Distinct Regulatory Changes Underlying Differential Expression of TCP Genes Associated with 32 Petal Variations in Zygomorphic Flowers of Petrocosmea (Gesneriaceae)

91 floral zygomorphy mainly through specific expression in dorsal petals of a flower. Their 93 expression changes are usually related to morphological diversity of zygomorphic flowers. However, it is still a challenge to elucidate the molecular mechanism underlying their expression differentiation. It is also unknown whether CINCINNATA ( CIN )-like TCP genes, locally 96 controlling cell growth and proliferation, are involved in the evolution of floral zygomorphy. To 97 address these questions, we selected two closely related species, i.e. Petrocosmea glabristoma and 98 P. sinensis with distinct petal morphology, to conduct expression, hybridization, mutant, and 99 allele-specific expression (ASE) analyses. The results show that the size change of the dorsal 100 petals between the two species is mainly mediated by the expression differentiation of CYC1C and 101 CYC1D , while the shape variation of all petals is related to the expression change of CIN1 . In 102 reciprocal F1 hybrids, the expression of CYC1C , CYC1D , and CIN1 conforms to an additive 103 inheritance mode, consistent with the petal phenotypes of hybrids. Through ASE analyses, we find 104 that the expression differentiation of these TCP genes is underlain by distinctly different types of 105 regulatory changes. We suggest that highly redundant paralogs with identical expression patterns 106 and interspecific expression differentiation may be controlled by remarkably different regulatory 107 pathways because natural selection may favor different regulatory modifications rather than 108 coding sequence changes of key developmental genes in generating morphological diversity. 109 110 111 112 113 114 115 116 117 118 119 whether different regulatory pathways or their interplay account the expression differentiation floral symmetry related genes therefore the diversification dorsal zygomorphic flowers. In this study, two Petrocosmea species, i.e. P. glabristoma and P. sinensis as the representatives of two clades, were selected to investigate possible regulatory pathways driving the morphological divergence of zygomorphic flowers by employing multiple approaches, including comparative expression analyses of CYC - and CIN -like TCP genes, hybridization, mutant, and ASE assays. The results showed that the expression differentiation of CYC1C , CYC1D and CIN1 coordinately promoted the morphological divergence of the dorsal petals that is characteristic of different types of zygomorphic flowers in the two species. In addition, both CYC - and CIN -like genes conformed to an additive inheritance mode in reciprocal F1 hybrids, 193 consistent with the phenotypes of the hybrids. We further found that cis -regulatory changes, 194 trans -regulatory changes, and a complex of both changes contributed to the expression differentiation of CYC1C , CYC1D , and CIN1 , respectively. This report will shed significant light on how regulatory changes underlie expression differentiation of key developmental genes and therefore morphological variations of the dorsal petals in zygomorphic flowers. Real-time PCR was also performed to investigate the expression patterns of all CIN -like 267 genes in all floral organs of the second whorl in both parents and F1 hybrids. The results showed 268 that CIN1 expression levels were similar in the dorsal, lateral, and ventral petals in both P.