The petals of angiosperm flowers, laminar organs occupying the second floral whorl, often share characteristics such as expression of B-function MADS genes, showy morphology, pigmentation and production of aromatic compounds. However, petals exhibit substantial diversity and organs with petaloid characteristics can be found outside of the second floral whorl, making the definition and identification of these organs rather challenging. The adaxial epidermal surfaces of petals are frequently composed of conical cells whereas those of other floral and vegetative cells are typically not . Identification of petaloid organs has been determined by observation of conical epidermal cells in cases of homeotic conversions involving petals [26, 27]. Analysis of micromorphological characteristics on the adaxial epidermal surfaces of first-whorl organs in the double-corolla species Clermontia parviflora reveals conical cells, a marker of petal identity. This supports the hypothesis of homeotic transformation of sepals to petals.
Based on the phylogenetic analysis presented here and the geographic distribution of Clermontia, the origin of this homeotic transformation was likely established via a single and geologically recent occurrence. The Hawaiian Islands were formed by the northwestward movement of the Pacific tectonic plate over a fixed volcanic plume, resulting in a layout where the islands progress from oldest to youngest in a northwest to southeast manner . C. fauriei is the only species found on Kauai, the oldest of the Hawaii Islands, and it displays the ancestral sepal-petal format. The neighboring island Oahu is home to five species, including C. fauriei and four double-corolla species . 5S-NTS data support previous findings suggesting Clermontia’s substantial sequence divergence from its sister genus Cyanea but low sequence divergence within the genus . Our data do indicate sequence divergence between C. fauriei and all remaining Clermontia species, including both standard sepal-petal and double-corolla species, perhaps reflective of a split age at least by the time of the origin of Kauai. The phylogenetic clustering of ancestral state and petal-petal format species observed suggests multiple potential reversals occurred during the radiation of Clermontia. Similar findings on Hawaiian lobelioid inter-relationships, including evolution of reversals in the Clermontia clade, have been reported by Givnish and collaborators [13, 31, 32].
The transformation of first-whorl sepal organs into organs bearing petal identity in double-corolla Clermontia species, which is often complete, led us to question whether this phenomenon may be regulated by ectopic expression of B-class genes. Our detection of ectopic expression of PI homologs in floral primordia indicates a likely role of PI in the differentiation and determination of outer whorl floral organs, resulting in the development of petal identity. Lacandonia schismatica (Triuridaceae) exhibits a homeotic transformation in which central stamens are surrounded by carpels. The simple displacement of the B-function has been shown to play a role in this morphological shift . In dove tree (Davidia), early B-class gene expression in petaloid bracts has been suggested to lead to a partial petaloid phenotype; however, the lack of late-stage expression makes the mechanism unclear . In Clermontia, not only is expression of PI homologs detected in floral primordia, but we have also shown continued expression in late-stage outer whorl floral organs, supporting the function of B-class genes in the maintenance of homeotic petal identity. Furthermore, we show that expression of a variety of other MADS-box gene homologs involved in various floral and non-floral functions, specifically AP3, TM6, SEP3, AGL6, SVP and SOC1, present expression patterns that are largely or completely consistent between standard sepal-petal and double-corolla species (Additional file 7).
Expression of a Clermontia AP3 homolog is detected in the outer whorl of both standard groundplan species and double-corolla species. Therefore, we hypothesize that ectopic expression of PI homologs would be sufficient to induce the obligate AP3-PI heterodimer autoregulatory feedback loop. In Arabidopsis, expression of PI alone is not able to induce petaloidy in vegetative organs; however, it is when co-expressed with AP3 in vegetative organs or expressed alone in outer whorl perianth . The expression of an AP3 homolog in the outer whorl perianth in Clermontia indicates that the genetic background condition required for ectopic PI expression to induce petaloidy is present. SEP3 is not able to induce petaloidy on its own, but has been shown to increase petaloid characteristics of ectopic petaloid organs, and the heterodimer AP3-PI forms a ternary complex with SEP3 [35, 36]. We hypothesize that in Clermontia, low-level expression of SEP3 and AP3 homologs in the ancestral condition would set up a context in which ectopic expression of PI would be enough to induce petal identity in the outer whorl perianth.
Gene duplications among B-class genes, resulting in the euAP3, TM6 and PI groups, have been shown to be of evolutionary significance in the production of floral diversity. It has been suggested that extension of the B-class gene model has played a diversity-generating role in cases with a history of gene duplication among PI homologs or AP3 homologs in taxonomic groups with otherwise undifferentiated first and second whorls . Here, we demonstrate ectopic and sustained expression of both Clermontia PI homolog duplicates, which clearly derive from a lineage-specific duplication within Campanulaceae (Additional file 8). This apparent subfunctionalization event following gene duplication may have played a key role in the events following PI duplication in Clermontia. The two homologs may be largely functionally redundant, or one may be expressed first and induce the expression of the other, consistent with an autoregulatory feedback loop when obligatory dimerization with AP3 occurs.
The precise regulation that restricts ectopic expression of PI homologs to the first-whorl primordia in early and late-stage organs, as seen in Clermontia parviflora, indicates that small regulatory changes may be responsible for the dramatic change in the floral groundplan established in eudicots. This tight spatiotemporal regulation allows for the deployment of a drastic homeotic mutation without causing potentially deleterious pleiotropic effects, such as transformation of the carpel whorl into stamens. In Arabidopsis, ectopic expression of B-function genes has been shown to be sufficient to transform the carpel whorl into staminoid organs . The specific regulation of this heritable mutation is necessary to produce viable organisms, since simple overexpression would be likely to cause infertility through the production of staminoid carpels. Other viable homeotic transformations within the angiosperm groundplan have occurred but have not led to radiations as seen in Clermontia.
The double-corolla phenotype may not have initially acted with adaptive advantage, its success perhaps relying instead on passive expansion of the mutation by random genetic drift in small populations in the unstable environments of the Hawaiian Islands. The sepal-petal ancestral status of Clermontia is supported by our phylogenetic analysis and the uniformly eudicot-standard floral morphology of its sister genus Cyanea and further closely related outgroup lobelioid species. While phylogenetic analysis demonstrates multiple possible reversals to the ancestral condition, the radiation of the double-corolla mutation indicates the long-term viability of the mutation. This gives insight into possible mechanisms that may have shaped diversity among floral groundplans in past unstable environments; small regulatory changes, as opposed to large coding-sequence differences, could account for morphological differentiation in other island groups such as the Hawaiian silverswords, Hawaiian mints and Canarian Crassulaceae.