Phylogenetic analysis of the "ECE" (CYC/TB1) clade reveals duplications predating the core eudicots

Abstract

Flower symmetry is of special interest in understanding angiosperm evolution and ecology. Evidence from the Antirrhineae (snapdragon and relatives) indicates that several TCP gene-family transcription factors, especially CYCLOIDEA (CYC) and DICHOTOMA (DICH), play a role in specifying dorsal identity in the corolla and androecium of monosymmetric (bilateral) flowers. Studies of rosid and asterid angiosperms suggest that orthologous TCP genes may be important in dorsal identity, but there has been no broad phylogenetic context to determine copy number or orthology. Here, we compare published data from rosids and asterids with newly collected data from ranunculids, caryophyllids, Saxifragales, and Asterales to ascertain the phylogenetic placement of major duplications in the "ECE" (CYC/TB1) clade of TCP transcription factors. Bayesian analyses indicate that there are three major copies of "CYC" in the ECE clade, and that duplications leading to these copies predate the core eudicots. CYC1 contains no subsequent duplications and may not be expressed in floral tissue. CYC3 exhibits similar patterns of duplication to CYC2 in several groups. Using RT-PCR, we show that, in flowers of Lonicera morrowii (Caprifoliaceae), DipsCYC2B is expressed in the four dorsal petals and not in the ventral petal. DipsCYC3B is expressed in flower and petal primordia, possibly most strongly in the ventral petal.

Fig. 1.

Published phylogenetic relationships of Arabidopsis and Dipsacales TCP genes. (A) Phylogeny of Arabidopsis TCP genes, modified from ref. , showing the major split between the CYC/TB1 subfamily and the PCF subfamily. ∗, Taxa with R domain. The ECE clade is enclosed in a dashed line. (B) Relationships of Dipsacales sequences modified from ref. . The major duplications are shown by using lines within a tube, representing the known species tree. Locating the phylogenetic position of the duplications leading to the three major copies (dashed lines) is the aim of this study.

Fig. 2.

Phylogeny of CYC-like genes using Bayesian analysis. Clades with >75% posterior probability are displayed with numbers above the lines. (A) Phylogeny with all taxa sampled. (B) Schematic showing just the major groups. The placements of previously named genes from Arabidopsis and Antirrhinum are labeled; and all included copies from these two model organisms are shown in bold. ∗, Polysymmetric species in CYC2 and CYC3. Species names are given in Table 1. virg., virginianum; amph., amphibium; cham., chamissoniana; aem., aemula.

Fig. 3.

Image of agarose gel electrophoresis of cDNA from L. morrowii flowers. CYC1 indicates expression of DipsCYC1, CYC2 indicates expression of DipsCYC2B, and CYC3 indicates expression of DipsCYC3B. G3pdh is included as a control. Bird’s-eye view of flower is included showing the differentiation between the four dorsal petals and the single ventral petal. Dotted circles indicate the portions of the flower used in each RNA extraction.

Fig. 4.

Phylogenies of floral MADS-box genes and the ECE clade, comparing the phylogenetic location of duplications. Each hypothesized duplication is indicated by a black dot. Non-rosid or -asterid eudicots are shown in bold. Trees of APETALA1, APETALA3, AGAMOUS, and SEPALLATA are modified from refs. –, respectively.