Recombination, balancing selection and adaptive evolution in the aflatoxin gene cluster of Aspergillus parasiticus

Aflatoxins are toxic and carcinogenic polyketides produced by several Aspergillus species that are known to contaminate agricultural commodities, posing a serious threat to animal and human health. Aflatoxin (AF) biosynthesis is almost fully characterized and involves the coordinated expression of approximately 25 genes clustered in a 70-kb DNA region. Aspergillus parasiticus is an economically important and common agent of AF contamination. Naturally occurring nonaflatoxigenic strains of A. parasiticus are rarely found and generally produce O-methylsterigmatocystin (OMST), the immediate precursor of AF. To elucidate the evolutionary forces acting to retain AF and OMST pathway extrolites (chemotypes), we sequenced 21 intergenic regions spanning the entire cluster in 24 A. parasiticus isolates chosen to represent the genetic diversity within a single Georgia field population. Linkage disequilibrium analyses revealed five distinct recombination blocks in the A. parasiticus cluster. Phylogenetic network analyses showed a history of recombination between chemotype-specific haplotypes, as well as evidence of contemporary recombination. We performed coalescent simulations of variation in recombination blocks and found an approximately twofold deeper coalescence for cluster genealogies compared to noncluster genealogies, our internal standard of neutral evolution. Significantly deeper cluster genealogies are indicative of balancing selection in the AF cluster of A. parasiticus and are further corroborated by the existence of trans-species polymorphisms and common haplotypes in the cluster for several closely related species. Estimates of Ka/Ks for representative cluster genes provide evidence of selection for OMST and AF chemotypes, and indicate a possible role of chemotypes in ecological adaptation and speciation.