The rapid rise and diversification of the angiosperms has puzzled biologists for centuries; processes leading to current angiosperm diversity remain a key question in evolutionary biology, with particular focus on the morphological diversity of flowers. The Zingiberales are an order of tropical monocots that represent an ideal group of plants to study the evolution of floral morphology. The order contains approximately 2,500 species, many of which form specialized pollination relationships with bees, birds, bats, dung beetles, moths, butterflies, and primates (lemurs) via alterations in floral form. After developing a technique for visualizing and then studying gene expression in floral apices, I investigated the role of two candidate gene families, the GLOBOSA (GLO)-like genes and the CYCLOIDEA/ TEOSINTE BRANCHED 1 (CYC/TB1)-like genes, in the evolution of floral morphology in the Zingiberales.

Evolutionary developmental biology often combines methods for examining morphology (e.g. Scanning Electron Microscopy) with analyses of gene expression (e.g. RNA in situ hybridization). Due to differences in tissue preparation for SEM and gene expression analyses, the same specimen cannot be used for both sets of techniques. I developed a method that couples extended-depth-of-field (EDF) epi-illumination microscopy to in situ hybridization in a sequential format, enabling both surface microscopy and gene expression analyses to be carried out on the same specimen (Chapter 1). I first created a digital image of inflorescence apices using epi-illumination microscopy and commercially available EDF software. I then performed RNA in situ hybridizations on photographed apices to assess expression of two developmental genes: Knotted1 (Kn1) in Zea mays (Poaceae) and a GLO homolog in Musa basjoo (Musaceae). I demonstrate that expression signal is neither altered nor reduced in the imaged apices as compared with unphotographed controls. The demonstrated method reduces the amount of sample material necessary for developmental research and enables individual floral development to be placed in the context of the entire inflorescence. While the technique presented is particularly relevant to floral developmental biology, it is applicable to any research where observation and description of external features can be fruitfully linked with analyses of gene expression.

The MADS box transcription factor family has long been identified as an important contributor to the control of floral development. It is often hypothesized that the evolution of floral development across angiosperms and within specific lineages may occur as a result of duplication, functional diversification, and changes in regulation of MADS box genes. In Chapter 2 I examine the role of GLO-like genes, members of the B-class MADS box gene lineage, in the evolution of floral development within the monocot order Zingiberales. I assessed changes in perianth and stamen whorl morphology in a phylogenetic framework. I identified GLO homologs from 50 Zingiberales species and investigated the evolution of this gene lineage. Expression of two GLO homologs was assessed in Costus spicatus Swartz (Costaceae) and Musa basjoo Siebold (Musaceae). Based on the phylogenetic data and expression results, I propose several family-specific losses and gains of GLO homologs that appear to be associated with key morphological changes. The GLO-like gene lineage has diversified concomitant with the evolution of the dimorphic perianth and the staminodial labellum. Duplications and expression divergence within the GLO-like gene lineage may have played a role in floral diversification in the Zingiberales.

In the Zingiberales, evolutionary shifts in symmetry occur in all floral whorls, making this an ideal group of plants in which to study the evolution of this important ecological and developmental trait. The CYC/TB1-like genes have been implicated in the development and evolution of floral symmetry in divergent angiosperm lineages, and I thus chose them as a candidate gene family to investigate their role in the evolution of floral symmetry within the Zingiberales (Chapter 3). I identified both Zingiberales-specific gene duplications and a duplication in the TB1-like (TBL) lineage that predates the divergence of the commelinid monocots. I examined the expression of two TBL genes in Costus spicatus (Costaceae) and Heliconia stricta (Heliconiaceae), two Zingiberales taxa with divergent floral symmetries. I found that TBL gene expression shifts concomitant with shifts in floral symmetry.

Through this body of work we have gained some insight into the mechanics of angiosperm evolution. Duplications in the GLO-like gene lineage in the Zingiberales may have allowed for gene sub- or neofunctionalization and the evolution of new morphologies; in particular, the evolution of differentiated sepals and petals and of the staminodial labellum. In addition, this study adds to the growing body of evidence that CYC/TB1-like genes have been repeatedly recruited through the course of evolution to generate bilateral floral symmetry (zygomorphy). Although this work certainly doesn't preclude the involvement of as yet uncharacterized genes and gene families, it adds to the growing body of evidence that angiosperms as a group do indeed have a genetic `toolkit': a core set of genes that have been variously deployed through evolutionary time to generate both convergent and divergent floral morphologies.