CYCLOIDEA paralogs function redundantly to specify dorsal flower development in Mimulus lewisii (Phrymaceae)

Premise: Duplicated genes (paralogs) are abundant in plant genomes and their retention may influence the function of genetic programs and contribute to evolutionary novelty. How gene duplication affects genetic modules, and the forces that contribute to paralog retention are outstanding questions. The CYCLOIDEA(CYC)-dependent flower symmetry program is a model for understanding the evolution of gene duplication, providing multiple examples of paralog partitioning and novelty. However, a novel CYC gene lineage duplication event near the origin of Higher Core Lamiales (HCL) has received little attention.

Methods: To understand the evolutionary fate of duplicated HCL CYC2 genes, we determined the effects on flower symmetry of suppressing MlCYC2A and MlCYC2B expression using RNA interference (RNAi). We determined flower symmetry phenotypic effects in single and double silenced backgrounds and coupled this with expression surveys of MlCYC2A, MlCYC2B, and a putative downstream RADIALIS (MlRAD5) ortholog.

Key results: MlCYC2A and MlCYC2B jointly contribute to bilateral flower symmetry. MlCYC2B exhibits a clear dorsal flower identity function and may additionally function in carpel development.  MlCYC2A functions in establishing dorsal petal shape. Further, our results suggest an MlCYC2A–MlCYC2B regulatory interaction which may affect pathway homeostasis.

Conclusions: Our results suggest that Higher Core Lamiales-specific CYC paralogs may be selectively retained for their joint contribution to bilateral flower symmetry, similar to the independently-derived CYC paralogs in the Lamiales model for bilateral flower symmetry research, Antirrhinum majus (snapdragon).

Methods
Methods for generating file: CYC_alignment.txt
Putative M. lewisii CYC orthologs were identified in Sengupta and Hileman (2018): Mimulus_lewisii_CYCLOIDEA_like_2b_sc2324_contig13781 (found on scaffold 2324 of the M. lewisii LF10 genome assembly v1.8 and as contig 13781 in the LF10 transcriptome v1.2, here MlCYC2A) and Mimulus_lewisii_CYCLOIDEA_like_2a_sc424_contig9407 (found on scaffold 424 of the M. lewisii LF10 genome assembly v1.8 and as contig 9407 in the LF10 transcriptome v1.2, here MlCYC2B). We recovered coding sequences for these genes from the M. lewisii LF10 draft genome (www.mimubase.org) and aligned them to known higher core Lamiales (HCL) CYC2A and CYC2B lineage genes (Appendices S1 and S2; see Supplemental Data with this article) in Geneious Prime v.2021.2.2. We manually aligned the coding sequences from start to stop codon with reference to amino acid translations and removed regions of high sequence divergence for which we could not confidently assign homology. We used these alignments to estimate gene relationships under Maximum Likelihood, GTR+Gamma model of molecular evolution in RAxML (Stamatakis, 2014) implementing 1000 bootstrap replicates. 

Methods for generating file: Mimulus_lewisii_floral_measurementsB.csv
To determine changes in flower morphology we characterized floral phenotypes of MlCYC2A, MlCYC2B and MlCYC2A:MlCYC2B RNAi lines. When possible, we collected the first several open flowers and photographed each flower from multiple viewpoints on a scaled stage. To photograph the inner corolla tube, we dissected flowers along the center of the ventral petal between the two wild-type nectar guide ridges. We measured 15 floral traits (Fig. 2; Appendix S4) per flower using Fiji (https://imagej.net/Fiji). Each measurement was taken 3x and we recorded the mean. For petal shape, we took the width-to-length ratio to account for variation in overall flower size.