Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories (Ipomoea) Ji Yang, 1 Hongya Gu, 1 Ziheng Yang 2 1 College of Life Sciences, Peking University, Beijing 100871, China 2 Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, England Received: 21 February 2003 / Accepted: 21 July 2003 Abstract. Chalconesynthase(CHS)isakeyenzyme in the biosynthesis of flavonoides, which are impor- tant for the pigmentation of flowers and act as at- tractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morningglories(Ipomoea),fivefunctionalCHSgenes (A–E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B,andCexperiencedacceleratedratesofaminoacid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories un- derwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/syn- onymous rate ratio (x = d N /d S ) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test sug- gested significant variation in selection pressure among amino acid sites, with a small proportion of themdetectedtobeunderpositiveselectionalongthe branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergenceofsubfamilyABCandsubfamilyDEand is at least partially responsible for a rate increase following gene duplication. Key words: Ipomoea — Chalcone synthase — Positiveselection— Maximumlikelihood—Codon models Introduction Plants of the morning glory genus (Ipomoea) are distributedworldwideandarecharacterizedbyarich diversity of flower colors. For example, the common morningglory(I. purpurea)haswhite,pink,andblue or dark-blue flowers (Clegg and Durbin 2000). The diversity in flower color is almost certainly due to differences in either the structural or the regulatory genes of the flavonoid biosynthetic pathway (Durbin et al. 1995), which culminates in the production of anthocyanins, the main pigments responsible for flower color. The presence or absence of these pig- ments affects the coloration of the floral display, whichattractspollinators.Theanthocyaninpigments are therefore important to reproductive success (Clegg and Durbin 2000). The first step in the flavonoid biosynthetic path- way, the formation of naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA, is catalyzed by the enzyme chalcone synthase (CHS). CHS is a typ- ical homodimeric plant polyketide synthase with two subunits of about 43 kDa (Ferrer et al. 1999). The chalcone synthase from Medicago sativa was first crystallizedforX-raydiffractionanalysis(Ferreretal. 1999). The three-dimensional structure of alfalfa CHS2 revealed that four chemically reactive residues (Cys164, Phe215, His303, and Asn336), which are J Mol Evol (2004) 58:54–63 DOI: 10.1007/s00239-003-2525-3 Correspondence to: Ziheng Yang; email: z.yang@ucl.ac.uk