Evolution of floral diversity in the genus Croton and related genera (Crotonoideae, Euphorbiaceae)
Croton, a mega-diverse genus with about 1,200 species, has great variation in floral and inflorescence structures. Flowers of Croton are unisexual with great dimorphism between male and female flowers. Previous studies revealed ambiguous interpretations of some characters between two flower sexes, e.g., female filamentous structures vs. male petals, and the nectary in male and female flowers. Great diversity in the androecium was reported but there no detailed investigation has ever been conducted. Knowledge from relatives of Croton in the tribe Crotoneae may help our understanding of floral morphology of Croton. However, their floral morphology is poorly known. Extensive examination of the inflorescence and floral morphology of all genera in the tribe will provide useful characters for taxonomic purpose. Moreover, our study will contribute to better understand the complex floral morphology in the whole family Euphorbiaceae which is under-explored. To explore developmental processes underlying androecial diversity in Croton and tribe Crotoneae, flower buds from about 100 species of Croton and related genera were collected from the wild, cultivated collections or extracted from herbarium sheets. Many techniques were used in the present study, e.g., light microscopy, thin section, scanning electron microscopy and micro-computer tomography (μCT). Morphological data were then mapped on the phylogenetic tree to study character evolution. The tribe Crotoneae is found to be heterogeneous with a combination of many characters. Stamen form, structure and arrangement are different in each genus. However, the synapomorphies of the whole tribe were identified, e.g., the presence of only male flowers on the distal part of the inflorescence, more than 10 stamens and reduced petals in female flowers. Within the tribe Crotoneae, two clades were recognised, i.e., a Sagotia-Sandwithia clade and a Acidocroton-Astraea- Brasiliocroton-Croton clade. In the Sagotia-Sandwithia clade, shared characters are a botryoid inflorescence, petals larger than sepals at anthesis and outermost stamens alternate with petals. In the Acidocroton-Astraea-Brasiliocroton-Croton clade, synapomorphies are the presence of thyrses or racemes, stellate trichomes and stellate- derived trichomes, e.g., lepidote and dendritic types, and the outermost stamen opposite petals. Inflorescence and floral morphologies of the tribe Crotoneae and other related groups in the Euphorbiaceae were also discussed. The question of common origin (homology) between organs in male and female flowers of Croton is a controversial subject. We examined floral development in unusual Croton species with fully developed petals in female flowers, i.e., C. alabamensis and C. schiedeanus, compared with C. chilensis which has filamentous structures in female flowers. Floral ontogeny reveals that filamentous structures should be considered a reductive form of petals. Moreover, we also question the interpretation of the nectary as of staminodial origin since its variable number and position suggest that it may have a receptacular origin. Interestingly, male floral morphology and development are highly diverse with the presence of a concave receptacle (hypanthium) in C. alabamensis, while other species of Croton have a convex receptacle. Stamens of Croton also show centrifugal development, which is highly unique among angiosperms because this condition is normally found in flowers with very high stamen number. There are many enigmatic groups within Croton with extreme variation of floral morphology. Many of these were thought to be distinct genera before the molecular phylogeny classification. Our study reveals great developmental diversity among them. In C. celtidifolius with very high stamen number (>100), stamen development is chaotic due to the breaking down of general whorl development. Several groups of Croton have a reduction of stamen number. In section Moacroton subgenus Quadrilobi, two to six stamens are present with the outermost stamen whorl alternate with petals, suggesting the loss of the centrifugal whorl. In Croton subgenus Geiseleria, several species, i.e., C. monanthogynus, C. michauxii, C. setiger and C. trinitatis, have a reduction of stamen number to three to eight evolving in parallel. They also lose the centrifugal stamen development, but the outermost stamen whorl is opposite petals differently from the condition in section Moacroton. Female flowers from some species also show variation in floral form. C. monanthogynus has a bicarpellate ovary contrary to the generally tricarpellate ovary in the genus and also in the family. Ovaries of C. setiger and C. michauxii have an unusual ovary with one carpel linked with a reduction of other floral organs. However, their detailed morphologies are different, suggesting parallel evolution combined with their distant position on the phylogeny. Our results reveal that the floral morphology in Croton and related genera is highly diverse with complex floral architectures. We use the relatively new technique of computer tomography to study rare and delicate samples. Synapomorphies of the tribe and its subclades and taxonomic informative characters were found which would improve identification of these plants in the field. We found that a combination of morphology, ontogeny and phylogeny facilitate the interpretation of origin and homology of several floral characters, e.g., inflorescence, male petals and female filamentous structures, floral nectary and androecial morphology, within Croton and the tribe Crotoneae. We hope the present study will be an important step towards better understanding floral morphology in the family Euphorbiaceae.