Abstract: Gene duplication and diversification is a significant aspect of gene and genome evolution, even though the evolutionary mechanism following duplication almost certainly varies by genes or gene families. We have identified three PISTILLATA (PI)-like genes, HoPI_1, HoPI_2, and HoPI_3, from basal angiosperm Hedyosmum orientale (Chloranthaceae) and found a functional divergence of these duplicate paralogs in the B-function process. However, the mechanism underlying the divergence of the three HoPI copies remains unclear. In this study, we carefully compared the coding sequence of the three duplicate HoPI paralogs, and investigated their evolutionary process after duplication. Molecular evolutionary analyses suggested that the coding sequence of the relatively recent duplicate paralogs HoPI_1and HoPI_3 may evolve under purifying selection, but the functional constraint that acted on the coding sequence of HoPI_2 may have been relaxed. Domain swapping and site-directed mutagenesis experiments further showed that HoPI_2 has lost class B-function activity through a gradual process of mutational degradation in MADS and I domains. Although we currently have no direct in vivo evidence that HoPI_2 has lost function in floral development, the relaxed selection, in combination with its inability to rescue pi-1 mutant phenotype, indicate that this distant paralog HoPI_2 might have been converted to be functionless in the B-function process. This study provides a detailed molecular characterization of duplicate PI lineage gene divergence in a single individual level, and our results suggest that mutations in the MI region are more likely to have negative effects on the biochemical activity of PI-like proteins.

Key words: B-function, coding sequence divergence, gene duplication, PISTILLATA-like genes, relaxed selection.