Gradual Disintegration of the Floral Symmetry Gene Network Is
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Gradual disintegration of the floral symmetry gene network is implicated in the evolution of a wind-pollination syndrome Jill C. Preston1, Ciera C. Martinez2, and Lena C. Hileman Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045 Edited by Enrico Sandro Coen, John Innes Centre, Norwich, United Kingdom, and approved January 3, 2011 (received for review July 30, 2010) Angiosperms exhibit staggering diversity in floral form, and evo- rical flowers of wind-pollinated Plantago are four-merous in all lution of floral morphology is often correlated with changes in three outer organ whorls (Fig. 1). In early development, both pollination syndrome. The showy, bilaterally symmetrical flowers of Aragoa and Plantago flowers are bilaterally symmetrical due to the model species Antirrhinum majus (Plantaginaceae) are highly the unidirectional emergence of organs in a ventral-to-dorsal se- specialized for bee pollination. In A. majus, CYCLOIDEA (CYC), quence (21). However, flowers at mid to late stages of de- DICHOTOMA (DICH), RADIALIS (RAD), and DIVARICATA (DIV) specify velopment in both genera are fully radially symmetrical (21, 26). the development of floral bilateral symmetry. However, it is unclear It is hypothesized that changes in the expression or bio- to what extent evolution of these genes has resulted in flower chemical function of genes that establish the ancestral condition morphological divergence among closely related members of Plan- of floral bilateral symmetry underlie the evolution of radial taginaceae differing in pollination syndrome. We compared floral symmetry in the Aragoa/Plantago clade (8, 21, 27). The floral symmetry genes from insect-pollinated Digitalis purpurea, which symmetry genetic pathway is best understood in A. majus, and has bilaterally symmetrical flowers, with those from closely related involves the action of three dorsal identity genes, CYCLOIDEA Aragoa abietina and wind-pollinated Plantago major, both of which (CYC), DICHOTOMA (DICH), and RADIALIS (RAD), and one have radially symmetrical flowers. We demonstrate that Plantago, ventral identity gene, DIVARICATA (DIV) (13, 28–32). CYC and but not Aragoa, species have lost a dorsally expressed CYC-like gene DICH are recently duplicated type II TCP transcription factors EVOLUTION and downstream targets RAD and DIV. Furthermore, the single P. that establish bilateral symmetry in both early and late flower major CYC-like gene is expressed across all regions of the flower, development, partly through their positive regulation of the similar to expression of its ortholog in closely related Veronica ser- MYB transcription factor RAD in the dorsal petals and stam- pyllifolia. We propose that changes in the expression of duplicated inode (13, 29, 31, 33). Although not expressed outside the dorsal CYC-like genes led to the evolution of radial flower symmetry in petals and staminode, RAD appears to negatively regulate the Aragoa/Plantago, and that further disintegration of the symmetry ventral identity protein DIV in the dorsal and lateral regions of gene pathway resulted in the wind-pollination syndrome of Plan- the flower (29, 30). Thus, ventral identity is confined to the tago. This model underscores the potential importance of gene loss ventral region of the flower (28, 30). in the evolution of ecologically important traits. Unlike close relatives Veronica serpyllifolia and D. purpurea, which have two and three CYC-like genes, respectively, only one volution of flower symmetry is tightly correlated with polli- CYC-like gene has been recovered from Plantago lanceolata Enator shifts and has been proposed as a key innovation within (ribwort plantain; subgenus Psyllium sensu lato) and P. major the angiosperms (1–6). In the plantain family (Plantaginaceae, (common plantain; subgenus Plantago) genomes, suggesting Lamiales), morphological and phylogenetic analyses suggest that a recent gene loss event in the Plantago lineage (14, 18, 27, 33– the ancestral flower was five-parted, bilaterally symmetrical 35). Furthermore, Reardon et al. recently demonstrated that (monosymmetrical and zygomorphic), and insect-pollinated (7, 8). expression of this gene does not appear to be confined to the For example, in Antirrhinum majus (snapdragon, Antirrhineae) and dorsal region of the P. lanceolata flower (27). Instead, evidence Digitalis purpurea (foxglove, Digitalideae), large bee-pollinated suggests expression in the region below the flower, correlating flowers are five-parted and establish bilateral symmetry due to with the loss of differentiated dorsal flower identity and a shift to differential growth rates along the dorsiventral axis. The dorsal radial symmetry. Interestingly, a