White clover (Trifolium repens) is naturally polymorphic for cyanogenesis (hydrogen cyanide release following tissue damage). The ecological factors favouring cyanogenic and acyanogenic plants have been examined in numerous studies over the last half century, making this one of the best-documented examples of an adaptive polymorphism in plants. White clover cyanogenesis is controlled by two, independently segregating Mendelian genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolysing enzyme, linamarase. In this study, we examine the molecular evolution and population genetics of Li as it relates to the cyanogenesis polymorphism. We report here that Li exists as a single-copy gene in plants possessing linamarase activity, and that the absence of enzyme activity in li/li plants is correlated with the absence of much or all of the gene from the white clover genome. Consistent with this finding, we confirm by reverse transcription-polymerase chain reaction that Li gene expression is absent in plants lacking enzyme activity. In a molecular population genetic analysis of Li and three unlinked genes using a worldwide sample of clover plants, we find an absence of nucleotide variation and statistically significant deviations from neutrality at Li; these findings are consistent with recent positive directional selection at this cyanogenesis locus.

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/ absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors. C YANOGENESIS (cyanide release following tissue damage) occurs in .2650 plant species, including ferns, gymnosperms, monocots, and dicots (Seigler and Brinker 1993). In its most common form, cyanogenesis involves the interaction of two compounds, cyanogenic glycosides and their hydrolyzing enzymes; these are separated in intact tissue and brought into contact with tissue disruption. White clover (Trifolium repens L., Fabaceae) is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in natural populations (Armstrong et al. 1913;Ware 1925). This polymorphism arises through the presence/absence of both of the underlying cyanogenic components. Inheritance of cyanogenesis in white clover follows a simple Mendelian two-locus, two-allele model (Coop 1940;Melville and Doak 1940;Corkill 1942): the gene Ac/ac controls the presence/absence of cyanogenic glucosides; and the independently segregating gene Li/li controls the presence/absence of linamarase, a cyanogenic b-glucosidase (reviewed by Hughes 1991). Plants that carry at least one dominant (functional) allele at both genes (Ac and Li) are cyanogenic, while the occurrence of two nonfunctional alleles at either gene confers the acyanogenic phenotype.White clover is a common legume of fields, lawns, and pastures in mesic temperate climates. It is a native of Eurasia but has been introduced worldwide as a forage crop and lawn plant. It is a perennial and an insectpollinated, obligate outcrosser; plants also spread vegetatively by stolons. Chromosome number and genetic map data indicate that white clover is an allotetraploid (e.g., Barrett et al. 200...

Understanding the molecular evolution of genes that underlie intraspecific polymorphisms can provide insights into the process of adaptive evolution. For adaptive polymorphisms characterized by gene presence/absence (P/A) variation, underlying loci commonly show signatures of long-term balancing selection, with gene-presence and gene-absence alleles maintained as two divergent lineages. We examined the molecular evolution of two unlinked P/A polymorphisms that underlie a well-documented adaptive polymorphism for cyanogenesis (hydrogen cyanide release with tissue damage) in white clover. Both cyanogenic and acyanogenic plants occur in this species, and the ecological forces that maintain this chemical defence polymorphism have been studied for several decades. Using a sample of 65 plants, we investigated the molecular evolution of sequences flanking the two underlying cyanogenesis genes: Ac/ac (controlling the presence/absence of cyanogenic glucosides) and Li/li (controlling the presence/absence of their hydrolysing enzyme, linamarase). A combination of genome walking, PCR assays, DNA sequence analysis and Southern blotting was used to test whether these adaptive P/A polymorphisms show evidence of long-term balancing selection, or whether gene-absence alleles have evolved repeatedly through independent deletion events. For both loci, we detect no signatures of balancing selection in the closest flanking genomic sequences. Instead, we find evidence for variation in the size of the deletions characterizing gene-absence alleles. These observations strongly suggest that both of these polymorphisms have been evolving through recurrent gene deletions over time. We discuss the genetic mechanisms that could account for this surprising pattern and the implications of these findings for mechanisms of rapid adaptive evolution in white clover.

Abstract. Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd 2÷. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40°C, first described by Zhang et al. (1991), is insensitive to Cd 2÷ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40°C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type fagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of fagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different fagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell-body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.

There is growing evidence for the convergent evolution of physically linked gene clusters encoding chemical defense pathways. Metabolic clusters are proposed to evolve because they ensure co-inheritance of all required genes where the defense is favored, and prevent inheritance of toxic partial pathways where it is not. This hypothesis rests on the assumption that clusters evolve in species where selection favors intraspecific polymorphism for the defense; however, they have not been examined in polymorphic species. We examined metabolic cluster evolution in relation to an adaptive polymorphism for cyanogenic glucoside (CNglc) production in clover. Using 163 accessions, we performed CNglc assays, BAC sequencing, Southern hybridizations and molecular evolutionary analyses. We find that the CNglc pathway forms a 138-kb cluster in white clover, and that the adaptive polymorphism occurs through presence/absence of the complete cluster. Component genes are orthologous to those in the distantly related legume Lotus japonicus. These findings provide empirical support for the co-inheritance hypothesis, and they indicate that adaptive CNglc variation in white clover evolves through recurrent deletions of the entire pathway. They further indicate that the shared ancestor of many important legume crops was likely cyanogenic and that this defense was lost repeatedly over the last 50 Myr.