DOI: 10.5061/dryad.qv9s4mwmg
title: CYCLOIDEA paralogs function redundantly to specify dorsal flower development in Mimulus lewisii (Phrymaceae)

description: 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).
description: Methods for generating file: CYC_alignment.txt
 Putative <em>M. lewisii </em>CYC orthologs were
 identified in Sengupta and Hileman (2018):
 Mimulus_lewisii_CYCLOIDEA_like_2b_sc2324_contig13781 (found on scaffold
 2324 of the <em>M</em>. <em>lewisii</em> 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 <em>M. lewisii </em>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
 <em>M. lewisii</em> 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. 
description: # CYCLOIDEA paralogs function redundantly to specify dorsal flower
 development in Mimulus lewisii (Phrymaceae)
 [https://doi.org/10.5061/dryad.qv9s4mwmg](https://doi.org/10.5061/dryad.qv9s4mwmg) CYC\_alignment.txt The DNA sequence alignment used to generate the *CYCLOIDEA* gene tree presented in Fig. 3. Mimulus\_lewisii\_floral\_measurementsB.csv The floral measurements gathered for comparison of RNAi lines to wild type presented in Figs. 6-8, Appendices S8-11. ## Description of the data and file structure CYC\_alignment.txt Fasta formatted alignment; document in .txt format. Mimulus\_lewisii\_floral\_measurementsB.csv spreadsheet of raw trait data in comma delimited format (.csv) <br>