nondorsal pattern of expression (adaxial), lateral, and ventral (abaxial) petals and stamens are easily for the V. serpyllifolia ortholog to the single Plantago CYC-like distinguishable based on differences in shape and/or ornamenta- gene was also recently demonstrated (34). The aim of this study tion, further reinforcing a single plane of symmetry within mature is to better understand the role of floral symmetry gene network flowers (Fig. 1; Fig. S1)(8–13). evolution in the shift from bilateral to radial symmetry in the Veroniceae is the sister tribe to Digitalideae, and includes the Veroniceae. Here we test three nonexclusive hypotheses: (i) loss large cosmopolitan genera Veronica (ca. 450 species) and Plantago of a dorsally expressed CYC-like paralog predates evolution of (ca. 200 species) and the small páramo endemic genus Aragoa (19 radial symmetry in the Aragoa/Plantago lineage; (ii) the non- species), each of which is monophyletic based on phylogenetic dorsal expression pattern of the single Plantago CYC-like gene analyses (14–19). In contrast to the ancestral flower form found in Digitalideae, several Veroniceae species have flowers that are four-parted, radially symmetrical (polysymmetrical and actino- Author contributions: J.C.P. and L.C.H. designed research; J.C.P. and C.C.M. performed morphic), and/or wind-pollinated (Fig. 1) (8, 15, 20–22). The small research; J.C.P. analyzed data; and J.C.P. and L.C.H. wrote the paper. flowers of Veronica are primarily fly- and/or self-pollinated, and The authors declare no conflict of interest. develop four sepals and four petals (23, 24). The enlarged dorsal This article is a PNAS Direct Submission. petal is inferred to develop from two dorsal petals that fuse early Data deposition: The sequences reported in this paper have been deposited in the Gen- in development (11, 25) and, consequently, Veronica flowers have Bank database (accession nos. HQ853598–HQ853667. retained bilateral symmetry within the petal whorl (11, 25). 1To whom correspondence should be addressed. E-mail: [email protected]. Whereas the radially symmetrical showy flowers of Aragoa have 2Present address: Section of Plant Biology, University of California, Davis, CA 95616. fi retained the ancestral number of ve sepals but have four identical This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. petals and stamens (11, 12, 21), the diminutive radially symmet- 1073/pnas.1011361108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011361108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Aragoa species, six Plantago species representing four of the five monophyletic subgenera (except the DIV intron), three Veronica species representing three of the twelve subgenera, and the Veronica outgroup Wulfenia carinthiaca (14–19) (Fig. 1; Fig. S2). DEL, ACT, and the DIV intron have been successfully used to resolve species relationships in eukaryotes and other plant groups (37–39). With the exception of ACT, individual analyses of aligned nucleotides strongly supported the monophyly of Veronica, Plan- tago,andAragoa, as previously suggested (14–19) (Fig. S2). Fur- thermore, consistent with combined analyses based on chloroplast gene markers (7, 20), all trees from individual analyses resolved a sister relationship between Aragoa/Plantago and Wulfenia/Veronica genes, with the genes from D. purpurea being more distantly related (Fig. S2). Results were concordant between maximum-likelihood and Bayesian reconstructions, and were supported by likelihood bootstrap (LB) and/or posterior probability (PP) values >70% and >90%, respectively (Fig. S2). Based on concordance between gene trees, DEL,ITS,andDIV intron sequences were concatenated for further analysis. The best trees based on combined analyses were not significantly different from independent analyses; the mono- phyly of each genus and the sister relationship between Aragoa/ Plantago and Wulfenia/Veronica was supported by LB (100% and 76%, respectively) and/or PP (100% and 86%, respectively) values (Fig. 1; Fig. S2). Floral Symmetry Gene Degeneration in Representative Species of Fig. 1. Maximum-likelihood phylogram based on combined nuclear mark- Plantago but Not Aragoa. To determine copy-number and orthology/ ers showing evolution of flower symmetry and its underlying genetic path- paralogy relationships among floral symmetry genes, multiple clones way in Plantaginaceae. Bold branches indicate supported (>70% bootstrap derived from PCR using various CYC-, RAD-, and DIV-like primer and/or >90% posterior probability) generic relationships based on the sets were sequenced from D. purpurea, V. serpyllifolia, P. major, P. chloroplast markers rps2, rbcL, ndhF, rps16, and matK-trnK (blue branch) lanceolata,andA. abietina. Consistent with previous studies, only (7, 15, 20), combined nuclear markers