Evolution and Expression Patterns of CYC/TB1 Genes in Anacyclus: Phylogenetic Insights for Floral Symmetry Genes in Asteraceae

Abstract

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development.

Keywords: Anacyclus; Asteraceae; CYC/TB1; CYC2 diversification; CYCLOIDEA; floral symmetry.

Figure 1

(A) Majority rule consensus tree from Bayesian analysis of the nucleotide dataset with excluded third codon positions (tree II) with the main CYC/TB1 gene lineages identified by colored boxes. Genes from Anthemideae are in bold. Branches affected by episodes of positive selection are indicated by black thick lines. Numbers in red represent posterior probabilities above 0.50. (B) Summary tree of the phylogeny of Asteraceae (modified from Panero et al., 2014) illustrating the relationships among the genera included in this study and their corresponding tribes.

Figure 2

Inflorescences of wild (A) and trumpet (B) individuals of Anacyclus clavatus. (C–F) Relative expression levels of A. clavatus genes AcCYC2a–AcCYC2d in vegetative and reproductive tissues of wild (colored bars) and trumpet (black bars) individuals. Bars represent relative differences in gene expression level in the tested tissues and are the average of three biological replicates. Error bars show the standard deviation. Cap 1, capitulum stage 1; Cap 2, capitulum stage 2; Ray, peripheric ray flower (wild type, bilateral) or tubular (“trumpet-type,” actinomorphic); Disc, disc flowers. Photographs by M.A. Bello (A) and R. Riina (B).

Figure 3

Morphology of Anacyclus capitulum and florets, and tissue-specific expression of AcCYC2 genes during capitulum development. (A) Section of a Anacyclus capitulum hybridized with a digoxigenin-labeled probe complementary to AcCYC2d. Youngest flower meristems (fm) can be seen at the top, older flowers to the left and right. AcCYC2d transcripts accumulate in young flower meristems and in meristems that are initiating corolla lobe primordia (*). In older flowers mRNA is detectable in developing stamens (st) and ovules (ov). No signal is detectable in developing corolla lobes (lb). (B) Scanning electron microscopy (SEM) image of a capitulum of an age comparable to that in (A). (C) Close up of an ovule displaying AcCYC2d expression. (D) Young disc floret showing AcCYC2a mRNA signal in stamens and ovule but not in corolla lobes. (E) SEM image of disc flowers comparable to those in (D).