey of the Great Lakes region of Canada to determine the extent of this mating system variation and how outcrossing rates are related to current population density, geographical distribution, and genetic diversity. Based on variation at microsatellite markers (progeny arrays to estimate multilocus outcrossing rates and population samples to estimate diversity measures) and controlled greenhouse pollinations, populations can be divided into two groups: (i) group A, consisting of individuals capable of setting selfed seed (including autogamous fruit set in the absence of pollinators), showing depressed outcrossing rates (T(m) = 0.2-0.6), heterozygosity (H(O) = 0.02-0.06) and genetic diversity (H(E) = 0.08-0.10); and (ii) group B, consisting of individuals that are predominantly self-incompatible (T(m) > 0.8), require pollinators for seeds set, and showing higher levels of heterozygosity (H(O) = 0.13-0.31) and diversity (H(E) = 0.19-0.410). Current population density is not related to the shift in mating system but does vary with latitude. Restricted gene flow among populations was evident among all but two populations (F(ST) = 0.11-0.8). Group A populations were more differentiated from one another (F(ST) = 0.78) than they were from group B populations (F(ST) = 0.59), with 41% of the variation partitioned within populations, 47% between populations, and 12% between groups. No significant relationship was found between genetic and geographical distance. Results are discussed in the context of possible postglacial expansion scenarios in relation to loss of self-incompatibility.     

Recent Loss of Self-Incompatibility by Degradation of the Male Component in Allotetraploid Arabidopsis kamchatica

The evolutionary transition from outcrossing to self-fertilization (selfing) through the loss of self-incompatibility (SI) is one of the most prevalent events in flowering plants, and its genetic basis has been a major focus in evolutionary biology. In the Brassicaceae, the SI system consists of male and female specificity genes at the S-locus and of genes involved in the female downstream signaling pathway. During recent decades, much attention has been paid in particular to clarifying the genes responsible for the loss of SI. Here, we investigated the pattern of polymorphism and functionality of the female specificity gene, the S-locus receptor kinase (SRK), in allotetraploid Arabidopsis kamchatica. While its parental species, A. lyrata and A. halleri, are reported to be diploid and mainly self-incompatible, A. kamchatica is self-compatible. We identified five highly diverged SRK haplogroups, found their disomic inheritance and, for the first time in a wild allotetraploid species, surveyed the geographic distribution of SRK at the two homeologous S-loci across the species range. We found intact full-length SRK sequences in many accessions. Through interspecific crosses with the self-incompatible and diploid congener A. halleri, we found that the female components of the SI system, including SRK and the female downstream signaling pathway, are still functional in these accessions. Given the tight linkage and very rare recombination of the male and female components on the S-locus, this result suggests that the degradation of male components was responsible for the loss of SI in A. kamchatica. Recent extensive studies in multiple Brassicaceae species demonstrate that the loss of SI is often derived from mutations in the male component in wild populations, in contrast to cultivated populations. This is consistent with theoretical predictions that mutations disabling male specificity are expected to be more strongly selected than mutations disabling female specificity, or the female downstream signaling pathway.     

Nucleotide variation at the myrosinase-encoding locus, TGG1, and quantitative myrosinase enzyme activity variation in Arabidopsis thaliana

The Arabidopsis thaliana TGG1 gene encodes thioglucoside glucohydrolase (myrosinase), an enzyme catalysing the hydrolysis of glucosinolate compounds. The enzyme is involved in plant defence against some insect herbivores, and is present in species of the order Capparales (Brassicales). Nucleotide variation was surveyed by sequencing c. 2.4 kb of the TGG1 locus in a sample of 28 worldwide A. thaliana accessions, and one Arabidopsis lyrata ssp. lyrata individual. Myrosinase activity was quantified for 27 of these same A. thaliana accessions, plus five additional others. Overall, estimated nucleotide diversity in A. thaliana was low compared to other published A. thaliana surveys, and the frequency distribution was skewed toward an excess of low-frequency variants. Furthermore, comparison to the outgroup species A. lyrata demonstrated that A. thaliana exhibited an excess of high-frequency derived variants relative to a neutral equilibrium model, suggesting a selective sweep. A. thaliana accessions differed significantly in total myrosinase activity, but analysis of variance detected no statistical evidence for an association between quantitative enzyme activity and alleles at the TGG1 myrosinase-encoding locus. We thus conclude that other, unsurveyed factors primarily affect the observed myrosinase activity levels in this species. The pattern of nucleotide variation was consistent with a model of positive selection but might also be compatible with a completely neutral model that takes into account the metapopulation behaviour of this highly inbreeding species which experienced a relatively recent worldwide expansion.